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Construction of pathogenic, biologic and genetic bases of the

1. 41 2 1 6 The soil borne plant pathogenic fungus Rhizoctonia solani K hn 42 2 2 Materials and Methods 55d roce eE T GUAE dra o irae 44 2 2 1 Sampling fungal isolation and DNA extraction from Rhizoctonia solani AG 344 2 2 2 Microsatellite genotyping eeeeeeeeeeeeeeeeen eee ener 46 2 2 3 Data analysis occisis ie eser bartasxa xix PrRaxa cic sas cdks Deskas uu aAA centel aiia ssec csl M IM XR IE 47 2 2 3 1 Microsatellite information content eeeeeeeeeereneneereennnnnnn 47 2 2 32 Genotype dlversily ini een ie oe 47 2 2 3 3 Gene diversity and population differentiation 47 2 2 3 4 Population differentiation eeeeeeeeeeeeeeeeeeeeeeeeen nennen 48 2 2 3 5 Reproductive mode Gametic and Hardy Weinberg Equilibrium tests 48 2 2 3 6 Test for Admixture and hidden population structure 49 2 2 3 7 Population bottleneck and demographic parameters 49 22 Rel Sari 50 2 3 1 Microsatellite information genotype and gene diversity 50 2 3 2 Gene diversity and population differentiation 50 2 3 3 Reproductive mode Gametic and Hardy Weinberg Equilibrium tests
2. Test Black scurf Municipality Temperature response Stem canker Fungicide sensitivity N N Subachoque 13 7 Cogua 14 7 Sibate 12 6 Silvia 7 6 Pasto 11 5 Ventaquemada 11 7 Soraca 15 6 LaUnion 10 6 Carcasi 10 6 Chitaga 8 4 Total 111 60 The isolates used for the pathogenicity tests were included in the test of infestation of sclerotia on tubers temperature response and fungicide sensitivity 3 2 3 Response to temperatures and sensitivity to the fungicide thifluzamide Fungal isolates from long term storage were plated on PDA dishes and grown for five days at room temperature 24 hours before the experiment each isolate was grown and the plates for the assay were prepared as follows i Plates with PDA were prepared for the temperature assays and ii plates with 20 ml of PDA amended with 14 2 ppm of the fungicide A plug of mycelia of 5 mm of diameter was took from the growing area with a cork borer and was transferred to the center of the plates Plates for each treatment were kept at each temperature evaluated for 15 and 25 C in a fitotron labline Biotronette without light The temperature used for the fungicide assay was 25 C Each isolate was replicated across three plates Each treatment was conducted by one person during the course of a single day After 48 hours the size of colonies from each plate were delimited with a permanent marker and all the isolates were kept in the previous conditions o
3. ooccccccccnncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnononnnnnnninnnininininss Table 2 1 Factors conditioning the evolutionary risk of plant pathogens ssssssss Table 2 2 Geographic origin of the populations of Rhizoctonia solani AG 3 used in this study Table 2 3 Overall measures of clonal diversity of Rhizoctonia solani AG 3 infecting potatoes in Colombia 3 ert re A date atr Mee aste cathe adeo Ret Ee ER dg Table 2 4 Hierarchical distribution of gene diversity among populations of Hhizoctonia solani AG 3 collected from potato symptomatic plants in Colombia oooooococccccccccococcnnnccncnccncnnnnnnnos Table 2 5 Measures of differentiation among populations of Rhizoctonia solani AG 3 in Colombian sso ee Nena AT AN Im MA eU Au eid Table 2 6 Tests for random association of alleles within each locus and between pairs of loci populations of Rhizoctonia solani AG 3 in ColOMblia ccccccccccoconcnncnnnncnonononcncnnononnnnnnnnnnconononnnnnonanos Table 2 7 Test for the presence of a historic populations is bottleneck sssssssssss Table 3 1 Number of isolates by municipality used for the analysis Table 4 1 Anastomosis Groups associated to Rhizoctonia diseases in potato crops around the 24 25 41 45 51 52 53 55 56 72 List of Figures Figure 1 1 Geographical populations of Rhizoctonia solani AG 3 sampled from infested potato fields from Colomb
4. 1 1 4 2 Infestation of tubers Infestation of tubers with sclerotia starts when the mother plant begins to senesce Sclerotia does not penetrate more than five or six cells deep in the periderm and does not result in physical damage of the tuber Banville et al 1996 Formation of sclerotia on tubers is higher during the time that follows vine kill or natural plant senescence this means that sclerotial formation is inhibited during active plant growth and the inhibition disappears gradually with the death of the mother plant and the tuber maturation Dijst et al 1986 Dijst 1988 Sclerotia may occur on progeny tubers from healthy uninfected plants as well on infected plants The absence of sclerotia does not mean that the pathogen is not present H solani is transmitted by contaminated seed tubers providing the most efficient mechanism for its long distance dispersal Once established in soil the mycelium and sclerotia of the pathogen are an additional source of primary inoculum 17 1 1 4 3 Symptoms Symptoms caused by A solani AG 3 on potato are observed on below and above ground organs in different stages throughout the crop season Carling and Leiner 1986 Before emergence the fungus attacks underground sprouts Lesions formed near the growing tip may kill them resulting in the inhibition or the delay of plant emergence causing poor and uneven stands with weakened plants Baker 1970 Banville 1989 Banville and Carling
5. 52 2 3 4 Admixture and hidden population structure eeeeeeseeeess 54 2 3 5 Population bottleneck and demographic parameters 56 Zu A BITLI o e CIT 56 2 4 1 Genetic and genotypic diversity ooooooonnncccnnnonaccnonnnancnc cnn cnn 57 2 4 2 Population differentiation eeeeeeeeeeeeeeeeeeeeeeeeeeeenenen nnne 57 2 4 3 Reproductive mod oiii derer dias 58 2 4 4 Demographic p rarmielers oci nisk ninos cea sk RYE Ra in non ku ind x Cr un ito Ee kar ada Sana Un 60 2 5 GODCIUSIODS occisus cL EIL E I 60 Lipi pell mere O 61 Chapter 3 Diversity and adaptability of Rhizoctonia solani AG 3 68 3 1 The state of the arti oue on cie ebore n e ei aa nanna XE Ga Cecwi NL ats Pa ER E HD Ea Ra a 68 3 1 1 Genetic diversity and the evolutionary forces eese 68 3 1 2 Variation in time NASA iii nana eua qe dci abel erra Pea Va PEa ka RII R E nnna 69 3 1 3 Differentiation of soil borne fungi in time and space 70 3 2 Materials and methods 5L LIED ance t twur Eta ta Rte sEREE E Ra Rs Raus ad 71 3 2 1 Fungal populations for study eeeeeeeeeeeeeeeeeeeeeeeeeen nennt 71 3 2 2 Prelimiliary Tests sincere oceani saei tn ex R id 71 3 2 3 Response to temperatures and sensitivity to the fungicide thifluzamide
6. Construction of pathogenic biologic and genetic bases of the Colombian populations of Rhizoctonia solani AG 3 necessary for the development of management strategies of stem canker and black scurf diseases of potato ROSA LILIA FERRUCHO Universidad Nacional de Colombia Facultad de Agronomia Bogota Colombia 2011 Construction of pathogenic biologic and genetic bases of the Colombian populations of Rhizoctonia solani AG 3 necessary for the development of management strategies of stem canker and black scurf diseases of potato ROSA LILIA FERRUCHO Tesis presentada como requisito parcial para optar al titulo de Ph D in Ciencias Agropecuarias Directora CELSA GARC A DOMINGUEZ M Sc Ph D L nea de investigaci n Gen tica de poblaciones de pat genos de plantas Grupo de Investigaci n Patolog a de papa Universidad Nacional de Colombia Facultad de Agronom a Bogot Colombia 2011 This work is dedicated to my grandfather for being the light of my life for his love and care he gave me the way to be a good human being and to Ursula for being a guide in a difficult moment Este trabajo hace parte de las investigaciones realizadas por la Facultad de Agronomia Universidad Nacional de Colombia Sede Bogota Sin embargo las ideas emitidas por el autor son de su exclusiva responsabilidad y no expresan necesariamente opiniones de la Universidad Art culo 14 de la Resoluci n No 00047 de 1981 El presiden
7. We checked for differences among genotypes from tuber seed and stems in the same plant Pairs of isolates collected on different stems in the same plant were commonly different 56 of the tested pairs and several isolates from stem canker and sclerotia on tuber seed 25 pairs had different genotypes 80 data not shown 2 3 2 Gene diversity and population differentiation All 295 MLMGs of R solani AG 3 were heterozygous at almost all loci but locus TC_AG3_8 was monomorphic for the populations Subachoque2 Sibate1 Pasto1 and Silvia and locus TC_AG3_10 was monomorphic for the population Silvia The expected heterozygosity HE Nei s unbiased gene diversity ranged from 0 45 to 0 66 across the populations Table 2 3 The overall allelic richness across populations was 3 3 51 Table 2 3 Overall measures of clonal diversity of Rhizoctonia solani AG 3 infecting potatoes in Colombia Allelic Siate Population Sample Number Ste specie Clonal goje EVENS pato ness Pria Subachoque1 28 23 15 8 0 18 85 22 0 85 0 6197 3 28 0 Subachoque2 18 12 6 6 0 33 84 38 0 84 0 6170 3 25 0 Cogua1 23 21 20 1 0 09 93 30 0 93 0 6373 3 57 3 Cundinamarca Cogua2 33 26 21 5 0 21 82 13 0 82 0 6200 3 33 2 Sibate1 21 17 17 0 0 19 89 45 0 90 0 5715 3 18 3 Sibate2 12 11 10 1 0 08 93 51 0 94 0 6600 3 88 0 Narifio Pasto1 18 17 17 0 0 06 95 29 0 95 0 5723 3 16 1 Pasto2 22 19 16 3 0 14 84 91 0 85 0 5574 3 17 1 Atioqul LaUnion1 17 14 10
8. The study of the adaptation of agricultural pathogens includes their ability to emerge in agro ecosystems The emergence occur through several mechanisms including host tracking host jumps hybridization and horizontal gene transfer Stukenbrock and McDonald 2008 The evolution of virulence and fungicide resistance genes in response to control strategies of pathogen populations have interest in evolutionary studies McDonald and Linde 2002 Stukenbrock and McDonald 2008 The base for evolution of organisms is their variability In pathogens characters with effect in fitness are a good option to evaluate adaptability Fitness can be assessed by measuring growth and meiotic or mitotic sporulation Pringle and Taylor 2002 The ability to improve fitness via adaptive evolution may be affected by environmental change that decreases individual and population fitness and may also impact genetic variation and the evolution of polygenic traits Hoffmann and Merila 1999 The evaluation of isolates of R solani from different geographical origin to different stress factors is the first step in the knowledge of the ability of the Colombian populations to be adapted to them Previous research has shown that isolates of this pathogen react differentially when they are challenged with fungicides in vitro Campion et al 2003 Lehtonen et al 2008 Willi et al 2011 or temperatures out of its optimal Willi et al 2011 References Bains P S
9. Altschul S F Madden T L Schaffer A A Zhang J Zhang Z Miller W D J L 1997 Gapped BLAST and PSI BLAST a new generation of protein database search programs Nucleic Acids Research 25 3389 3402 Andersen T F 1996 A comparative taxonomic study of Rhizoctonia sensu lato employing morphological ultrastructural and molecular methods Mycological Research 100 1117 1128 Anderson N A Stretton H M 1982 The Genetics and Pathology of Rhizoctonia solani Ann Rev Phytopathology 20 329 347 Anguiz R Martin C 1989 Anastomosis groups pathogenicity and other characteristics of Rhizoctonia solani isolated from potato in Peru Plant disease 73 99 201 Arrieta J M 2000 Manejo Integrado de malezas en el cultivo de la papa In Herrera C A Fierro L H Moreno J D Eds Manejo integrado del cultivo de la papa Manual t cnico Produmedios Bogot Colombia pp 42 158 Bains P S Bisht V S 1995 Anastomosis group identity and virulence of Rhizoctonia solani isolates collected from potato plants in Alberta Canada Plant disease 79 241 242 Baker K F 1970 Types of Rhizoctonia diseases and their occurrence In Parmeter Jr J R Ed Rhizoctonia solani biology and pathology California Univ California press pp 125 148 Balali G R Neate S M Kasalkheh A M Stodart B Melanson D L Scott E S 2007 Intraspecific variation of Rhizoctonia solani AG 3 isolates recovered from pot
10. Mall S 1982 Anastomosis Groups of Potato Isolates of Rhizoctonia solani in India Mycologia 74 337 338 Talbot M 2003 Effects of biological control and a ryegrass rotation on Rhizoctonia disease of potato University of Maine p 108 Taylor J Jacobson D Fisher M 1999 The evolution of asexual fungi Reproduction Speciation and Classification Annual Review of Phytopathology 37 197 246 Truter M Wehner F C 2004 Anastomosis grouping of Rhizoctonia solani associated with black scurf and stem canker of potato in South Africa Plant Disease 88 83 Tsror L 2010 Biology Epidemiology and Management of Rhizoctonia solani on Potato Journal of Phytopathology 158 649 658 Tsror L Peretz Alon l 2005 The Influence of the Inoculum Source of Rhizoctonia solani on Development of Black Scurf on Potato Journal of Phytopathology 153 240 244 Virgen Calleros G Olalde Portugal V Carling D E 2000 Anastomosis groups of Rhizoctonia solani on potato in central M xico and potential for biological and chemical control American Journal of Potato Research 77 219 224 Woodhall J W Lees A K Edwards S G Jenkinson P 2007 Characterization of Rhizoctonia solani from potato in Great Britain Plant Pathology 56 286 295
11. two monomorphic loci b Population specific FIS indices and p values calculated based on 10 100 permutations using ARLEQUIN 3 5 1 2 Excoffier and Lischer 2010 c lA is an index of multilocus gametic disequilibrium for the random association of alleles among distinct loci pairs Maynard Smith et al 1993 d Testing for complete panmixia based on 10 000 randomizations HO for diploid data the two alleles at a locus are shuffled together associations between alleles at a locus are maintained in the randomized data sets This test is purely for associations between loci Agapow and Burt 2001 e Number of pairs of loci with significant disequilibrium according to Fisher exact test probability test using both a Markov chain with 10 000 batches and 1 000 iteration batch implemented by Genepop Raymond and Rousset 1995 after Bonferroni correction for multiple comparisons Rice 1989 56 2 3 5 Population bottleneck and demographic parameters The test for bottlenecks or founder events was no significant in any of the tested populations Table 2 7 Populations Pasto2 and Ventaquemada1 presented high number of loci with significant heterocigote deficit this is consistent with population expansion Table 2 7 Test for the presence of a historic population bottleneck Population HD HE Exp HE Sign Test One tail for deficit Subachoque1 4 7 6 22 0 43 0 81 Subachoque2 2 8 5 73 0 12 0 92 Cogua1 7 4 6 44 0 11 0 05 Cogua2 4 7 6 32 0 46
12. 1995 Balali et al 1995 Virgen Calleros et al 2000 Banville and Carling 2001 Ceresini et al 2002 Campion et al 2003 Justesen et al 2003 Truter and Wehner 2004 Woodhall et al 2007 Lehtonen et al 2008a Cede o et al 2001 However isolates of this AG have been reported to cause target spot of tobacco tomato and eggplant Analysis based on DNA sequence has lead to the subdivision of the AG 3 in subgroups potato PT Johnk et al 1993 Kuninaga et al 2000 Tobacco TB Kuninaga et al 2000 and tomato TM Kuninaga et al 2000 Kuninaga et al 2007 Isolates belonging to AG 2 1 Carling and Leiner 1986 Cede o et al 2001 AG 4 Anguiz and Martin 1989 Kuninaga et al 1997 Virgen Calleros et al 2000 Truter and Wehner 2004 and AG 5 Bains and Bisht 1995 Balali et a 1995 Virgen Calleros et al 2000 are found frequently associated to stem canker on potato Also AG 7 in Mexico Carling et al 1998 is reported to be pathogenic to potato AG 1 has been isolated from potato roots Bandy et al 1988 and AG 8 has been reported infecting and pruning potato roots leading 16 to damping off and reducing foliage although no classical signs of Rhizoctonia disease were apparent Carling and Leiner 1990 Truter and Wehner 2004 AG 9 has been found associated with potato and potato growing soils but it seems to be limited to the infection of young seedlings Carling and Leiner 1986 Artificial inocu
13. 2 1 1 4 Reproduction and matting system Organisms can transmit genes to the next generation by clonal reproduction or via mating and recombination For clonal organisms their genomes would be an exact copy of the parental as consequence all parts of the genome of the offspring s will have the same evolutionary history In contrast the progeny that result of the mating and meiotic recombination of genetically different parental will have different evolutionary histories Taylor et al 1999 Recombination in plant pathogenic fungi occurs either through sexual reproduction or somatic hybridization Burdon and Silk 1997 Somatic hybridization may increases genotypic diversity in a pathogen population but their importance varies both within and among species Burdon and Silk 1997 Reproduction and mating systems can determine how the gene diversity is distributed within and among individuals in a population McDonald and Linde 2002 Mating systems determines the crossability between individuals Pathogen species can range from strict inbreeding to obligate outcrossing with intermediate species with mixed reproduction Outcrossing organisms put together new combinations of genes increasing genotype diversity and the potential for rapid adaptation in a changing environment McDonald 2004 de Meeus et al 2007 Asexual and inbreeding pathogens have the potential to keep together well co adapted combination of alleles leading to low genotypic
14. 2001 Infection of growing plants lead to the formation of cankers on stems and roots typically they are brown dry and sunken lesions Banville et al 1996 Jeger et al 1996a Agrios 2005 Cankers formed in the stem base may girdle the stem interfering with the normal upwards water and downwards carbohydrate movements thus reducing plant vigor weakening plants and lowering stands or stem numbers or both as a consequence tuber yield is reduced Jeger et al 1996a Agrios 2005 Aboveground symptoms include chlorosis and purpling of leaves and stunting Hartill 1989 In severe infection small green aerial tubers may be formed on the stems above the soil The stems become resistant to infection after emergence it is possible that new compensatory sprouts emerge successfully with no significant damage from previously affected stems Lehtonen et al 2008b As consequence of stolon infection the number size and quality of tubers is reduced due to malformation and cracking Hide and Horrocks 1994 Agrios 2005 Fox 2006 Black scurf which is the most conspicuous sign of Hhizoctonia disease on potato is characterized by the formation of black irregular sclerotia of various sizes on the tuber surface they are formed at the end of the crop cycle particularly after vine death Those structures are essential for the surviving and dispersal of H solani Banville 1989 Jeger et al 1996b Agrios 2005 Tsror 2010 An alteration in th
15. 72 3 2 4 Pathogenicity tests da 73 3 2 4 1 Pathogenicity on StOMS ccccesesssseeeseeceeeeeenssseeeeeecoeseseesseneeneneeseeeeeenssananoeees 73 3 2 4 2 Infestation ON TUDO ic a 73 3 2 5 Data ANALY SIS nda 74 A O 74 3 3 T Preliminary tesis cn bisce lk i ana aaa aaa aaa Kaaa aaan Uie fus 74 3 3 2 Temperature response ii ii 75 3 3 3 Sensitivity to Thifluzamide eese eee cnn renace 75 3 3 4 Pathogenicity tests iii dba RN d HER 77 3 4 DISCUSSION ii da dd 79 e O A A S 82 liii IC m 82 Chapter 4 Final Considerations eeeeeeeeeeseeeeeeeeeeeeee nennen nennen nnn 95 4 1 ANASTOMOSIS gFOUDS ii nece cn ida ai 95 4 2 Genotypic and physiologic variability of R solani AG 3 96 4 3 Variability of R solani AG 3 and the control of the diseases black scurf and SUSI CAN GT de TEE 98 4 9 1 Legal COnWON aiii a 98 4 3 2 Cultural Practices conan 99 4 3 3 Chemical control e aerae ai 100 4 3 4 Resistant varieties a is 100 4 3 5 Biological CONTO a is 101 References iia s 101 List of tables Table 1 1 Number of isolates belonging to each anastomosis group of Rhizoctonia solani associated to symptoms of stem canker and black scurf on potatoes in Colombia Table 1 2 Symptoms caused by Rhizoctonia solani AG 2 1 and AG 3 recovered from potato crops in Colombia on different plant species
16. FSTAT Version 1 2 A computer program to calculate F statistics Journal of Heredity 86 485 486 Gr nwald N J Goodwin S B Milgroom M G Fry W E 2003 Analysis of genotypic diversity data for populations of microorganisms Phytopathology 93 738 746 Guo S W Thompson E A 1992 Performing the exact test of Hardy Weinberg proportions for multiple alleles Biometrics 48 361 372 Halkett F Simon J C Balloux F 2005 Tackling the population genetics of clonal and partially clonal organisms Trends in Ecology amp Evolution 20 194 201 Hartl D L Clark A G 2007 Principles of Population Genetics Sinauer Associates Inc Publishers Sunderland MA 653 pp Hedrick P W 2005 Genetics of Populations Jones amp Bartlett Sudbury MA 737 pp Hubisz M J Falush D Stephens M Pritchard J K 2009 Inferring weak population structure with the assistance of sample group information Molecular Ecology Resources 9 1322 1332 Hurlbert S H 1971 The non concept of species diversity a critique and alternative parameters Ecology 52 577 586 Justesen A F Yohalem D Bay A Nicolaisen M 2003 Genetic diversity in potato field populations of Thanatephorus cucumeris AG 3 revealed by ITS polymorphism and RAPD markers Mycological Research 107 1323 1331 Karaoglu H Lee C M Y Meyer W 2004 Survey of Simple Sequence Repeats in Completed Fungal Genomes Molecular Biology and Evolution 22
17. H Fry W E 2006 Selection for fungicide resistance within a growing season in field populations of Phytophthora infestans at the center of origin Phytopathology 96 1397 1403 Hoffmann A A Merila J 1999 Heritable variation and evolution under favourable and unfavourable conditions Trends in Ecology amp Evolution 14 96 101 Huelsenbeck J P Andolfatto P Huelsenbeck E T 2011 Structurama Bayesian inference of population structure Bioinformatics Evolutionary Bioinformatics 7 55 59 Jeger M J Hide G A Boogert P H J F Termorshuizen A J Baarlen P 1996 Pathology and control of soil borne fungal pathogens of potato Potato Research 39 437 469 Justesen A F Yohalem D Bay A Nicolaisen M 2003 Genetic diversity in potato field populations of Thanatephorus cucumeris AG 3 revealed by ITS polymorphism and RAPD markers Mycological Research 107 1323 1331 Lehtonen M J Ahvenniemi P Wilson P S German Kinnari M Valkonen J P T 2008 Biological diversity of Rhizoctonia solani AG 3 in a northern potato cultivation environment in Finland Plant Pathology 57 141 151 McDonald B A Linde C 2002 Pathogen population genetics evolutionary potential and durable resistance Annual Review of Phytopathology 40 349 379 Milgroom M G 1996 Recombination and the multilocus structure of fungal populations Annual Review of Phytopathology 34 457 477 Naito S 2006 Ecological studi
18. H Garcia R 2001 Identificaci n y virulencia de grupos de anastomosis de Rhizoctonia solani Kuhn asociados con papa en m rida Venezuela Interciencia 26 296 300 31 Ceresini P C Fenille R C Souza N L 1996 Associac o de Rhizoctonia spp binucleadas e de R solani K hn GA 4 HGI vagens de amendoinzeiro Arachis hypogaea no estado de Sao Paulo Summa Phytopathologica 22 145 155 Ceresini P C Shew H D Vilgalys R J Cubeta M A 2002 Genetic diversity of Rhizoctonia solani AG 3 from potato and tobacco in North Carolina Mycologia 94 437 449 Cubeta M A Vilgalys R 1997 Population Biology of the Rhizoctonia solani Complex Phytopathology 87 480 484 Demirci E D ken M T 1998 Host penetration and infection by the anastomosis groups of Rhizoctonia solani Kuhn isolated from potatoes Transactions of Journal of Agriculture and Forestry 22 609 613 Demirci E Eken C Zengin H 2002 First report of Rhizoctonia solani and binucleate Rhizoctonia from Johnson grass in Turkey Plant Pathology 51 391 Dijst G 1988 Effect of periderm and water soluble exudates of potato tubers on black scurf formation before and after haulm destruction Netherlands Journal of Plant Pathology 94 257 266 Dijst G Bouman A Mulder A Roosjen J 1986 Effect of haulm destruction supplemented by cutting off roots on the incidence of black scurf and skin damage flexibility of harvest period and yield o
19. Piry et al 1999 The number of alleles at one locus is reduced faster than the gene diversity when the size of the population is reduced this because rare alleles are more readily affected by drift than more frequent ones When population size is restored the average number of alleles increases faster than the gene diversity until reaching mutation drift equilibrium The test evaluates the heterozygosity excess or deficiency under the assumption of mutation drift equilibrium for each sample and for each locus using an approach based on coalescence Piry et al 1999 The program was run using the strict stepwise mutation model SMM the deviations from mutation drift equilibrium across all loci in each population were assessed for statistical significance using the sign test and Wilcoxon s rank test which is suggested when using less than 20 polymorphic loci Cornuet and Luikart 1996 50 2 3 Results 2 3 1 Microsatellite information genotype and gene diversity From 32 microsatellite loci evaluated 11 were used to characterize the genetic diversity in 18 populations of R solani AG 3 in Colombia South America All loci were polymorphic except for the loci TC_AG3_8 and TC_AG3 10 that were monomorphic in some populations 99 alleles were revealed by the analysis of the microsatellite loci The number of alleles at each locus varied from two TC_AG3_0 to 14 TC_AG3_16 with an average of 9 per loci Annex 2 A Allele frequencies
20. are interested mainly in selection and gene flow as factors controlling the amount and distribution of variability into the populations Selection is a directional process that leads to changes increasing or decreasing in allele or genotypes frequencies in response to a specific environmental factor McDonald and Linde 2002 de Meeus et al 2007 Selection is a frequency dependent process the fitness of particular genotypes depend on its frequency into the population de Meeus et al 2007 Changes in genotype or gene frequencies are evolutionary changes that occur on micro evolutionary scales of time The short generation times and large population sizes of microbial populations are ideal conditions for natural selection A pathogen can evolve over time to be adapted to the host in many ways Mutation provides the natural variability upon which selection can act An organism with an inherently high mutation rate would be more likely to give rise to mutations that avoid recognition without loss of fitness Leach et al 2001 3 1 2 Variation in time and space Actually ecologists and population geneticists recognize that the arrangement of the environment landscape is connected to the maintenance of diversity They have started to relate spatial variability with disease risk and its intensity Ostfeld et al 2005 The variability in the amount and distribution of diseases in time space is caused by the diversity and the distribution of
21. 3 65 6F2P 1 51 2 56 1 50 Average Sibate 0 45 2 71 2 15 7A4P 0 26 0 69 1 91 7B1P 0 45 1 18 2 16 Silvia 7B2P 0 00 3 04 2 50 7B5P 0 00 0 00 2 31 7C2P 0 00 3 02 1 38 7P 0 00 0 12 3 60 Average Silvia 0 12 1 34 2 31 eres 12A4P 0 00 1 69 3 85 12A6P 0 00 5 44 1 08 89 12B2P 0 00 1 51 2 33 12BTOYA 0 00 1 41 1 56 12C4P 0 00 2 94 0 60 12C5P 2 0 00 2 33 2 36 12D1P 0 00 1 07 3 97 Average Soraca 0 00 2 34 2 25 1A11P 0 40 1 23 2 34 1B2P 0 00 1 67 2 79 1D2P 0 00 0 00 1 98 Subachoque 1E1P 2 0 00 2 19 4 15 1E3P 0 00 1 66 3 92 1E4P 0 00 2 03 1 42 1E9S 0 00 3 54 2 89 Average Subachoque 0 06 1 76 2 78 11B2P 0 00 1 31 2 27 11B5P 1 1 23 2 43 1 05 11B6P 0 00 0 00 4 17 Ventaquemada 11C4P 0 00 1 97 2 88 11D1P 0 00 0 00 4 60 11D3P 0 00 1 29 2 23 11F4P 0 00 2 56 4 46 Average Ventaquemada 0 18 1 37 3 10 Control i 0 00 0 00 0 00 Average Control 0 00 0 00 0 00 General Average 0 11 1 57 2 27 Plants inoculated with a plug of PDA 90 Annex 3 E Test for statistic differences among the variables evaluated in pathogenicity tests with isolates of Rhizoctonia solai AG 3 Size of lesions in Size of lesions in Incidence of black Severity of black stolons roots scurf scurf Municipality Group Average Group Average Group Average Group Average Subachoque B 2 70 ABC 1 75 C 29 66 B 0 89 Cogua A 4 08 AB 2 28 BA 57 57 BA 1 26 Sibat
22. Africa Plant disease 88 83 Tsror L 2010 Biology Epidemiology and Management of Rhizoctonia solani on Potato Journal of Phytopathology 158 649 658 Tsror L Peretz Alon 2005 The Influence of the Inoculum Source of Rhizoctonia solani on Development of Black Scurf on Potato Journal of Phytopathology 153 240 244 Virgen Calleros G Olalde Portugal V Carling D E 2000 Anastomosis groups of Rhizoctonia solani on potato in central M xico and potential for biological and chemical control American Journal of Potato Research 77 219 224 Weinhold A R Sinclair J 1996 Rhizoctonia solani Penetration colonization and host response In Sneh B Jabaji Hare S Neate S Dijst G Eds Rhizoctonia Species Taxonomy Molecular Biology Ecology Pathology and Disease Control Kluwer Academic Publishers The Netherlands pp 163 174 35 White T J Burns T Lee S Taylor J 1990 Amplification and direct sequencing of fungal ribosomal genes for phylogenetics In Innis M A Gelfand D H Shinsky J White T J Eds PCR protocols A Guide to Methods and Applications Academic Press San Diego pp 315 322 Windels C E Nabben D J 1989 Characterization and pathogenicity of anastomosis groups of Rhizoctonia solani isolated from Beta vulgaris Phytopathology 79 83 88 Woodhall J W Lees A K Edwards S G Jenkinson P 2007 Characterization of Rhizoctonia solani from potato in Great
23. Bisht V S 1995 Anastomosis group identity and virulence of Rhizoctonia solani isolates collected from potato plants in Alberta Canada Plant Disease 79 241 242 Banville G J Carling D E Ostysko B E 1996 Rhizoctonia disease on potato Rhizoctonia species taxonomy molecular biology ecology pathology and disease control Springer pp 321 330 Campion C Chatot C Perraton B Andrivon D 2003 Anastomosis Groups Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France European Journal of Plant Pathology 109 983 992 Carling D E Leiner R H 1990 Virulence of isolates of Rhizoctonia solani AG 3 collected from potato plant organs and soil Plant Disease 74 901 903 Ceresini P C Shew H D Vilgalys R J Cubeta M A 2002a Genetic diversity of Rhizoctonia solani AG 3 from potato and tobacco in North Carolina Mycologia 94 437 449 Ceresini P C Shew H D Vilgalys R J Rosewich U L Cubeta M A 2002b Genetic Structure of Populations of Rhizoctonia solani AG 3 on Potato in Eastern North Carolina Mycologia 94 450 460 de Meeus T McCoy K D Prugnolle F Chevillon C Durand P Hurtrez Bouss s S Renaud F 2007 Population genetics and molecular epidemiology or how to d busquer la b te Infection Genetics and Evolution 7 308 322 Grunwald N J Sturbaum A K Romero Montes G Garay Serrano E Lozoya Salda a
24. D Size of lesions on stems stolons and roots generated by the artificial inoculation with isolates of R solani AG 3 collected on different municipalities in Colombia Municipali Average Size of Average size of Average size of unicipality Isolate lesions on stems lesions on roots lesions on stolons 19A2SKH 2 20 2 80 1 84 19B1P 0 00 6 26 3 24 19C2S 0 00 1 02 2 83 Carcasi 19D2P 0 00 0 00 2 34 19D4P 0 00 3 86 2 12 19D5S 0 00 3 22 4 72 19E1SKH 0 00 3 55 2 79 Average Carcasi 0 31 2 96 2 84 22A3P 0 00 3 46 4 31 22B25P 0 00 0 00 1 69 Chitaga 22C3P 0 00 7 94 2 89 22C4P 0 80 2 49 2 65 22H3P 0 00 1 91 2 09 2212P 0 00 2 29 2 51 Average Chitaga 0 13 3 01 2 69 4A11P 0 00 0 48 5 98 4A12P 0 00 1 42 6 22 4A14P 0 00 2 90 4 45 Cogua 4A3P 0 00 0 74 2 41 4A7P 0 00 0 49 2 50 4AP 0 00 4 78 3 57 4B6P 0 00 6 48 2 54 Average Cogua 0 00 2 47 3 95 16A1P 0 00 0 00 3 66 16B10P 2 0 00 1 91 4 31 LaUnion 16C10P 0 00 1 18 1 19 16C2P 0 00 0 00 1 77 16D7P 0 00 0 00 2 50 16E7S 0 00 0 00 3 43 Average La Union 0 00 0 52 2 81 10A3P 1 0 44 0 91 0 74 10B8P 0 00 0 00 3 03 Pasto 10C1P 2 0 00 0 61 3 41 10C3P 0 00 1 85 2 94 10E3P 2 0 00 0 57 2 58 10F5P 0 00 0 55 2 80 Average Pasto 0 07 0 75 2 58 6A2S 0 00 5 70 2 23 6A5P 0 76 2 37 2 49 6C4S 0 87 1 51 2 18 Sibate 6D4P 0 00 3 64 1 12 6D6P 0 00 2 89 1 91 6F1P 0 00 0 31
25. Neate S Dijst G Eds Rhizoctonia Species Taxonomy Molecular Biology Ecology Pathology and Disease Control Kluwer Academic Publishers The Netherlands pp 149 162 Kellens J T C Peumans W J 1991 Biochemical and serological comparison of lectins from different anastomosis groups of Rhizoctonia solani Mycological Research 95 1235 1241 Ko W Hora F 1979 A selective medium for the quantitative determination of Rhizoctonia solani in soil Phytopathology 61 707 710 Kuninaga S Carling D E Takeuchi T Yokosawa R 2000 Comparison of rDNA ITS Sequences between Potato and Tobacco Strains in Rhizoctonia solani AG 3 Journal of General Plant Pathology 66 2 11 Kuninaga S Natsuaki T Takeuchi T Yokosawa R 1997 Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani Current Genetics 32 237 243 Kuninaga S Sayama A Yokosawa R 2007 Rhizoctonia solani strains associated with a leaf blight of tomato are classified into anew subgroup within AG 3 Annals of the Phytopathologicial Society of Japan 73 7625 7630 33 Kuninaga S Yokosawa R Ogoshi A 1979 Some properties of anastomosis Group 6 and B in Rhizoctonia solani Kuhn Annals of the Phytopathologicial Society of Japan 45 207 214 Lees A K Cullen D W Sullivan L Nicolson M J 2002 Development of conventional and quantitative real time PCR assays for the detection an
26. a simulation study Molecular Ecology 14 2611 2620 Excoffier L Lischer H E L 2010 Arlequin suite ver 3 5 a new series of programs to perform population genetics analyses under Linux and Windows Molecular Ecology Resources 10 564 567 Excoffier L Smouse P E Quattro J M 1992 Analysis of Molecular Variance Inferred From Metric Distances Among DNA Haplotypes Application to Human Mitochondrial DNA Restriction Data Genetics 131 479 491 Falush D Stephens M Pritchard J K 2003 Inference of population structure using multilocus genotype data linked loci and correlated allele frequencies Genetics 164 1567 1587 63 Ferrucho R L Zala M Zhang Z Cubeta M A Garcia Dominguez C Ceresini P C 2009 Highly polymorphic in silico derived microsatellite loci in the potato infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes Molecular Ecology Resources 9 1013 1016 Frank J A Leach S S 1980 Comparison of tuber borne and soilborne inoculum in the Rhizoctonia disease of potato Phytopathology 70 51 53 Garnier Gere P Dillmann C 1992 A computer program for testing pairwise linkage disequilibria in subdivided populations The Journal of Heredity 83 239 239 Glaubitz J C 2004 Convert A user friendly program to reformat diploid genotypic data for commonly used population genetic software packages Molecular Ecology Notes 4 309 310 Goudet J 1995
27. compatibility reactions and ii to determine the aggressiveness of each AG on potato and other plant species 1 2 Materials and methods 1 2 1 Population sampling and establishment of a fungal isolate collection Stem canker diseased and early sprouting potato plants were sampled from the main producing areas in Colombia as follows six distinct fields in Cundinamarca four fields in Boyac two fields in Nari o Antioquia and Santander and one field in Cauca and Norte de Santander Figure 1 1 In each field six to eight rows were sampled and between 20 to 30 symptomatic plants were collected Rows were separated six meters between them The infected plants were brought to the Laboratorio de Biotecnologia Antonio Angarita Zerda LBAAZ of the Facultad de Agronomia at the Universidad Nacional de Colombia Plants were washed with tap water and infected stem segments and sclerotia from tubers were plated on selective medium for R solani Ko and Hora 1979 modified Castro et al 1988 Ceresini et al 1996 and stored at room temperature in the dark After 24 to 48 h pure cultures of R solani were established by transferring hyphal tips from the growing colonies on the selective medium onto potato dextrose agar medium PDA Oxoid 19 Venezuela Pacific Ocean Wy hor gt w Ecuador Figure 1 1 Geographical populations of Hhizoctonia solani AG 3 sampled from infested potato fields from Colombia The following d
28. contain a reservoir of virulence or fungicide resistant genes McDonald and McDermott 1993 Burnett 2003 The consequences of genetic drift are the decrease of diversity into the populations and the increase on the differentiation among populations both of them due to loss of variation in the random sampling of genotypes McDonald and McDermott 1993 2 1 1 3 Gene Flow Commonly referred as migration gene flow is the movement individuals or their gametes among sub populations The main consequences of gene flow are i Introduction of novel genetic material ii homogenization among populations iii foundation of new populations founder effect McDonald and McDermott 1993 McDonald and Linde 2002 Burnett 2003 de Meeus et al 2007 Hartl and Clark 2007 Gene flow is a simple evolutionary force however it has a great relevance in the genetic diversity of the populations If the process of migration leads to the foundation of new pathogen populations the immigrants can be established causing large epidemics If the pathogen already exists the recognition of gene 38 flow events usually is restricted to instances that lead to obvious shifts in the genetic aggressiveness of the local populations Burdon and Silk 1997 The average of gene flow over generations will determine the degree of differentiation between populations McDermott and McDonald 1993 McDonald and Linde 2002 de Meeus et al 2007 Hartl and Clark 2007
29. disease control Springer pp 321 330 Bennett A F 1997 Adaptation and the evolution of physiological characters In Dantzler W H Ed Handbook of Physiology Oxford Univ Press New York pp 3 16 Bernardes de Assis J Storari M Zala M Wang W Jiang W ShiDong L Jin M McDonald B A Ceresini P C 2009 Genetic structure of populations of the rice infecting pathogen Rhizoctonia solani AG 1 IA from China Phytopathology 99 1090 1099 Brewer M T Larkin R P 2005 Efficacy of several potential biocontrol organisms against Rhizoctonia solani on potato Crop Protection 24 939 950 Campion C Chatot C Perraton B Andrivon D 2003 Anastomosis Groups Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France European Journal of Plant Pathology 109 983 992 Carling D E Leiner R H 1986 Isolation and characterization of R solani and binucleate A solani like fungi from aerial stems and subterranean organs of potato plants Phytopathology 76 725 729 Carling D E Leiner R H Westphale P C 1989 Symptoms signs and yield reduction associated with Rhizoctonia disease of potato induced by tuber borne inoculum of Rhizoctonia solani AG 3 American Potato Journal 66 693 701 Cede o L Carrero C Quintero K Araujo Y Pino H Garcia R 2001 Identificaci n y virulencia de grupos de anastomosis de Rhizoctonia solani Kuhn asoc
30. diversity and allelic richness we used FSTAT v 2 9 3 2 Goudet 1995 based on 1 000 permutations h Calculated according to El Mousadik and Petit El Mousadik and Petit 1996 i Alleles occurring in only one population calculated with Convert 1 3 52 AMOVA estimates showed that variation among states represented only 1 57 of the total variance The variation among fields into states represents 0 82 of the total and the variation within fields accounts for the 93 5 of the total variability Table 2 4 Pairwise analysis for population differentiation showed that the evaluated populations are genetically similar with low genetic distance among them The overall As value was 0 026 p value lt 0 01 Of 153 of pairs of populations evaluated 43 were significant for differentiation when the Hs index was used p value 0 05 3 pairs showed low differentiation Rs lt 0 05 36 pairs have moderate differentiation Rs between 0 05 and 0 15 and four pairs with high differentiation Rsr from 0 15 to 0 25 Annex 2 B Table 2 4 Hierarchical distribution of gene diversity among populations of Rhizoctonia solani AG 3 collected from potato symptomatic plants in Colombia Distance method sum of squared size differences Ast 2 Sum of Variance Percentage Fixation p Source of variation df eU aim squares components of variation indices value Among groups 10 1 301 656 0 96822 1 57 FCT 0 0157 0 0743 Among populations within groups 7 553 7
31. en la estructura gen tica de la poblaci n La evaluaci n de la respuesta del pat geno a dos temperaturas la sensibilidad al fungicida thifluzamide y la agresividad en diferentes rganos de las plantas de papa mostraron que los aislamientos de todas las localidades geogr ficas var an en la respuesta los factores evaluados Los niveles de costra negra y de chancros variaron entre aislamientos sobre el cultivar de papa evaluado Palabras clave Hongos del Suelo Diversidad Biol gica Estructura de la Poblaci n Evoluci n Adaptabilidad Content Pag Abstract Resumen Ege tds 5 List f Figures ainia ada 6 B ciego nuo Cl RR 7 Iri Od UC LOB oes eon roa Osa eS TATS 8 Chapter 1 Characterization of Anastomosis Groups associated to stem canker in potato crops In Colombla e io 13 T I THE state OF UG APU cassis catu iis dia 13 1 1 1 Rhizoctonia solani K hN ocio oe en ee eee 13 1 1 2 Grouping of R solani isolates esses nennen nnns 13 1 1 3 Stem canker and black scurf on potato Solanum tuberosum L 15 1 1 4 Dis as CY Cle e S 16 1 1 4 1 Infection of stems and stolons eeceeeeeeeeeeeeenneee 16 1 1 4 2 Infestation of TUDGTS ia do rx nnmnnn nnna 16 1 143 SV IML ONS es cscs sec eS as sce cect cece tae 17 11 44 sube 17 1 2 Materials and Methods ccccccsssseeeessseeeesesesensnneneeeeeeseensnseenneeeoeseenseese
32. from strictly recombining to a mixed reproductive 43 system in which there are recombination events followed by clonal expansion during the growing season Bernardes de Asis et al 2009 R solani AG 3 has been extensively studied Ogoshi 1987 however many aspects of its biology and epidemiology still remain unresolved R solani AG 3 has been considered asexual producing mycelia and sclerotia as the main structures for dispersion and surviving Cubeta and Vilgalys 1997 In the fields the sexual stage T cucumeris is observed however the role of basidiospores on the cycle life of this pathogen and in the epidemiological cycle of the disease is unknown Ceresini et al 2002b analyzing data from PCR RFLP molecular markers found evidence of both colonality and recombination defining the population structure of R solani AG 3 in North Caroline USA Studies using molecular markers Ceresini et al 2002a Ceresini et al 2002b Justesen et al 2003 Balali et al 2007 biochemical markers Balali et al 2007 and phenotypic traits Campion et al 2003 Lehtonen et al 2008 have shown high diversity levels in R solani AG 3 populations around the world The study on five North American populations showed that the gene flow is the main force shaping the geographic structure of this pathogen the lack of population differentiation is consequence of the tuber borne genotypes introduced each cycle to the fields Ceresini et a 2
33. isolates to which the AG 3 specific PCR assay resulted in negative amplifications the ITS rDNA region from these isolates and also from few positive controls were amplified using universal primers ITS5 5 GGAAGTAAAAGTCGTAACAAGG 3 and ITS4 5 TCCTCCGCTTATTGAT ATGC 3 White et al 1990 Sequencing reactions were prepared using BigDye TM v3 0 cycle Sequencing Kit Applied Biosystems Sequencing reactions were run on an ABI Prism 3100 genetic analyzer Sequences were visualized and edited using the program Sequencher 4 0 5 Gene Codes Corp All the sequences were analyzed with BLAST Altschul et al 1997 against the NCBI sequence database National Center for Biotechnology Information GenBank http www ncbi nlm nih gov to detect similar sequences of known Anastomosis Groups Sequence data of the individuals tested were imported and assembled using BioEdit v 5 0 9 Hall 1999 Three sequences of R solani AG 3 from the NCBI database EF370434 AB19015 and AB19021 were used as references Nucleotide identity among sequences was determined for each AG identified 1 2 3 Microscopic and macroscopic hyphal interactions Microscopic somatic compatibility reactions were determined on an essay using 60 isolates collected in our study and its AG previously identified by ITS rDNA sequencing Pairings were made on slides containing a thin layer of water agar WA Oxoid Mycelial plugs 0 5 cm in diameter from actively growing cultures 4
34. off rot on roots shoots and fruits canker lesions on sprouts and stolons and sclerotial diseases Ogoshi 1996 1 1 2 Grouping of R solani isolates Classification of Rhizoctonia spp has evolved mainly from studies of isolates obtained from diseased plants Affinities for hyphal fusion anastomosis was the first methodology used to group isolates in Rhizoctonia spp Matsumoto 1921 proposed four categories to group strains based on hyphal anastomosis perfect imperfect contact and no reaction The cell death was included as criteria to evaluate relation between individuals Categories currently used involve the previous criteria and are defined as C3 Perfect reaction this shows a very 14 close relationship indicating the same AG C2 more distant reaction related isolates which are in the same AG but belong to different vegetative compatibility population VCG C1 represents a more distant relationship as can be found in AGs with subgroups and CO No reaction different AG Carling 1996 Studies using restriction fragment length polymorphism RFLP of nuclear DNA Andersen 1996 RFLPs Jeon et al 2010 and sequencing of ribosomal DNA rDNA Kuninaga et al 1997 Gonz lez et al 2006 Sharon et al 2006 Sharon et al 2008 and P tubulin Gonz lez et al 2006 analysis of soluble proteins Reynolds et al 1983 pectic zymograms Balali et al 2007 lectins Kellens and Peumans 1991 Hamshou et al 2007 profi
35. on PDA and grown for five days at room temperature 24 hour before the inoculation each isolate was transferred to a new plate with PDA One plug of mycelia of 5 mm of diameter was used to inoculate each growing plant in the stem base Each isolate was inoculated on five plants Plants were kept on a growing room at 20 C with 12 hours of photoperiod and were watered whenever the substrate was dry and fertilized twice a week using nutrient solution Murashige and Skoog 1962 Two months after the inoculation plants were harvested then washed and the disease was evaluated as average of the lesion size mm on stems roots and stolons 3 2 4 2 Infestation on tubers After a month of sprouting uniform tubers were took and transferred to bags with soil The soil used was collected in a not growing potato field and was evaluated for R solani AG 3 by PCR using specific primers Lees et al 2002 Two weeks after sow plants were inoculated with two plugs of mycelia of 5 mm of diameter close to the stem base Each isolate was inoculated on five plants Plants were kept in an open area fertilized with 15 15 15 twice 30 grams per plant the first fertilization at sow and the second at hilling and 74 foliar aspersion with micronutrients three times in the cycle The control of pests and diseases was effectuated whenever was necessary After six months tubers were collected washed and the black scurf evaluated using a severity scale James 197
36. on plant pathogen populations have increased our knowledge regarding their evolutionary potential in the agro ecosystems and have allowed the characterization of the most important processes governing pathogen evolution McDonald and Linde 2002 Along with epidemiology population genetics help to describe the dynamic of the diseases on time and space looking for the geographical origin of the pathogens mechanism of dissemination and the scale of distribution of genetic variation from lesion into the plants to plants hosts fields and countries McDonald 2004 This knowledge is important to establish control strategies of pathogens according to the amount and distribution of the genetic diversity of the pathogen populations 2 1 1 Sources of genetic variability in fungi 2 1 1 1 Mutation Mutation is the ultimate source of genetic variation in plant pathogenic fungi Mutation is any heritable change in the genetic material This includes changes in single nucleotides as well 37 as the rearrangement of chromosomes such as inversions or translocations Hartl and Clark 2007 Changes in the DNA sequence create new alleles McDonald and Linde 2002 The main consequences of mutation on plant pathogen populations are i the evolution of avirulence genes that break major resistance genes on the plant and ii the generation of fungicide or antibiotic resistant strains McDonald and Linde 2002 McDonald 2004 In plant pathogens muta
37. plant pathogenic fungi Population genetics study the processes that lead to genetic changes in populations over time and space McDonald 1997 The main goal of population genetics is to understand the evolutionary processes shaping and maintaining genetic variation within and among populations over time and space Changes in genotype or allele frequencies in populations are considered evolutionary changes they are fundamental for genetic analysis Milgroom and Peever 2003 McDonald 2004 Pathogen populations constantly have to be adapted to changes in their environment In agricultural ecosystems environmental changes include resistant varieties applications of fungicides and fertilizers irrigation and crop rotation McDonald 1997 Control strategies if they are expected to be effective must target a population of pathogen instead of an individual Thus plant pathologists should focus more effort on the genetics of populations to understand how populations will evolve in response to different control strategies 2 1 4 Genetic structure of populations Genetic structure is defined as the amount and distribution of genetic variation within and among populations this attribute is determined by the evolutionary history and the potential of change of the organisms in a population McDonald 1997 McDonald and Linde 2002 There are two types of diversity contributing to genetic structure gene and genotype diversity Gene diversity is the n
38. suggest that they could be potentially exploited as bio control although an in depth study of their effects on potatoes is necessary 1 5 Conclusions PCR and sequence analysis were useful techniques to discriminate among AGs associated to stem canker and black scurf in Colombia Only two anastomosis groups AG 2 1 and AG 3 were associated with potato stem canker and black scurf diseases in Colombia The most common was AG 3 The AGs identified not only infected potatoes but also other plant species such as tomato lulo bean carrot and pea Those plants sometimes are used as rotation crops which can help to the increase and survival of fungal inoculum The grasses were not infected with any of the AGs this suggest that crop rotation with grasses is the best alternative to diminish the amount of inoculum in potato fields 29 Sequence analysis showed high uniformity in nucleotide sequence among AG 3 isolates corroborating that those genomic regions are not suitable for diversity analysis in the Rhizoctonia complex Macroscopic somatic compatibility tests showed high variability among the isolates tested this suggest that the Colombian population of this pathogen is more diverse that previously was though References Adams G C Butler E E 1979 Serological Relationships Among Anastomosis Groups of Rhizoctonia solani Phytopathology 69 629 629 Agrios G N 2005 Plant Pathology Fifth Edition Academic Press 920 p
39. temperature was high in the populations of Pasto La Union and Ventaquemada Figure 3 2 3 3 3 Sensitivity to Thifluzamide All the isolates were affected by the fungicide The isolates less affected were those from the municipalities Cogua and Subachoque Figure 3 3 In Cogua there was an isolate that was poorly affected this increased the average of the size of the colony The differences were located mainly among isolates in each municipality not among municipalities particularly in isolates from Ventaquemada Subachoque Soraca and Silvia 76 o q g o ES a Eg NS o gt 2 o o 4 Subachoque Ventaquemada Municipalities Figure 3 2 Proportion of growth of the isolates relative to the optimal temperature under n vitro conditions Colony size Cm Subachoque Ventaquemada Municipalities Figure 3 3 Average of colony size Cm in isolates collected on different municipalities and grown in presence of thifluzamide 14 2 ppm Area of isolates in Petri dishes without fungicide was around 20 Cm 77 All the isolates were sensitive to thifluzamide at 14 ppm All the isolates showed a small colony in presence of the fungicide at 48 hours and the proportion of growth relative to the control was less than 5 Figure 3 4 however the isolates continued its growth and were less inhibited in time The test in vitro with 22 isolates growing in presence of the fungicide an
40. well adapted genotypes of the pathogen Each environment has its own constraints to the pathogens this mean that the environment selects for the organisms in a region Heterogeneous environments are composed by many niches that may vary in either space or time Kassen 2002 Kassen and Rainey 2004 As consequence different types may be favored in each niche so diversity is maintained according to the niche exclusion principle Natural selection eliminates the fittest types under any given set of growth conditions leading to a loss of diversity Kassen and Rainey 2004 Evolutionary theory predicts that in a spatially heterogeneous environment selection allows the emergence of ecological specialists or different types being adapted to different niches Kassen and Rainey 2004 Migration of plant pathogens forces them to survive and be adapted to novel environments Local adaptation is consequence of adaptation to particular host genotypes 70 and selection pressures Sicard et al 2007 Local adaptation or the enhanced performance on sympatric host populations can result on rapidly evolving parasites adapted to the most common host genotypes and to the environment Belotte et a 2003 Sicard et al 2007 Many problems in evolutionary biology are focused on phenotypic variation Phenotypic variation allows the differentiation among species and among individuals into species Traits that are often measured and given in quantitative
41. 0 41 Sibate1 4 6 5 76 0 57 0 78 Sibate2 6 5 6 36 0 29 0 23 Pasto1 6 4 5 73 0 21 0 18 Pasto2 8 3 6 37 0 04 0 03 La Union2 5 6 6 40 0 52 0 71 La Union2 7 4 6 13 0 16 0 38 Silvia 6 3 5 18 0 13 0 15 Ventaquemada1 9 2 6 39 0 003 0 04 Ventaquemada2 4 7 6 27 0 45 0 68 Soraca1 8 3 6 28 0 03 0 06 Soraca2 6 5 6 34 0 30 0 16 Carcasi1 6 5 6 30 0 30 0 41 Carcasi2 5 6 6 44 0 50 0 26 Chitaga 6 5 6 57 0 25 0 23 HD and HE deficit and excess heterozygosity at 11 unlinked loci HE is generated by simulations under the assumption of mutation drift equilibrium Data from BOTTLENECK V 1 2 2 4 Discussion The study of population structure and dynamics of R solani AG 3 requires differentiation of the relative influence of life history traits and historical processes in shaping present day populations This is the first study describing deeply the population structure of R solani AG 3 in Colombia A wide sampling was realized including all the main producing areas and some counties with low production We hypothesized that the main evolutionary force shaping the population structure of this pathogen is the gene flow considering that R solani AG 3 is dispersed long distance through human mediated movement of contaminated tuber seed Simons and Gilligan 1997 57 2 4 1 Genetic and genotypic diversity The Colombian population is genetic and genotypically diverse Our results supports the high variability detected in pathogenicity and morphology Cam
42. 000 Despite of the importance of black scurf and stem canker in Colombia the pathogen biology still remains poorly understood and the most important questions about the genetic structure of R solani AG 3 populations remain unanswered The main objective of this research was to evaluate the genetic diversity of R solani AG 3 using SSR markers The analysis of the data will allow us to answer the following questions 1 Are the Colombian populations of R solani AG 3 genetically diverse This is a confirmatory question considering de variability found on populations around the world in previous studies 2 Are those populations genetically subdivided or gene flow is shaping their genetic structure In Colombia there is a continuous trade of tuber seed and the ruling for low inoculum content maximum 10 of sclerotia on tuber seed was established only 2003 this situation surely has contributed to the no differentiation of the populations of R solani AG 3 in Colombia however the hypothesis is that populations close to Venezuela and to Ecuador are genetically different from those in the Colombian Central Andes And 3 Is the population structure consistent with a random mating hypothesis How frequently recombination occurs The hypothesis is that although recombination can occur this process is not enough strong and frequent in the Colombian population of R solani AG 3 44 2 2 Materials and Methods 2 2 1 Sampling fungal isolation and DN
43. 000 batches and 10 000 iterations per batch Raymond and Rousset 1995 and implemented in GENEPOP version 3 4 To avoid the error type the significance levels were adjusted following the Bonferroni correction method for multiple test Rice 1989 The multilocus index of association A Maynard Smith et al 1993 and r an alternative measure of A were tested for its significance with 10 000 randomizations Agapow and Burt 2001 Those tests are implemented in MULTILOCUS version 1 3 The hypothesis of complete panmixia was tested comparing the observed data set to data sets in which sexual recombination is imposed on the data by randomly shuffling the alleles among individuals independently for each locus An A significantly different from zero means disequilibrium 7 ranges from zero when there is no disequilibrium to one when there is disequilibrium among tested loci 49 The inbreeding coefficient Fis across loci was calculated to test for a significant deficit or excess of heterozygote when compared with HWE expectations Weir and Cockerman 1984 this test was based on 10 000 permutations using the program ARLEQUIN 3 5 1 2 When significant this test can explain deviation from HWE and GE as inbreeding in the population 2 2 3 6 Test for Admixture and hidden population structure A possible cause of the departure from HWE and gametic disequilibrium between pairs of loci into the populations is the Wahlund effect A Bayesi
44. 06 0 095 0 018 0 019 0 023 0 002 0 026 0 036 Pasto2 0 034 0 030 0 054 0 085 0 024 0 103 0 036 0 029 0 000 0 009 0 009 0 200 0 028 0 029 0 220 0 002 0 148 0 061 Ventaq1 0 040 0 009 0 013 0 009 0 110 0 028 0 170 0 114 0 076 0 000 0 133 0 206 0 602 0 881 0 152 0 371 0 417 0 810 Soraca1 0 013 0 004 0 006 0 011 0 071 0 029 0 099 0 083 0 061 0 020 0 000 0 865 0 718 0 223 0 268 0 348 0 195 0 111 Soraca2 0 019 0 003 0 005 0 004 0 016 0 029 0 033 0 040 0 020 0 018 0 017 0 000 0 993 0 300 0 811 0 511 0 775 0 434 Ventaq2 0 006 0 011 0 027 0 014 0 069 0 034 0 136 0 075 0 058 0 011 0 014 0 033 0 000 0 759 0 331 0 408 0 547 0 354 LaUnion1 0 033 0 004 0 018 0 014 0 093 0 032 0 163 0 083 0 060 0 027 0 012 0 010 0 019 0 000 0 187 0 248 0 627 0 783 LaUnion2 0 004 0 010 0 001 0 001 0 033 0 018 0 045 0 046 0 010 0 016 0 006 0 013 0 004 0 014 0 000 0 078 0 227 0 412 Carcasi1 0 035 0 009 0 001 0 013 0 137 0 003 0 181 0 165 0 138 0 001 0 004 0 003 0 004 0 013 0 042 0 000 0 182 0 108 Carcasi2 0 026 0 007 0 001 0 023 0 087 0 019 0 124 0 076 0 024 0 009 0 013 0 016 0 010 0 020 0 011 0 020 0 000 0 283 Chitaga 0 019 0 020 0 019 0 002 0 062 0 033 0 138 0 060 0 039 0 024 0 022 0 002 0 004 0 024 0 003 0 032 0 001 0 000 Distances were computed as the sum of squared size differences between two haplotypes using ARLEQUIN 3 5 1 2 Excoffier and Lischer 2010 The Ast values are located below the diagonal and above diagonal its signific
45. 1 Additionally incidence of the disease on tubers in each plant was recorded 3 2 5 Data analysis Data from preliminary tests were analyzed with excel The optimal dose of the fungicide was estimated using the procedure NLIM SAS V9 0 Temperature response was estimated as the proportion of the growth relative to the optimal 25 C and the fungicide sensitivity as the proportion of the colony size in the presence of fungicide relative to the colony size in the absence of fungicide Descriptive analysis of the evaluated variables was executed using excel Analysis of variance was carried out to test for the effects of temperature and fungicide on radial growth of the fungi and on the pathogenicity of the isolates on potato plants SAS V9 0 A cluster analysis was executed using the software SAS V9 0 in order to identify groups of individuals associated to geographical regions with similar behavior in the evaluated variables 3 3 Results 3 3 1 Preliminary tests The preliminary tests showed that R solani AG 3 has a wide capability to react to different factors of stress imposed to the individuals The higher growth rate was found at temperatures among 20 and 30 C and was close zero at 4 C and at 40 C for almost all the isolates tested Annex 3 A In average the formation of sclerotia started at 6 days for all the temperatures tested The formation of sclerotia was inhibited at 4 and 40 C Annex 3 B The fungicide thifluzam
46. 22 DowAgrosciences Thifluzamide Technical bulletin Product Brochure http www dowagro com England P R Osler G H R Woodworth L M Montgomery M E Briscoe D A Frankham R 2003 Effects of intense versus diffuse population bottlenecks on microsatellite genetic diversity and evolutionary potential Conservation Genetics 4 595 604 Ettema C H Wardle D A 2002 Spatial soil ecology Trends in Ecology amp Evolution 17 177 183 83 Franklin R Mills A 2007 The importance of microbial distribution in space and spatial scale to microbial ecology In B F R Mills A L Eds The Spatial Distribution of Microbes in the Environment Springer 1 30 pp Gill J S Sivasithamparam K Smettem K R J 2000 Soil type with different texture affects development of Rhizoctonia root rot of wheat seedlings Plant and Soil 221 113 120 Gilligan C A Bailey D J 1997 Components of pathozone behaviour New Phytologist 136 342 358 Hartl D L Clark A G 2007 Principles of Population Genetics Sinauer Associates Inc Publishers Sunderland MA 653 pp Hedrick P W 2005 Genetics of Populations Jones and Bartlett Sudbury MA 737 pp James W C 1971 A Manual of Assessment Keys for Plant Diseases APS Press The American Phytopathological Society St Paul Minnesota USA Kareiva P Watts S McDonald R Boucher T 2007 Domesticated nature shaping landscapes and ecosystems for hu
47. 25 0 50378 0 82 FSC 0 0083 0 2111 Among individuals within populations 277 17 381 131 253 659 4 11 FIS 0 0421 0 0977 Within individuals 295 17 014 000 5 767 458 93 50 FIT 0 0649 0 0195 Total 589 36 250 512 6 168 317 Analysis of molecular variance performed using the program ARLEQUIN version 3 5 1 2 Excoffier and Lischer 2010 Distance method based on the sum of squared size differences Rsr between two haplotypes for microsatellite data 10100 permutations were used The groups correspond to the number of counties The highest level of subdivision was observed when comparing populations from different states The groups of populations located at the south of the country Nari o and Cauca states are differentiated from those located in the Cundinamarca Boyac Santander and Norte de Santander states The samples from different fields in each municipality were pooled in one single population and the previous results for population differentiation were corroborated Table 2 5 Geographically close populations presented Ast values close to zero or their values were not significantly different from zero 2 3 3 Reproductive mode Gametic and Hardy Weinberg Equilibrium tests HWE and gametic disequilibrium between pairs of loci were tested with the clone corrected data set The exact test for HWE showed that average 78 4 of loci were in HWE in the populations Table 2 6 The highest number of loci in HWE were present in populations Ventaqu
48. 3 Ceresini et al 2002b 59 Moreover we found unique MLMGs that might be the products of sexual reproduction that creates unique individuals by recombination during meiosis Halkett et a 2005 Clonal reproduction also was detected Some loci deviated from HWE and a significant deficit of heterozygotes was observed in almost all populations indicating nonrandom mating The departure from HWE of some loci may be due to inbreeding Additionally almost all the populations were characterized by some loci on gametic disequilibrium and significant values of lA and 7 Non random associations among alleles at different loci can be generated by asexual reproduction nonrandom mating linkage selection population structure isolated populations low gene flow or genetic drift founder events small populations Milgroom 1996 Taylor et al 1999 Halkett et al 2005 Populations of R solani AG 1 from different countries present a reproductive mode varying from strictly recombining to a mixed reproductive system in which there are recombination events followed by clonal expansion during the growing season Rosewich et al 1999 Ciampi et al 2008 Bernardes de Assis et al 2009 The evidence for clonal reproduction and recombination suggest a structure named epidemic structure this was described on bacteria Maynard Smith et al 1993 As in bacteria fungi can exhibit population epidemic structure in which sexual recombinatio
49. 4 0 18 82 57 0 83 0 6379 3 39 1 LaUnion2 35 28 22 6 0 20 85 79 0 86 0 6210 3 39 4 Soraca1 22 18 16 2 0 18 89 63 0 90 0 5742 3 24 0 Bonds Soraca2 23 17 13 4 0 26 75 89 0 76 0 6343 3 52 2 Ventaq1 20 17 16 1 0 15 90 50 0 91 0 5785 3 29 0 Ventaq2 21 15 10 5 0 29 89 09 0 89 0 5824 3 07 0 Carcasi1 10 9 7 2 0 10 92 59 0 93 0 6535 3 83 0 Santander Carcasi2 11 11 8 3 0 00 100 00 1 00 0 6082 3 26 0 Norte de Santander Chitaga 14 13 7 6 0 07 94 23 0 94 0 5604 3 04 1 Cauca Silvia 7 7 6 1 0 00 100 00 1 00 0 4545 2 74 0 Overall 355 295 237 58 0 15 89 36 0 89 0 5978 3 31 18 a N sample size of each population b Number of genotypes shared with other s populations are showed in brackets Go Stoddart and Taylor s genotypic diversity scaled to sample size Stoddart 1983 Stoddart and Taylor 1988 values scaled by the maximum number of expected genotypes d Mean followed by the same letter are not significantly distinct p 0 05 based on pairwise bootstrap test for differences in clonal diversity indices between populations calculated with GenoDive Meirmans and Van Tienderen 2004 1000 permutations with subsampling to match the size of the smallest population e an evenness value 1 0 indicates that all genotypes have equal frequencies f Nei s unbiased gene diversity Nei 1978 also known as expected heterozygosity averaged over all loci corrected for sample size g To test whether pairwise samples differed for Nei s unbiased gene
50. 6 642 650 Slatkin M 1995 A measure of population subdivision based on microsatellite allele frequencies Genetics 139 457 462 Stoddart J A 1983 A genotypic diversity measure Journal of Heredity 74 489 490 Stoddart J A Taylor J F 1988 Genotypic Diversity Estimation and Prediction in Samples Genetics 118 705 711 Strange R N Scott P R 2005 Plant disease a threat to global food security Annual Review of Phytopathology 43 83 116 Taylor J Jacobson D Fisher M 1999 The Evolution Of Asexual Fungi Reproduction Speciation and Classification Annual Review of Phytopathology 37 197 246 Tsror L 2010 Biology Epidemiology and Management of Rhizoctonia solani on Potato Journal of Phytopathology 158 649 658 Weir B S Cockerman C C 1984 Estimating F statistics for the analysis of population structure Evolution 38 1358 1370 Wilson P S Ketola E O Ahvenniemi P M Lehtonen M J Valkonen J P T 2008 Dynamics of soilborne Rhizoctonia solani in the presence of Trichoderma harzianum effects on stem canker black scurf and progeny tubers of potato Plant Pathology 57 152 161 Woodhall J W Lees A K Edwards S G Jenkinson P 2007 Characterization of Rhizoctonia solani from potato in Great Britain Plant Pathology 56 286 295 Zhan J Pettway R E McDonald B A 2003 The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by hig
51. 639 649 Ko W H Hora F 1971 A Selective Medium for the Quantitative Determination of Rhizoctonia solani in Soil Phytopathology 61 707 707 Kuninaga S Natsuaki T Takeuchi T Yokosawa R 1997 Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani Current Genetics 32 237 243 Lees A K Cullen D W Sullivan L Nicolson M J 2002 Development of conventional and quantitative real time PCR assays for the detection and identification of Rhizoctonia solani AG 3 in potato and soil Plant Pathology 51 293 302 64 Lehtonen M J Ahvenniemi P Wilson P S German Kinnari M Valkonen J P T 2008 Biological diversity of Rhizoctonia solani AG 3 in a northern potato cultivation environment in Finland Plant Pathology 57 141 151 Manly B F J 1990 Randomization and Monte Carlo Methods in Biology Chapman and Hall 2 ed Chapman and hall and CRC London pp 21 30 Martin F English J 1997 Population Genetics of Soilborne Fungal Plant Pathogens Phytopathology 446 446 Maynard Smith J Smith N H O Rourke M Spratt B G 1993 How clonal are bacteria PNAS 90 4384 4388 McCartney H A Foster S J Fraaije B A Ward E 2003 Molecular diagnostics for fungal plant pathogens Pesticide Management Science 59 129 142 McDermott J M McDonald B A 1993 Gene flow in plant pathosystems Annual Review of Phytopathology 31 353 373 Mc
52. 8 hours in PDA were paired with mycelial plugs of tester isolates separated 1 5 cm each other The slides were incubated at room temperature for 24 hours and the and the interaction was evaluated under light microscope checking the type of interaction for 15 points of contact in each pairing using safranin O staining Bandoni 1979 The hyphal anastomosis categories evaluated were i CO when paired hyphae grow without recognition this indicates that the paired isolates belong to different AGs ii C1 contact reaction there is only contact between hyphae Apparent cell wall fusion but not membrane fusion this reaction indicates that the paired isolates are distantly related although may belong to the same AG iii C2 killing reaction fusion of cell walls and cytoplasmatic membrane occurs but exchanged cytoplasm does not remain viable and death at the fused cells occur This indicates that the paired isolates belong to the same AG but are genetically distinct and iv C3 perfect fusion in this case fusion of cell walls and membranes occurs This reaction type indicates that the paired isolates are clones or genetically identical Cubeta and Vilgalys 1997 McCabe et al 1999 A pair of separate isolates was considered 21 to being to the same AG if more than 50 of fusion contacts were C1 C2 and C3 reactions Sneh et al 1998 An additional set of 21 AG 3 PT isolates was tested for macroscopic hyphal interactions to determ
53. 98N 77 31053N 3084 S tuberosum Parda Pastusa 2007 Antiquis Tu LaUnion1 5 58 22 N 75 22 59 W 2505 S tuberosum Capiro 2008 LaUnion2 6 00 26 N 75 21 40 W 2504 S tuberosum Capiro 2008 Ventatuemada Ventaq1 5 c N 73 e W 2918 S tuberosum Parda Pastga 2007 Boyac Ventaq2 5 20 OE N 73 2725 3 W 2907 S tuberosum ICA Unica 2009 Sbata Soracal 5 30 09 N 73 2022 09 W 2809 S tuberosum Gr phureja 2007 Soraca2 5 30 45 N 73 19 33 W 2849 S tuberosum Capiro 2009 Santander C reasi Carcasil 6 39 418 N 72 34610 W 2950 S tuberosum Parda Pastusa 2008 Carcasi2 6 4 470 N X 72 33 181 W 3296 S tuberosum Parda Pastusa 2008 Norte de Santander Chitaga Chitaga 7 03 062 N 72 40 638 W 2989 S tuberosum Parda Pastusa 2008 Cauca Silvia Silvia 2 31 30N 76 19 09W 3238 S tuberosum Gr phureja 2007 a masl meters above sea level 46 2 2 2 Microsatellite genotyping Tandem repeats were brought by the leader of the sequencing project of R solani AG 3 Twenty two trinucleotide four tetranucleotide and seven pentanucleotide microsatellite motifs were selected for developing the markers Primers were designed from flanking regions using the software Primer 3 Rozen and Skaletsky 2000 Polymorphism was initially assessed on agarose gels using 16 isolates randomly chosen from two populations of R solani AG 3 Subachoque1 and Cogua1 Then they were evaluated by capillary electrophoresis and the best conditions were established
54. A Watts C W Dexter A R Hall D 1999 Continuity of air filled pores and invasion thresholds for a soil borne fungal plant pathogen Rhizoctonia solani Soil Biology and Biochemistry 31 1803 1810 Otten W Hall D Harris K Ritz K Young I M Gilligan C A 2001 Soil physics fungal epidemiology and the spread of Rhizoctonia solani New Phytologist 151 459 468 Ritchie F Bain R A McQuilken M P 2009 Effects of nutrient status temperature and pH on mycelial growth sclerotial production and germination of Rhizoctonia solani from potato Journal of Plant Pathology 91 589 596 Ritz K 2007 Spatial Organisation of Soil Fungi In Franklin R B Mills A L Eds The Spatial Distribution of Microbes in the Environment Springer 179 202 pp Sicard D Pennings P S Grandcl ment C Acosta J Kaltz O Shykoff J A 2007 Specialization and local adaptation ff a fungal parasite on two host plant species as revealed by two fitness traits Evolution 61 27 41 Simons S A Gilligan C A 1997 Factors affecting the temporal progress of stem canker Rhizoctonia solani on potatoes Solanum tuberosum Plant Pathology 46 642 650 Strange R N Scott P R 2005 Plant disease a threat to global food security Annual Review of Phytopathology 43 83 116 Willi Y Frank A Heinzelmann R Kalin A Spalinger L Ceresini P C 2011 The adaptive potential of a plant pathogenic fungus Rhizoctonia solani A
55. A extraction from Rhizoctonia solani AG 3 Seven of the main producing potato states in Colombia were sampled Nari o and Cauca in the south of the country Antioquia in the west Cundinamarca and Boyaca in the middle Santander and Norte de Santander in the north of the country Table 2 2 Those States are responsible for approximately 85 of the potato production in Colombia Espinal et a 2006 Paired sympatric populations were sampled six in Cundinamarca four in Boyaca two in Antioquia two in Santander and two in Nari o and one population in Cauca and one in Norte de Santander Table 2 2 Samples from plants between two and three months of age exhibiting symptoms of stem canker were collected from naturally infected potato plants Solanum tuberosum Gr andigena and Gr Phureja in commercial farms In each field 20 30 symptomatic plants were collected distributed along the field normally in each field seven rows and five plants per row were sampled with ten meters between rows sometimes the tuber seed was collected and the isolation of the fungus from sclerotia was made when they were present The fungus was isolated transferring fragments of infected stems and sclerotia into plates containing selective media Ko and Hora 1971 and incubated at room temperature 20 C in the dark Pure cultures of R solani were established by transferring hyphal tips from de colonies growing in the selective medium for 24 to 48 h to plates with potat
56. Biological diversity of Rhizoctonia solani AG 3 in a northern potato cultivation environment in Finland Plant Pathology 57 141 151 Lehtonen M J Somervuo P Valkonen J P T 2008b Infection with Rhizoctonia solani Induces Defense Genes and Systemic Resistance in Potato Sprouts Grown Without Light Phytopathology 98 1190 1198 McDonald B A Linde C 2002 Pathogen population genetics evolutionary potential and durable resistance Annual Review of Phytopathology 40 349 379 Naz F Rauf C A Abbasi N A Haque l Ahmad l 2008 Influence of Inoculum Levels of Rhizoctonia solani and Susceptibility on New Potato Germplasm Pakistan Journal of Botany 40 2199 2209 Ogoshi A 1987 Ecology and Pathogenicity of Anastomosis and Intraspecific Groups of Rhizoctonia solani Kuhn Annual Review of Phytopathology 25 125 143 Olanya O M Lambert D H Reeves A F Porter G A 2009 Evaluation of potato clones for resistance to stem canker and tuber black scurf in field studies following artificial inoculation with Rhizoctonia solani AG 3 in Maine Archives Of Phytopathology And Plant Protection 42 409 418 Otten W Gilligan C A 2006 Soil structure and soil borne diseases using epidemiological concepts to scale from fungal spread to plant epidemics European Journal of Soil Science 57 26 37 Otten W Gilligan C A Watts C W Dexter A R Hall D 1999 Continuity of air filled pores and invasion threshold
57. Britain Plant Pathology 56 286 295 36 Chapter 2 Genetic diversity and population structure of Rhizoctonia solani AG 3 in Colombia 2 1 The state of the art Plant pathogenic fungi are a large and heterogeneous group of organisms with the ability to cause disease in natural and agricultural ecosystems Their life history and the ways in which they interact with their hosts are diverse ranging from species that establish biotrophic associations with hosts to species that can survive for long periods of time on dead host tissue or saprophytically in soil Burdon and Silk 1997 Their effects on plants are diverse ranging from mild symptoms to catastrophes in which large areas of plant crops are destroyed Strange and Scott 2005 Plant pathogens have great impact on the food production around the world they are very difficult to manage as consequence of their variability on time and space Crop plants instead have a narrow genetic base and continuously are challenged by pathogens Strange and Scott 2005 One of the main goals of plant pathology is to formulate strategies for disease control Methods are mainly based on genetic resistance in the host and application of chemical fungicides Usually the control practices loss effectiveness quickly due to the ability of the pathogens to evolve and to be adapted to external factors McDonald and McDermott 1993 Martin and English 1997 McDonald 1997 McDonald and Linde 2002 Studies
58. C_AG3_9 and TC_AG3_ 18 Group C TC_AG3_8 TC AG3 10 and TC_AG3_19 Individual PCR amplifications were conducted for loci TC_AG3_0 and TC_AG3_16 For fragment analyses 1 2 uL of fluorescent labeled PCR product were mixed in three distinct sets set 1 Group A Group B set 2 Group C TC_AG3_16 and set 3 TC_AG3 _0 Each set was electrophoresed using an ABI PRISM 3100 Applied Biosystems automated sequencer along with a fluorescently labeled size standard GeneScan 500 LIZ Applied Biosystems In each set the PCR products were mixed with sterile distilled water to a final volume of 30 uL The isolate RHOO2 isolated from sclerotia on tuber was included as control in every run of 94 samples The genotyping of one of the two populations used for describing the microsatellites was repeated The statistical binning of the alleles was conducted using the software GeneMapper version 4 0 ABI 47 2 2 3 Data analysis 2 2 3 1 Microsatellite information content For the data analysis was assumed that R solani AG 3 is a functional diploid Ceresini et al 2002b Justesen et al 2003 and the data support this supposition Except for a few cases one or two alleles for each SSR loci were found The isolates with more than two peaks in the chromatogram were eliminated of the analysis The initial analysis for the 11 microsatellite markers included the range of repeats for each locus the total and average number of alleles per locus and per s
59. DB4PBICZP 1 IGASAZE RA ISRAI GERD P 1E1P IDB4P2 13A4R3BSPTA4P TIF P TED MAT PIPERA 2EP 3T TAFBBP 1D3R2A4RA 14P 166 10P2 150 MRS ESPS E PRES D 5 F 2FRASBESP 1803P 128 TOUBSRA RAI GHSELPIESSASP4AIZP 4ATSR2B2R2DITP 1902A3C3PP GAZBASBASBA 138 1 RAFAS3B3P13E2P 22513 ZDB25A301P 11 BEBBOGEC BASED amp BF TP 180522058 X3P TIEZ2P Dimension 1 Dimension 2 ocu las 93 Annex 3 H Principal component analysis for the scores of the disease on stems roots and stolons i2aep epp 1205P 2 12A6P rege pe e 1P ize lesions on roots AP FIA GAZS TAP TBgr R x 19052 vc ud toric P e p ARPT BSP CH AEn Lars P TE IFISA Fee x1E1P 2 1602 10C3P T ESA 110K FS m MS EEE 2 BER Aat x 19D2P al A hg E D4 MS P 4ATIP3C2S CO d ith net IM inion Ord e AA 2 rr rr IATIP Er xd ISEISIEUC 3 752PF 106 AREA IOE3PHIBBBA E rei IOFSAZB2A2ETOYA 128 TO TRA 151 DEPASPE 1P 12D 1A66 IOP 2 11C APIBZRABFIBDABF2R BSP IOASPISAZSKH 11F4A ID TPIGAGEGZP IDASPHIDSESDAP SZ e lgsions_ 12D IF2C 4F2ASF2A f2B2A2B TONA N he sti 10CIBD2f5A 1 ADC SPEC 1050 1ESBASP 190 285 E7 SEE f8D2FGC 2P ISDSEBD2P 19D Q2B25P 220 HIDE P CONTAEIBASP Dimension 3 ocultar 94 Annex 3 1 Principal component analysis for the scores of the disease on tubers q 5D 1P2 HDP AAT a IBA3P KH 553P 1503P mypeldence lg __Tubers_witfsclerotia y IBB UP x 15D1P AAGAGP 13D5P 223 P 14E3P 2 20314 K3C 3P SE3E AGP TUNBEBR MIFSP Dm
60. Donald B A 1997 The population genetics of fungi tools and techniques Phytopathology 87 448 453 McDonald B A 2004 Population Genetics of Plant Pathogens The Plant Health Instructor aps online http www apsnet org edcenter advanced topics PopGenetics Pages default aspx McDonald B A Linde C 2002 Pathogen population genetics evolutionary potential and durable resistance Annual Review of Phytopathology 40 349 379 McDonald B A McDermott J M 1993 Population genetics of plant pathogenic fungi Bioscience 43 311 319 Bioscience 43 311 319 Meirmans P G Van Tienderen P H 2004 Genotype and Genodive Two Programs for the Analysis of Genetic Diversity of Asexual Organisms Molecular Ecology Notes 4 792 794 Milgroom M G 1996 Recombination and the multilocus structure of fungal populations Annual Review of Phytopathology 34 457 477 Milgroom M G Peever T L 2003 Population Biology of Plant Pathogens The Synthesis of Plant Disease Epidemiology and Population Genetics Plant Disease 87 608 617 Nei M 1978 Estimation of Average Heterozygosity and Genetic Distance from a Small Nmumber of Individuals Genetics 89 583 590 Ogoshi A 1987 Ecology and Pathogenicity of Anastomosis and Intraspecific Groups of Rhizoctonia solani Kuhn Annual Review of Phytopathology 25 125 143 Petit R J El Mousadik A Pons O 1998 Identifying populations for conservation on the basis of genetic marke
61. European Journal of Plant Pathology 109 983 992 Carling D E 1996 Grouping in Rhizoctonia solani by hyphal anastomosis In Senh B Jabaji Hare S Neate S Dijst G Eds Rhizoctonia Species Taxonomy Molecular Biology Ecology Pathology and Disease Control Kluwer Academic Publishers The Netherlands pp 37 47 Carling D E Baird R E Gitaitis R D Brainard K A S K 2002 Characterization of AG 13 a newly reported Anastomosis Group of Rhizoctonia solani Phytopathology 92 893 899 Carling D E Brainard K Virgen Calleros G Olalde Portugal V 1998 First report of Rhizoctonia solani AG 7 on potato in Mexico Plant Disease 82 127 Carling D E Leiner R H 1986 Isolation and characterization of Rhizoctonia solani and binucleate R solani like fungi from aereal stems and subterranean organs of potato plants Phytopathology 76 725 729 Carling D E Leiner R H 1990 Virulence of isolates of Rhizoctonia solani AG 3 collected from potato plants and soil Plant disease 74 901 903 Carling D E Leiner R H Westphale P C 1989 Symptoms signs and yield reduction associated with Rhizoctonia disease of potato induced by tuber borne inoculum of Rhizoctonia solani AG 3 American Potato Journal 66 693 701 Castro C Davis J R Wiese M V 1988 Quantitative estimation of Rhizoctonia solani AG 3 in soil Phytopathology 78 1287 1292 Cede o L Carrero C Quintero K Araujo Y Pino
62. G 3 under heat and fungicide stress Genetica 139 903 908 85 Annex 3 A Growth in vitro of isolates of Rhizoctonia solani AG 3 in response to four temperatures after 48 hours of incubation Colony size pixeles o B BulaLBm L LLL BE N 9 b 9 HG N 9 b N b N 9 11D6P 12F2P 15E1P 15E8P 15M2P 18B4P 19D2P 22A3P m4oC M100C M200C M300C B400C 1 2 and 3 are the number of repetitions for each isolate 86 Annex 3 B Time to sclerotia formation in isolates of Rhizoctonia solani AG 3 growing in four temperatures 30 wn gt c gt wwqia lt lt _ _ i _ Days to sclerotia formation m042C m109C m202C m302C m402C 1 2 and 3 are the number of repetition for each isolate 87 Annex 3 C Growth of isolates of Rhizoctonia solani AG 3 in vitro in presence of different doses of the fungicide Thifluzamide 300000 4 250000 4 200000 4 2 v X ES 150000 a gt 2 o Y 100000 50000 o la dac k Le L L nk L k W L L 24 48 72 96 24 48 72 9 24 48 72 96 24 48 72 96 24 48 72 9 6 24 48 72 9 6 24 48 72 96 24 48 72 96 24 48 72 96 5A2P 5A4S 5A5S 5B1P 5B5P 5B5S 5C3P 1 5D1P 5F2P mOppm MO 1lppm Mmippm m10ppm m100ppm m1000ppm Evaluations of colony size were done at 24 48 72 and 96 hours after inoculation on Petri dishes 88 Annex 3
63. GACI N PROYECTO DE TESE TESIS DEL PROGRAMA DE MAESTR AS EN CIENCIA AGRARIA procadi a calificar APROBADO i REPROBADO el trabajo de lesi como sigug a CALIFICACION DEL TRABAJO ESCRITO b CALIFICACION SUSTENTACION P BLICA d t Fi MeL PAULO CERESIHI PASAPORTE No 49304 ca A cos G GE Ho po IT EO clencia 7 traesalagia amp para sb pole Darm 2D Fin 42403 FACULTAD DE AORCNDMA Edificio E00 Piso Sr Oficina 338 Tanina BT 11316 ES Cavrsiadsc 57 1 310 5006 Exi 00060 Fax finder Casas alocir nicor iicp bagira edu co Bogoti Colormbla Sur Ameria Acknowledgments First of all want to thank to my academic supervisor Professor Celsa Garcia Dominguez Thank you for your guidance support and interest along the development this project since its conception to the materialization She took many risks and hope not have disappointed want to express my gratitude to all my fellow researchers and interns especially to Johan Cifuentes for his dedication he assumed many responsibilities and was confident that things were doing well Thanks to my academic advisers Paulo Ceresini Universidade Estadual Paulista UNESP Alba Marina Cotes Corpoica Maria Isabel Chacon Facultad de Agronom a Universidad Nacional de Colombia and Ursula Ram rez Escobar Instituto de Gen tica Universidad Nacional de Colombia for their useful and valuable suggestions to improve the research To Angela M
64. L raygrass Poa pratensis L Wild radish Raphanus raphanistrum L and mustard Brassica sp L ii Grass plantlets Pennisetum clandestinum Hochst ex Chiov rooting on peat Potato sprouts Solanum tuberosum L cv capiro rooting on peat were used as susceptible host plant Seedlings were transferred to pots containing autoclaved quartz sand five days before inoculation Each plant was inoculated with a 5 mm PDA plug obtained from the margins of the fungal colonies Carling and Leiner 1986 Plants were transferred to a growth chamber and incubated at 20 C with 12 hour photoperiod watered with distilled water every two days and fertilized with nutrient solution Murashige and Skoog 1962 once a week Fifteen days after inoculation the symptoms on stems and roots were evaluated The following categories of symptoms were considered i on stems symptoms were classified as small superficial lesions SS mild symptoms with lesions size 5 mm large superficial lesions LSL lesions gt 5mm cankers C ii On roots besides determining the levels of disease incidence on roots lesions categories were characterized similarly as on stems Symptomatic and non symptomatic plant tissue was transferred to selective media to check for the presence of the fungus 22 1 3 Results Symptoms observed in the field were the previously described in the literature In the stem base there were cankers of different size depending on the age o
65. McDonald and Linde 2002 McDonald 2004 de Meeus et al 2007 Selection is imposed to each pathogen deme by a range of biotic and abiotic factors Host related factors major resistance genes variation in presence quantity and relative balance of phenolic compounds variations in morphology etc frequently are seen as important but their effect on population diversity is unpredictable In a uniform environment selection may favor particular genotypes thus reducing its diversity The survival of mutant genotypes requires variation in fitness among individuals The evolutionary potential of a population is proportional to the amount of genetic diversity in genes that have an effect on fitness McDonald 1997 McDonald and Linde 2002 de Meeus et al 2007 2 1 2 Dynamics of diversity The interplay of selection genetic drift migration and mutation has a major effect on the genetic structure and diversity of all pathogen populations McDonald and Linde 2002 The relative roles of these factors may change between different pathogen host associations between stages in the epidemiological cycle and between associations in agricultural and natural ecosystems Burdon and Silk 1997 The evolutionary forces are continuously intermeshing to generate the overall variation encountered within a species in a continuous and dynamic process In the agroecosystems pathogens may be considered as colonizing species subject to frequent local extinc
66. O C1 and C3 reactions and when AG 3 was paired with AG 2 1 CO and C1 reactions were observed In this work the AG differentiation by somatic compatibility was not a reliable methodology However the macroscopic somatic compatibility test allows to infer death reactions between pairs tested Previously was shown that C2 and C3 reactions are highly correlated with the macroscopic vegetative reactions tuft and merge respectively MacNish et al 1997 For instance macroscopic vegetative reactions can be used to predict microscopic anastomosis reactions and vice versa Dead reaction was the common reaction into the isolates tested and was evident due to the barrage formation Figure 1 3 a macroscopic indicator of vegetative incompatibility between isolates belonging to the same AG MacNish et al 1997 In Basidiomycetous fungi the somatic incompatibility is commonly restricted to secondary multicariotic mycelia in R solani has been associated to differences between the products 27 of the genes involved Burnett 2003 and has been proposed as a mechanism to prevent the horizontal transmission of malign elements like myco viruses in groups like aspergilli Pal et al 2007 Variation in the interaction among isolates was observed in some pairings the barrage was strong with a huge amount of aerial mycelia Figure 1 3 a b whereas in other cases the line was soft but well defined 91 31 of the pairs formed barrage indic
67. RC INE 7 PF Sg3P 1InA3PKK 1902P 188 4AGETSSE ASD 1 PIDESBE 1 BB3R B260 4344 782P xK SC2PIIBSPTSD2P A14P TIBGR IAGP 761 2L PH Z2ER I3DSP ne SFP 11A5P gt see Z2B25E2C 4P yop S S030481 62hserasb 4P 2 waist a 2098 13C3R3D5BT 12P Z2B25I6E733ESA D3ROESRDESPPI BS Mea SIGA F SF3P1E3P 37 122P iur rir DR P IEF IN B y bes 1607P XK 3AcF 14D WE TEE LSA 2P Ea E ut Gey 3B5BF3P LIAGFABBR 1DZP I3GIP Z2CSRZRGDGEB3P 19 19D age A 1BREP 11F4PIBOSPASASS 14D 1R3E2RBD En es M 5 10C3R 1D 1R2F2PIGEBRSD53 3A5P TC IR BIED3RAFZI n GP IDBBRIBSPIESR3DSRBDSGISEIGKH SFE SP 41 CS P 5D4313C BASP 4A Boems 2l x OC 1P2 ESF IP AATSIR I 5 150398 1 5C 43A53B 1 E 13G 1 ASBSW2BALSST 1 14B3R3F3363P GAZZA PSB 16BIDFaE3ROBSHE3P TESKFI 19D2P IGASP14D IR TBGIE3P 13053793P SF3P3H IP I2A4R5D2E2C3E2E13 1BA33165 IOPEDSAGASBE IP GFZI 1DF5R3C3RBB4EZ2ETZZH3P 1E1P486 R6D7 ROFSAOBSADAJES Z3 11B2A6D7P I4C2FZ2C 4R5D3P 2 132A S SERRE SP HIBZP 11 100354 IGAGIEINBPHSP 1168 1OPRESPISAZSSKH 19053 14A3FIF P TOF P TOC 4R2ERI i12F2P 1IBSPTC2PESD3RTB2P Z2B25E2A3RE2DPIZFZMZD IR2A4P 128 TUZEN 3H SC an 1BBRDOE3PERC ASDSAGE 34D I28TOYA Z2B25EZA3E2H3R2E4P 220 4P ISEJSKHIDFSRDC 1PPIAGR 1C A 4C2P 1507 E2E I3 ZOCSEZAGR3GIFE4P I3F3H3G1P IDC3RSE TRIBES ZEZ2ERNDIEZ E 2SBESF I2ABRZETO YA ISD5315A1R A3P 14E3P 1BB 4E2C3READEE3EBESP JaABNSD E MERA AGP Z2C 4R5D02R OZ I3HF3PPBZIBF2PBIRCTIP 1ASP 1902340 2P 1 IB2P13F3P iGEHSERSH A4 55D3BIA2ERRE EC ZI B P 1
68. Silvia N 7 2 5 4 3 1 4 1 4 3 2 4 33 Lora Jum por 3of 2 11 12 9 7 13 4 11 14 7 9 99 alleles N Sample size for each population Data without cloning correction 67 Annex 2 B Measures of differentiation among populations of Rhizoctonia solani AG 3 infecting potato in based on As values Subach1 Subach2 Cogua1 Cogua2 Sibate1 Sibate2 Silvia Pasto1 Pasto2 Vent1 Soracal Soraca2 Vent2 LaUnion1 LaUnion2 Carci Carc2 Chitaga Subach1 0 000 0 229 0 284 0 487 0 193 0 512 0 214 0 043 0 054 0 042 0 243 0 931 0 398 0 093 0 592 0 152 0 162 0 199 Subach2 0 011 0 000 0 659 0 417 0 013 0 182 0 024 0 004 0 070 0 557 0 305 0 511 0 696 0 445 0 724 0 284 0 476 0 832 Cogua1 0 007 0 010 0 000 0 701 0 003 0 555 0 009 0 005 0 014 0 679 0 532 0 579 0 993 0 803 0 396 0 396 0 325 0 803 Cogua2 0 002 0 001 0 015 0 000 0 018 0 463 0 044 0 011 0 015 0 282 0 611 0 564 0 651 0 260 0 407 0 578 0 197 0 405 Sibate1 0 017 0 060 0 067 0 078 0 000 0 093 0 172 0 466 0 131 0 000 0 005 0 246 0 016 0 004 0 050 0 001 0 012 0 020 Sibate2 0 001 0 019 0 009 0 000 0 042 0 000 0 012 0 021 0 004 0 141 0 893 0 912 0 911 0 146 0 179 0 435 0 214 0 106 Silvia 0 027 0 093 0 111 0 104 0 029 0 138 0 000 0 121 0 148 0 001 0 008 0 223 0 009 0 004 0 083 0 007 0 004 0 000 Pasto1 0 041 0 081 0 073 0 100 0 001 0 088 0 053 0 000 0 136 0 004 0 0
69. Size lesion average on potato plants cv Capiro caused by isolates of H solani AG 3 collected on different municipalities in Colombia The sclerotia infestation on tubers was mild The highest value was present in plants inoculated with isolates collected on Silvia with 1 75 96 of the surface of tubers covered by sclerotia Figure 3 6 The test for differences among municipalities was significant Annex 3 E however the values were small falling in one category in the severity scale Severity of black scurf Carcasi Chitaga Cogua La Union Pasto Sibate Silvia Subachoque Ventaquemada Control Municipalities Figure 3 6 Percent of sclerotia on the tuber surface of the cv Capiro caused by isolates of R solani AG 3 collected on different municipalities in Colombia There were differences in the incidence of black scurf on tubers among isolates collected on different fields P value lt 0 05 and among localities The isolates from Cogua Sibate and Ventaquemada showed the highest levels of incidence of sclerotia on tubers and the lowest was found in plants inoculated with isolates from Subachoque Figure 3 7 0 000 000 00 Control 79 The pathogenicity of the isolates was not dependent on their origin stems or sclerotia from tubers Isolates obtained from sclerotia did not produced higher levels of black scurf In each municipality there were a few isolates that produce high levels of sclerotia The same result were obtai
70. a rbarD p value disequilibrium Jo Subachoque1 23 9 11 0 0439 0 6510 1 3979 0 1439 0 0010 9 55 16 36 Subachoque2 12 10 10 0 1385 0 2141 1 2592 0 1430 0 0010 2 45 4 44 Cogua1 21 7 11 0 1755 0 0655 0 2851 0 0293 0 3100 1 55 1 82 Cundinamarca Cogua2 26 7 11 0 0162 0 4321 1 4109 0 1429 0 0010 14 55 25 45 Sibate1 17 7 10 0 0477 0 3098 0 9931 0 1125 0 0010 3 55 5 45 Sibate2 11 7 11 0 0128 0 4604 0 5048 0 0510 0 4190 0 55 0 00 ee Pasto1 17 6 10 0 2487 0 0186 0 7759 0 0885 lt 0 0010 0 45 0 00 arino Pasto2 19 9 11 0 0908 0 2033 0 6503 0 0670 0 0020 0 55 0 00 oa LaUnion1 14 9 11 0 0294 0 5503 1 1763 0 1225 lt 0 0010 3 55 5 45 ntioquia a LaUnion2 28 8 11 0 0254 0 5806 0 8859 0 0916 lt 0 0010 8 55 14 55 Soraca1 18 8 11 0 2764 0 0088 0 3726 0 0387 0 2650 0 51 0 00 Soraca2 17 7 11 0 1142 0 1975 0 5130 0 0519 0 0530 0 55 0 00 BOYACA Ventaquemada1 17 8 11 0 0348 0 6011 1 0026 0 1032 0 0010 0 55 0 00 Ventaquemada2 15 11 11 0 1050 0 7448 1 2943 0 1338 0 0010 0 55 0 00 pe Carcasi1 9 10 11 0 0276 0 4594 0 6409 0 0654 0 4270 0 48 16 36 antander Carcasi2 11 11 11 0 2143 0 8407 0 6972 0 0724 0 2300 0 55 0 00 Norte de Santander Chitaga 13 9 11 0 1881 0 8407 0 9325 0 0982 0 0010 0 55 0 00 Cauca Silvia 7 9 9 0 0731 0 6314 0 7317 0 0930 0 0230 0 36 0 00 a HWE Hardy Weinberg test performed according to an exact test analogous to Fisher exact test using a Marcov chain with forecasted length of 100 000 Guo and Thompson 1992 one and
71. al 2003 Lehtonen et al 2008 and genotypic diversity Kuninaga et al 1997 Ceresini et al 2002a Ceresini et al 2002b Justesen et al 2003 60 Balali et al 2007 Woodhall et al 2007 which is not consistent with inbreeding or a selfing organism 2 4 4 Demographic parameters The Colombian populations did not present evidence of bottlenecks or founder events Genetic drift acts more quickly in small populations reducing genetic variation bottlenecks can reduce drastically the genetic variation of the populations even if the bottleneck does not last for very many generations In plant pathogen populations the control of the diseases are the most important force causing reduction in their size The management of R solani AG 3 in Colombia is recent and it uses Thifluzamide in the soil and applications of Trichoderma sp however the results show that this activity has not affected substantially the population size of this pathogen in the localities evaluated Populations Pasto2 and Ventaquemada1 presented gene diversity deficiency across loci this show that those populations have experienced recent population expansion which is the result of a high increasing in the population size Modern plant pathogen populations are shaped by the evolutionary forces that have acted on their ancestral populations McDonald and Linde 2002 Genetic polymorphism may be indicative of evolutionary adaptation which plays a key role for survi
72. al propagules produced asexual propagules produced 5 Efficient directional selection R gene deployed in genetically uniform monoculture and continuously over large areas 5 Disruptive selection R genes deployed in mixtures multilines R genes deployed as rotations in time or space Source McDonald and Linde 2002 2 1 5 Molecular markers and the study of population genetics Population genetics deals mainly with genetic processes To understand the influence of selection inbreeding genetic drift gene flow and mutation in population genetics is necessary to describe and quantify the amount of genetic variation in a population and the pattern of genetic variation among populations Nei 1978 Hedrick 2005 The most common approach to study population genetics is through molecular markers Actually there is a wide range of molecular markers available The choice of the genetic marker depends mainly on the question to answer on the experience and competence of the researchers and on laboratory facilities McCartney et al 2003 In population genetics the molecular markers must help to test ecological and epidemiological hypothesis and to distinguish biological processes in the populations of pathogens not only to quantify its diversity 42 Genetic markers represent genetic differences between individual organisms or species All genetic markers occupy genomic positions within chromosomes like genes Collar
73. ampled field the identification of private alleles i e those present in only one population all them derived from the information of allele frequencies and computed by the program CONVERT version 1 31 Glaubitz 2004 2 2 3 2 Genotype diversity Each isolate was assigned to a multilocus microsatellite genotype MLMGs using GENODIVE Meirmans and Van Tienderen 2004 isolates with the same MLMG were treated as clones Indices of clonal diversity were calculated as follows i number of MLMGs per population ii site specific MLMGs iii clonal fraction or the proportion of isolates originated from asexual reproduction calculated as 1 number of different multilocus genotypes total number of isolates Zhan et al 2003 iv Stoddart and Taylor s genotypic diversity calculated as Gy 1 2pi2 where pi is the frequency of the ith genotype Stoddart and Taylor 1988 and v its evenness an indicator for how the genotypes are distributed within the population Gr nwald et al 2003 These measures were calculated using the program GENODIVE If pairs of populations differed in their genotype diversity indices was tested using a bootstrapping approach resampling with replacement where the individuals were resampled from the populations and the diversity indices were compared after every replicate Manly 1990 using 1 000 permutations with subsampling matching the size to the smallest population Gr nwald et al 2003 Finally the c
74. an statistical procedure implemented in STRUCTURE version 2 3 2 was used Falush et al 2003 Hubisz et al 2009 Pritchard et al 2000 The test determines whether there are individuals immigrants in a sample with respect to their reference geographical population This program calculates the membership coefficients Q of every MLMG sampled and assign individuals probabilistically to populations based on their genotypes Additionally identifies admixed individuals whose genetic composition is drawn from more than one population Hubisz et a 2009 Pritchard et al 2000 An initial run was executed considering all the geographical populations and a simulation of the best K was made Evanno et al 2005 Then three runs of MCMC simulations were performed with a burn in period of 2 000 000 generations followed by 1 000 000 iterations each run Parameters were set to the admixture model with the prior number of clusters corresponding to the number of fields sampled K 18 and using prior information of the populations as the initial condition for assign MLMGs The prior As values used were those previously calculated with ARLEQUIN 3 5 1 2 In a second analysis the same priors were used except that individuals were not assigned to the sampled populations and k varied from two to 18 2 2 3 7 Population bottleneck and demographic parameters The test for recent founder effects or historical bottlenecks was made with the program BOTTLENECK V 1 2
75. ance Significant values at p lt 0 05 are shown in red Test based on 1 100 permutations 68 Chapter 3 Diversity and adaptability of Rhizoctonia solani AG 3 3 1 The state of the art Fungi are one of the most diverse groups of organisms in nature Approximately 70 000 species have been described but is estimated to exist about 1 5 million of fungal species Kiffer and Morelet 1997 Most of them obtain nutrients living in close association with other organisms Many fungi are pathogenic and have strong impact on human health or lead to severe economic losses due to crop and animal diseases Strange and Scott 2005 The emergence of fungal diseases of crop plants during the past century probably is due to human activities that have modified the ecosystems at global scale e g climate warming widespread deforestation habitat fragmentation urbanization changes in agricultural practices and global trade Kareiva et al 2007 The intensification and globalization of agriculture as well as the increases of international trade and travel have broken many natural barriers to pathogen dispersal causing the redistribution of many organisms Kolar and Lodge 2001 Plant pathogens are very successful in agro ecosystems causing serious diseases They produce great deal of inoculum their latent period is very short have very efficient and fast dispersion means water wind vectors propagation material and produce different kind of subs
76. and crisp colour in cv Record Potato Research 37 43 49 Jager G Hekman W Deenen A 1982 The occurrence of Rhizoctonia solani on subterranean parts of wild plants in potato fields Netherlands Journal of Plant Pathology 88 155 161 James W C McKenzie A R 1972 The effect of tuber borne sclerotia of Rhizoctonia solani Kuhn on the potato crop American Potato Journal 49 296 301 Jeger M J Hide G A Boogert P H J F Termorshuizen A J Baarlen P 1996a Pathology and control of soil borne fungal pathogens of potato Potato Research 39 437 469 Jeger M J Hide G A Van den Booger P H Termorshuizen A J Van Baarlen P 1996b Soilborne fungal pathogens of potato Potato Research 39 437 469 Jeon Y A Kim W G Kim D H Kwon S W Hong S B 2010 Taxonomic Position of Korean Isolates of Rhizoctonia solani Based on RAPD and ITS Sequencing of Ribosomal DNA The Plant Pathology Journal 26 83 89 Johnk J S Jones P K Shew H D Carling D E 1993 Characterization of Populations of Rhizoctonia solani AG 3 from Potato and Tobacco Phytopathology 83 854 858 Justesen A F Yohalem D Bay A Nicolaisen M 2003 Genetic diversity in potato field populations of Thanatephorus cucumeris AG 3 revealed by ITS polymorphism and RAPD markers Mycological Research 107 1323 1331 Keijer J 1996 The initial steps of the infection process in Rhizoctonia solani In Sneh B Jabaji Hare S
77. anya et a 2009 though the responses are weak and no resistant cultivars have been identified or developed Jeger et al 1996 In a recent study systemic induction of resistance in sprouts upon infection with a virulent isolate of R solani was demonstrated and the secondary infection of the sprouts was inhibited Lehtonen et al 2008b The high variability of R solani and the effect of gene flow and sexual reproduction situate this fungus in the category of high evolutionary risk which need an approach for host resistance based on more than one resistance gene Until the R solani S tuberosum interaction is completely elucidated breeding for resistance does not seem to be an option for the management of the black scurf and stem cankers on potato 101 4 3 5 Biological control Different organisms have been used to suppress R solani including bacteria actinomycetes Bacillus and fluorescent Pseudomonas and fungi Trichoderma Verticillium binucleated Rhizoctonias and hypovirulent isolates of R solani all of them reduce the disease or inhibit the pathogen in controlled assays however in natural conditions usually fails to reduce disease severity or to stimulate plant growth Tsror 2010 Combination of organisms have been reported to give better results in the control of stem canker severity by 40 49 in greenhouse trials Brewer and Larkin 2005 Microbial communities are evolving entities Groups of individuals in a p
78. ar a Vargas Natalhie Mart nez and to the emeritus professor Enrique Torres for their revision of the document and helpful comments Thanks to all the members of the Group of Potato Pathology for their continuous emotional and labor support Dayana Irene Carol Edgar and Ricardo To my professors Oscar Oliveros and Teresa Mosquera with them always found open the door and a proper answer to my frustrations Thanks to my friends for their support especially to Nayibe Linda Diana Martha Andr s Augusto and Isabel they gave me the reasons to go on when the forces fall spent nice time during my years in the Antonio Angarita Zerda Laboratory have been privileged to work and share time and thoughts with remarkably skilful talented and friendly people Thanks to all of them for their advices friendship and care Thanks to the ETH and all the people in the Plant Pathology Group especially to Bruce McDonald for his help and support Special acknowledges to the founding institutions Universidad Nacional de Colombia Division de Investigaci n de la sede Bogot y Escuela de Posgrados de la Facultad de Agronom a Ministerio de Agricultura y Desarrollo Rural de Colombia Asohofrucol and ETH Swiss Federal Institute of Technology Zurich Switzerland All they provided funds for this research Thanks to my mother for her care and love and for being my most unconditional friend Thanks to the life for guiding my steps in the be
79. as proposed that the infection of potato plants by R solani AG 3 may be initiated by soil borne as well as tuber borne inoculum and their relative importance depends upon inoculum density and the environmental factors Frank and Leach 1980 However tubers are exposed to the soil inoculum more time than stems as consequence it is more likely that isolates sampled from tubers mainly represent the population present in the soil Justesen et al 2003 AMOVA and Fsr results were confirmed by the analysis with STRUCTURE low differentiation among populations additionally those results showed that there are migrant individuals in populations and that many individuals have a genetic background from different populations This is explained because the pathogen moves frequently through tuber seed contaminated and supports the occurrence of sexual recombination generating admixed genotypes as previously was shown Ceresini et al 2002b 2 4 3 Reproductive mode The sexual stage of R solani AG 3 is known however for a long time was proposed that this pathogen was prevalent asexual in the fields The hypothesis of clonality for this pathogen in Colombia was tested The high genotypic diversity along with the low clonal fraction the high proportion of loci on HWE gametic equilibrium for most of the pairs of microsatellite loci and Fis values not significant different from zero confirm previous findings for random mating in populations of R solani AG
80. ating genetic diversity among the isolates tested at least in the genes related with somatic compatibility 8 69 of the pairings showed merge reaction those isolates came from different fields except for one isolate from Nari o1 that showed merge reaction with five isolates belonging to different geographical origin AG 3 is the major and most aggressive group of R solani on potato in Colombia The symptoms were the typical canker reported on stems Roots did not show symptoms probably the environment was not suitable for root infection considering that there are reports of symptoms on this organ Our results about the prevalence of AG 3 on potato are in accord with previous worldwide reports Carling and Leiner 1986 Anguiz and Martin 1989 Bains and Bisht 1995 Balali et al 1995 Virgen Calleros et al 2000 Campion et al 2003 Truter and Wehner 2004 Woodhall et al 2007 Cede o et al 2001 The AG 2 1 was also detected on stem cankers but in lower frequency and it caused only mild symptoms on potato Our results of the pathogenicity tests on different plants species showed that AG 3 is able to infect different hosts but there were differences among plants in severity and affected organ Potato tomato and lulo exhibited stem cankers pea bean corn and carrot showed infection on the roots Those results point at the limited efficiency of plant rotation as a disease management practice because plants that usually are f
81. ation subdivision of H solani AG 3 was found among isolates collected on different varieties indicating that there is a strong force that prevents the differentiation The movement of infected tuber seed among counties is the most plausible cause of the low differentiation The highest level of subdivision was observed among groups of populations located at the south of the country Nari o and Cauca states and those located in the Cundinamarca Boyac Santander and Norte de Santander states Those populations are geographically distant and probably have a low interchange of tuber seed with the other populations Close populations were not differentiated Lack of population structure has been described previously in populations of R solani AG 3 sampled from stem potato plants in USA and Denmark Ceresini et al 2002b Justesen et al 2003 On previous studies was proposed the genetic differentiation among isolates found in soil from those from plants on rice Banniza et al 1999 and potato Justesen et al 2003 however our results show that there are no specific MLMGs associated to black scurf on tubers or to stem canker In some cases the MLMG on tuber seed was the same on stem canker showing that tuber borne inoculum could start the infection The opposite situation also was found the MLMG on sclerotia on tuber seeds was different from MLMG on stem canker which is evidence that the lesion was initiated by soil borne inoculum Previously w
82. ato fields in Central Iran and South Australia Mycopathologia 163 105 115 Balali G R Neate S M Scott E S Whisson D L Wicks T J 1995 Anastomosis group and pathogenicity of isolates of Rhizoctonia solani from potato crops in South Australia Plant Pathology 44 1050 1057 30 Bandoni R J 1979 Safranin O as rapid nuclear stain for fungi Mycologia 71 873 874 Bandy B P Leach S S Tavantzis S M 1988 Anastomosis group 3 is the major cause of Rhizoctonia disease of potato in Maine Pant Disease 72 596 598 Banville G B Carling D E 2001 Rhizoctonia canker and black scurf In Stevenson W R Loria R Franc G Weingartner D P Eds Compendium of Potato Diseases APS Press St Paul Minnesota pp 36 37 Banville G J 1989 Yield losses and damage to potato plants caused by Rhizoctonia solani Kuhn American Potato Journal 66 821 834 Banville G J Carling D E Otrysko B E 1996 Rhizoctonia disease on potato In Baruch S Suha J H Neate S Dijst G Eds In Rhizoctonia Species Taxonomy Molecular Biology Ecology Pathology and Disease Control Kluwer Academic Publishers The Netherlands pp 321 330 Burnett J 2003 Fungal Populations and Species Oxford University Press USA 368 p Campion C Chatot C Perraton B Andrivon D 2003 Anastomosis Groups Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France
83. between populations Genetic differentiation between populations was considered significant when Ps0 05 The AMOVA was conducted to partition the variance components due to among group states effect among populations within group effect and within population effect Significance of the fixation indexes was tested using 10 100 permutations by a nonparametric approach Excoffier et al 1992 using the program ARLEQUIN 3 5 1 2 Excoffier and Lischer 2010 2 2 3 5 Reproductive mode Gametic and Hardy Weinberg Equilibrium tests To assess the contribution of sexual recombination to the genetic structure of the Colombian populations of R solani AG 3 the tests of Hardy Weinberg Equilibrium HWE and multilocus association were performed to determine the associations within and among loci respectively Tests for HWE were executed for each locus within each population with the program ARLEQUIN 3 5 1 2 This test is analogous to Fisher s exact test on a two by two contingency table but extended to a contingency table of arbitrary size Guo and Thompson 1992 Raymond and Rousset 1995 P values were obtained using a Markov Chain Monte Carlo MCMC approach generating an exact probability distribution not biased by rare alleles or low sample size Raymond and Rousset 1995 The hypothesis of independence among locus of different MLMGs was tested using the Fisher s exact test Garnier Gere and Dillmann 1992 based on an MCMC algorithm with 10
84. bility The population structure was studied by characterizing and evaluating microsatellite markers which were obtained from the genome of R solani AG 3 Polymorphisms at the SSR markers were analyzed by capillary electrophoresis followed by data analysis using several software in order to study the frequencies of the alleles in the populations and how was this related to the evolutionary forces SSR are effective markers that have been used in many species for studies of population genetics SSR markers have many advantages over other markers such as ease analysis high polymorphism rate high reliability and codominance Actually the availability of genomes sequenced makes their development more easy and achievable Schl tterer 2000 The analysis of multilocus data allows determining the genotypic variability into the populations and inferring the relative importance of each evolutionary force in shaping the genetic structure of the populations The calculation of genetic distances between populations gives an estimate of isolation or relationship among populations Mutation and genetic drift at selectively neutral loci lead to differentiation in the allele frequencies between populations and gene flow give cohesiveness to them McDonald and Linde 2002 Gr nwald et al 2006 Multivariate analysis is a convenient way to represent the overall organization of genetic data making statistic inferences about the spatial distribution of genetic di
85. ce of chlorophyll in the autotrophic phase Carling and Leiner 1990 and to the physical resistance of the tissue The second hypothesis is related to the substrate quartz sand allow higher expansion of the fungus Otten et al 2001 and because is an inorganic substrate that does not bring any nutrient pressed to the fungus to move inside and parasite young roots and stolons The evolutionary forces mutation migration genetic drift selection and reproduction system have influence in the amount of variation in the populations of organisms but he selective factors causing differentiation among populations are natural selection and random genetic drift Comparative studies of quantitative genetic and neutral marker differentiation assess for the relative roles of natural selection and random genetic drift in the divergence among populations Leionen et a 2008 Most of the phenotypic evolution likely is driven by natural selection however in plant pathogen fungi the genetic drift is common as bottlenecks caused fungicides and founder events Small Ne increases the rate of genetic drift providing more scope for non adaptive differentiation England et al 2003 The populations of R solani AG 3 evaluated in this study have a high effect of migration among populations the permanent gene and genotype flow hide the effects of natural selection and genetic drift in the populations this can be the main reason why association among isolates to geog
86. continuous values are referred as quantitative traits Hartl and Clark 2007 Quantitative traits include size fitness components and rate of growth they are usually attributed to polygenic effects and are the result of the interaction among the genes and the environment Hedrick 2005 3 1 3 Differentiation of soil borne fungi in time and space Soil is a diverse and complex environment Soil composition has strong effect on ecological and evolutionary processes into the populations in scales ranging from the soil pores until landscape Ettema and Wardle 2002 The composition microbial communities in the soil changes along environmental gradients such as temperature soil properties and geographical locations if selection in these different environments is often strong can lead to ecological specialization Kassen and Rainey 2004 Local environment modify the activity of the organisms i e nutrients uptake predation risk competence and indirectly the growth reproduction movement and surviving Some environmental factors i e temperature water potential can be the same at macro and micro scales others nutrient contents moist and pH are not uniform and do not reflect conditions affecting individuals or microbial communities Franklin and Mills 2007 Ritz 2007 then more detailed studies are necessary to identify microenvironments regulating populations of microorganisms in the soil In soil borne diseases the factors allowin
87. d causing stem cankers from 0 4 to 10mm but not on roots However these isolates produced small spots on a large proportion of roots from pea bean corn and carrot The two grasses P clandestinum and P pratense evaluated did not show any symptoms when inoculated with AG 2 1 nor AG 3 showing that they are not suitable host for those AGs Symptoms caused by binucleate Rhizoctonia isolates AG A AG E and AG l were characterized by the presence of many small superficial lesions on roots Soft symptoms are characteristic of some binucleate isolates of R solani and they have been used as biocontrol organisms 1 4 Discussion This is the first large scale population study carried out in Colombia aiming at determining the distribution of AGs associated with Rhizoctonia diseases on potatoes and assessing the influence of the various AGs in symptom expression on different hosts Previously was reported that the main AG associated to stem canker is AG 3 and the Colombian population was not an exception The most proportion of isolates belonged to this AG Those results were confirmed by PCR with specific primers sequencing of the rDNA and somatic compatibility The most efficient and accurate methodology to identify AGs was PCR and sequencing The somatic compatibility tests confirmed the results about the AG however the differentiation between perfect fusion and death reactions were not possible Pairings amongst AG 3 isolates resulted in C
88. d AG 3 recovered from potato fields in Colombia AG 2 1 AG 3 Stem Size Root Diseased Stem Size Root Diseased lesion Cm lesion roots lesio Cm lesion roots n Pisum sativum SSL lt 0 01 SSL 34 5 NI 0 SSL C 18 6 Phaseolus vulgaris NI 0 SSL 45 3 NI 0 SSL 31 2 Solanum quitoense SSL 0 40 NI 0 NI C 0 70 NI 0 Zea mays NI 0 SSL 40 SSL 0 40 SSL 60 0 Raphanus raphanistrum C 0 83 NI 0 NI 0 NI 0 Brassica spp C 0 61 NI 0 NI 0 NI 0 Solanum tuberosum cv capiro C 1 03 NI 0 C 1 05 NI 0 Penissetum clandestinum NI 0 NI 0 NI 0 NI 0 Poa pratense NI 0 NI 0 NI 0 NI 0 Solanum lycopersicum NI 0 C 0 NI C 0 62 NI 0 Daucus carota C 0 01 NI C 7 4 NI 0 NI C 49 9 Stem and root lesions were categorized as follows Non infected NI SSL Superficial Small Lesion and C Canker Size of the lesions is the average of five plants Isolates of R solani AG 3 significantly affected stems of solanaceous hosts such as potato tomato and lulo causing large cankers gt 5mm No symptoms were observed on roots of 26 these plants AG 3 isolates caused abundant small lesions on roots of peas beans corn and carrots but they did not cause stems lesions The other plants Brassica spp H raphanistrum P clandestinum and P pratense inoculated with AG 3 did not present symptoms Besides potatoes isolates of R solani AG 2 1 affected the Brassicaceae hosts yellow mustard and purple mustar
89. d among localities In Colombia the regulation for seed production of H solani on tubers allow until 10 of tuber surface with sclerotia additionally farmers exchange seed informally and only few of them use certified seed tubers providing the means for homogenization of whole population which was confirmed by the presence of two genetic populations from the 18 geographical populations sampled The second force that acts in the structure of the Colombian populations of R solani AG 3 is its mode of reproduction The data revealed cycles of sexual reproduction followed by asexual multiplication This condition gives an advantage to those populations Meiotic recombination puts together new combinations of alleles and the asexual multiplication increases the frequency of those new combinations of alleles in the populations Pathogen populations with mixed reproduction show particular genetic structure called epidemic structure The sexual recombination gives raise an excess of recombinant genotypes the most fit then dominates in the asexual cycles Many fungi that have being assumed mitosporic exhibit characteristics of recombination in their population genetic structure and that truly asexual lineages if they exist do not appear to persist for evolutionarily significant lengths of time Taylor et al 1999 Evidence for epidemic structure has been found in populations of R solani AG1 IA suggesting a reproductive mode varying from strictly reco
90. d et al 2005 In fungi the markers used include i morphological phenotypic traits or characters ii physiological mating types somatic incompatibility and fungicide resistance iii pathogenic iv cytological and v molecular biochemical and DNA based Burnett 2003 The most widely used are DNA markers predominantly due to their abundance in genomes Schl tterer 2004 The preferred markers in population genetics are characterized by its neutrality codominance and polymorphism Collard et a 2005 Hartl and Clark 2007 Microsatellites are widespread markers They are tandemly repeated sequences of 1 6 nucleotides found at high frequency in the nuclear genomes of most taxa They are also known as simple sequence repeats SSR variable number tandem repeats VNTR and short tandem repeats STR A microsatellite locus typically varies in length between 5 and 40 repeats Dinucleotide trinucleotide and tetranucleotide repeats are the most common motifs choice in molecular genetic studies Schl tterer 2000 2004 Typically microsatellites display high mutation rate among 10 and 10 mutations per locus per generation generating the high levels of allelic diversity Schl tterer 2000 Karaoglu et al 2004 The mutation rate of SSR markers varies depending on the locus the length of the repeated motif the specie and sometimes the allele Benali et a 2011 Microsatellites gain and lose repeat units by DNA replication sl
91. d evaluated for 96 hours showed that the fungicide inhibits the growth in the first 48 hours after that the fungus diminish its sensitivity At 3 ppm the response is quicker than at 14 2 ppm in the first case at 24 hours the fungus started slowly to grow and in presence of 14 2 ppm of the fungicide at 48 hours 3 3 4 Pathogenicity tests Only a few isolates produced cankers on the stems of potato sprouts planted on quartz sand The infection of different organs was independent so amount and size of lesions on stolons were not related to amount or size of lesions on roots Figure 3 5 Annex 3 D and Annex 3 1 There were statistically significant differences in the size of lesions caused by isolates collected on different municipalities Annex 3 E There were significant differences in the number and in size of lesions in stolons and roots among isolates into municipalities Proportion of colony size relative to the control La Union Subachoque Ventaquemada Municipalities Figure 3 4 Proportion of growth of the isolates in presence of 14 2 ppm of fungicide relative to the growth without the fungicide 78 2 70 4 09 S02 257 287 e ea 2 53 3 234 d 2 2 216 231 17 1 07 aa 0 57 04 Carcasi Chitaga Cogua LaUnion Pasto Sibate Silvia Soraca Subachoque Ventaquemada E 3 z E ri 3 9 pa o 9 0 wAverageSizelesionsonstems mAveragesizelesionsonroots Average size lesions on stolons Figure 3 5
92. d identification of Rhizoctonia solani AG 3 in potato and soil Plant Pathology 51 293 302 Lehtonen M J Ahvenniemi P Wilson P S German Kinnari M Valkonen J P T 2008a Biological diversity of Rhizoctonia solani AG 3 in a northern potato cultivation environment in Finland Plant Pathology 57 141 151 Lehtonen M J Somervuo P Valkonen J P T 2008b Infection with Rhizoctonia solani Induces Defense Genes and Systemic Resistance in Potato Sprouts Grown Without Light Phytopathology 98 1190 1198 MacNish G C Carling D E Brainard K A 1997 Relationship of microscopic vegetative reactions in Rhizoctonia solani and the occurrence of vegetatively compatible populations VCP in AG 8 Mycological Research 100 61 68 Martin S B 1988 Identification isolation frequency and pathogenicity of anastomosis groups of binucleate Rhizoctonia spp from strawberry roots Phytopathology 78 379 384 McCabe P A Gallagher M P Deacon J W 1999 Microscopic observation of perfect hyphal fusion in Rhizoctonia solani Mycological Research 103 487 490 Murashige T Skoog F 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiologia Plantarum 15 473 497 Naito S 2006 Ecological studies on teleomorphic and anamorphic stages in Rhizoctonia fungi Journal of General Plant Pathology 72 400 403 Ogoshi A 1987 Ecology and Pathogenicity of Anastomosis and Intraspecific Groups o
93. d of this pathogen and actually it is present in all the production areas of potato This pathogen is recognized only by few farmers in Colombia basically because there is no information about its importance distribution and biology This research was proposed with the aim to go in deep in the knowledge of this pathogen and the disease in potato crops in Colombia The main goals were i to identify the Anastomosis Groups associated with symptoms in the main producing potato areas in Colombia ii to characterize the genetic population structure of this pathogen and iii to study the response of this fungus to stress factors Each main objective is organized as a chapter of this document The information generated can be used for basic and applied purposes for example to know the evolutionary and adaptive history of this fungus as well as to propose management strategies of the disease To obtain the isolates commercial potato fields were visited on seven of the main potato producing states in Colombia Plants were reviewed for aboveground symptoms and for the presence of cankers at the base of the stems The farmers were inquired about their knowledge of the disease and symptoms in their fields Symptomatic plants were transported to the laboratory to do the isolation of the fungus The Anastomosis Groups AG were identified by PCR using specific primers for AG 3 sequencing the ribosomal DNA using universal primers for fungi and by somatic compati
94. diversity Taylor et al 1999 McDonald and Linde 2002 McDonald 2004 Pathogenic fungi with mixed reproduction systems take advantage of both conditions Meiotic recombination puts together new combinations of alleles that are tested in the local environment then both asexual reproduction and selection operate to keep together combinations of alleles beneficial to the organism in the local environment in this way a large population of well adapted individuals is created As consequence populations with mixed reproduction are predicted to be more successful in nature McDonald and Linde 2002 McDonald 2004 The contribution of sexual and asexual reproduction determines how fast new combinations of genes are generated If those combinations involve virulence or fungicide resistance genes the pathogen will be successful in breaking resistance and overcoming the effect of fungicides McDonald and McDermott 1993 McDonald and Linde 2002 39 2 1 1 5 Selection Selection is a directional process that leads to changes in allele frequencies or genotypes McDonald and Linde 2002 de Meeus et al 2007 There are two kind of selection 1 Directional selection is the selection for or against a specific gene or gene combination which decrease the genetic variation in populations and 2 Disruptive or balancing selection that is the selection for or against several genes or gene combination this increases genetic variation in populations
95. e B 2 16 A 2 74 BA 54 58 BA 1 31 Silvia B 2 30 ABCD 1 34 BC 41 49 A 1 73 Pasto B 2 53 BCD 0 72 BA 51 49 BA 1 11 Ventaquemada AB 3 02 ABCD 1 33 A 58 77 BA 1 21 Soraca B 2 25 AB 2 34 ABC 46 03 BA 1 14 La Union B 2 87 CD 0 57 BC 41 61 BA 1 51 Carcasi B 2 87 A 3 02 ABC 44 40 B 0 81 Chitaga B 2 71 A 2 56 ABC 43 62 BA 1 09 Control C 0 D 0 D 0 C 0 Values with the same letter show no differences among average values Test of Duncan of pairwise comparisons among means a 0 05 91 Annex 3 F Symptoms induced by Rhizoctonia solani AG 3 in plants of the cv Diacol capiro 45 4 08 3 5 58 77 51 49 43 62 27 61 2 5 1 51 1 26 1 09 0 5 3 0 25 2 2 05 Carcasi Chitaga m Average severity of black scurf 96 m Average Size of lesions in stolons mm 1 21 Cogua La Union Pasto Sibate Silvia Average Size of lesions in roots mm 1 Soraca Subachoque Ventaquemada mAverage incidence of black scurf 00 00 Control 70 60 50 40 30 20 10 92 Annex 3 G Principal component analysis for the test in vitro SOLERA COLOR TION AREA_AT_48_HOURS_Cm JOC3FE P Z2I2P 2EP 2PS 1902388 243 A 1BF2P ISAZGIGO3RGA TPTGB 10505 Z2DRGEBRGAGRSE N3F3R3BSR3A4R3CHR3EZPISE2RSURP I2ETOYA 1SEIGIGAIR3E2R3A RZBZRZA RZDIBZDIRZE P IDEXPIDE3PZ 1 186R 1BSP 1 GMGRSD2R D 1R2A4PTIF PTDFSROC IBRI
96. e evolutionary processes leading to the adaptation by selection affecting the frequencies of races or fungicide resistant phenotypes of pathogens Milgroom and Peever 2003 Actually biologists are aware on adaptation of different phenotypic and physiological traits particularly those that represent an advantage for the parasites populations This forces us to think in selection and adaptation of plant pathogenic fungi as a highlight in research especially under climate change perspective In Colombia the potato production is distributed along the high lands in the Andean mountains from 2400 to 3200 masl representing variable environments including climate agricultural practices and soil properties that can influence to the populations to react differentially to stress factors For that reason the evaluation of the responses of isolates of R solani AG 3 to two factors causing stress temperature and one fungicide and the study of differences in aggressiveness among isolates collected on different geographical regions was proposed in this part of the research 3 2 Materials and methods 3 2 1 Fungal populations for study Individuals from 10 geographical populations of R solani AG 3 were chosen from the collection of the Potato Pathology Group of the Agronomy Faculty at National University in Bogota The isolates tested included individuals of the main producing areas of potato in Colombia The number of isolates tested in the different experiment
97. e low pairwise population subdivision of H solani AG 3 in Colombia indicates that migration prevents the differentiation of the geographical populations this is due to the movement of infected tuber seed among counties Populations of Pasto and Cauca had a moderate differentiation with the last populations this is probably due to a low interchange of tuber seed among them and a higher inoculum movement from Ecuador Those results support observations about the efficient dispersion of this pathogen in contaminated tuber seed among geographical distant populations Long distance dispersal of sclerotia and mycelia is suggested by the presence of the same multilocus genotypes in different counties According to this 58 MLMGs were shared among populations and eight MLMGs were found more than once in geographically distant populations MLMGs one three and 13 were found in geographically separated populations however the common situation was to find at least one MLMG shared between fields in the same municipality or state This show a higher interchange of tuber seeds among fields into the same state and more restricted among states In Colombia there are 30 registered varieties however five are mainly cultivated Diacol capiro Parda pastusa Pastusa suprema Sabanera and Criolla Espinal et al 2006 this brings to the fungus a uniform agro ecosystem and could be a cause for the 58 differentiation among populations However low pairwise popul
98. e size and number of infected tubers can occur and in severe infections tubers can be malformed and cracked Hide et al 1973 Frank and Leach 1980 Banville 1989 Carling et al 1989 Jeger et al 1996a Another symptom of the Rhizoctonia disease complex is a superficial white collar on the stem base this symptom is caused by the teleomorph 7 cucumeris Banville and Carling 2001 Despite its frequency in the field the role of the sexual form of the fungus in the disease epidemiology is unclear Jeger et al 1996b 1 1 4 4 Epidemiology H solani spreads to new growing areas on tuber seed infested by sclerotia and contaminated plant debris in soil Keijer 1996 Tsror and Peretz Alon 2005 Tuber borne inoculum is the main source of primary infection leading to stem canker symptoms on the underground plant parts Soil borne inoculum of R solani include mycelia and sclerotia already inhabiting soil where the potato is planted Balali et al 1995 Soil borne infection emerges later in the season since the fungus needs some time to grow into proximity with its potato host Carling and Leiner 1986 18 Alternative hosts can support the long survival of R solani AG 3 in potato producing areas R solani AG 3 has been isolated from the underground organs of crop and weed plants Jager et al 1982 Windels and Nabben 1989 In pathogenicity trials buckwheat carrot cauliflower lucerne oats radish clover tobacco tomato whea
99. efore planting reduces the rate of growth of the fungus Banville et a 1996 Additionally selection of soils with low organic matter low porosity and moderate density can contribute to diminish the colonization of the substrate and delay the contact of the fungus with the host Successive cropping of potato in the same field will increase the inoculum potential in the soil and long term rotations diminish the viability of that inoculum The continuous cropping of potato may enhance the incidence and severity of stem canker due to the increase in soil borne inoculum density Honeycutt et al 1996 Rotations of three years or longer are suggested to reduce damage caused by R solani Banville et al 1996 However farmers must be cautious about the plant species used for rotation avoiding those that can host AG 3 Some studies have shown that diseases can increase in potato fields with certain crop rotations Rhizoctonia disease incidence and severity is reduced in most rotations 100 compared with the continuous cropping of potato Canola barley or sweet corn prior to potato had the lowest levels of Rhizoctonia disease and the best tuber quality in the opposite soybean green bean broccoli and clover increased the diseases in potato Larkin and Honeycutt 2006 Tsror 2010 Our results showed that not only potato but carrot tomato lulo pea bean and corn were infected by isolates of R solani AG 3 therefore those species are
100. eirbi 1 2P t Eya 12C 4603P 1248 2LP Dd K APT Me 1 LAP 16S 22H3P 1463920 1P1 ERES BOP 1 GB 4P211F L SE P 14FORAFSP 1 PDA dp ode 168 1082 22B2Sf6E1HD 1 16C2HF3P 1 HA Bg TLF3P 18A3P 1 1103081 BALA LISD 1756 4A9B 122625P Z2c3pDas 12F A LIA 15BB 1 GF 1 11P SEP Em za JPFSHBSP Z2DP 14E3PE3BSI2B2P MASP i JN a E3p Picks 1305P ISETP2C 4P AE TB ARA EF TP 2 BOHGBZBC SI3BSEID QOASPISD2P 4A15P 1SD2AQCSPHESP 1 1ACKABGP P cu 13D5C25 1904P a te ge utes hie eal npud 22D PI9C 23 LE3P 2 YD 3PTARERZSD 1 epet 1245RE4P X Z2BZ3BDITRAP LAI HEZP3B 11P pep SIL 12E4P 1904P 1502P 1 rei 1 6F2BD 1 19D2A2CS5P 2 are dd JE Pe 2P 0C 1 B322625P D7 I3E2HC OF SD TP3B 1TB 1P11D32A5PI4E3PHD 1ABDACONTROL i 18D5fBB iNA3PIBE2A2F 20ER 10E3P 202 3830 EQ EZ PISR2P ZOC3RGAUM DSB 1 BB3BASO 3AGEPPZZPZPLP 24 E TBSP TIC TE 1RASBE 1EXI ES Vis 4 E2RSDZfoC 10A5D3P 14FORA 123 m 9 D EBF2PSC2PI2C LPLD 122H3P 12010168P WITH IP LAGPLATSEZP 10F5MD TTGA4PIGB 10P2 16D7f0C 3B 1BASP GASECZPBSPIDC3IZAGK3G 1f5E1P5D7P 18DST3C 3f8B5 I2 F2HG2HGZf5M2IPA 1 PSC 106 1FCONTROL Z2A3IGA3RGCZf5D 1P 2168 T RZD 7 P5E7 1320 NTL CONTRO LIGA 2 EB3IGB LP ISD 3059S 1E3P CO MA 1902C ONTROL 4 3 2 1 o 1 2 3 4 Dimers lor 2 ocultar 95 Chapter 4 Final Considerations R solani AG 3 is a soil borne fungus widely distributed everywhere potato is cultivated This fungus causes the diseases stem canker and black scurf on tubers Hide et al 1985 Damage on sprouts diminis
101. emada2 11 11 Subachoque2 10 10 and Silvia 9 9 and the lowest number of loci in HWE were present in populations Cogua1 Cogua2 Sibate2 and Soraca2 with four loci out of equilibrium each one 53 Table 2 5 Measures of differentiation among populations of Rhizoctonia solani AG 3 in Colombia Contrasts between counties Fist p value Sibate versus Subachoque 0 0375 0 016 Sibate versus Ventaquemada 0 0438 0 001 Sibate versus Antioquia 0 0286 0 021 Pasto versus Subachoque 0 0712 0 001 Pasto versus Cogua 0 0390 0 000 Pasto versus Ventaquemada 0 0718 0 000 Pasto versus Soraca 0 0496 0 003 Pasto versus Antioquia 0 0330 0 010 Pasto versus Carcasi 0 0901 0 000 Pasto versus Chitaga 0 0436 0 044 Silvia versus Subachoque 0 1068 0 014 Silvia versus Ventaquemada 0 1216 0 001 Silvia versus Antioquia 0 0770 0 015 Silvia versus Chitaga 0 1377 0 000 Contrast between states Cundinamarca versus Narino 0 024 0 033 Only pairs of populations with significant differences are presented Distances were computed as the sum of squared size differences between two haplotypes Slatkin 1995 based on 10100 permutations Loci TC AG3 9 was out of HWE in nine populations and TC AGS3 6 in seven of the 18 populations and in most of the cases it was due to heterocigote excess Heterocigote deficit was found in populations Sibate2 four loci Pasto1 and Soraca2 with three loci with heterocigote deficit each one Cogua2 Pasto2 Ventaquemada1 LaUn
102. enase enzyme within the tricarboxylic acid cycle DowAgrosciences and although is supposed to have a long residual control of Rhizoctonia diseases from these results is clear that the fungus recovers its ability to grow actively after 48 hours of being exposed to the fungicide in vitro showing actual ability of the fungus to be adapted to this fungicide or fungistatic effect 81 It has been postulated that there are differences in virulence among isolates AG 3 collected from soil tubers and potato stems Carling and Leiner 1990 However in this research there was no association the size of the lesions on the plants was independent on the origin of the isolate stem canker or sclerotia on tuber seed This shows that the virulence of isolates of R solani AG 3 does not depend on the origin of the isolate stems tubers or soil but on the isolate in self Carling and Leiner 1990 Under controlled environments the growth and expansion of the colonies of H solani is controlled by the physical chemical and biological properties of the substrate Ritz 2007 In this experiment we have more infection in stolons and roots than in stems as usually is expected There are two hypothesis associated to this response the first associated to the plant the sprouts used were planted superficially in this condition the sprouts were green all the time Previously was postulated that the resistance of the stems to the pathogen is associated to the presen
103. eneeeooesneeees 18 1 2 1 Population sampling and establishment of a fungal isolate collection 18 1 2 2 Anastomosis group identification eeeeeeeeeeeeeerene nennen 19 1 2 3 Microscopic and macroscopic hyphal interactions 20 1 2 4 PathogenicHy tests 5 uses amori Bes reca tapa oon tn Eo Cx Ia eskain annann aisanana ineen vada 21 pA NIIT re 22 1 3 1 Anastomosis group identification by PCR and sequencing of the ribosomal A A A uatteatate 23 1 3 2 Hyphal interactions EE 24 1 3 3 Pathogenicity tests iia sasha aa Sa aaa 25 1 4 DISCUSSION A o 26 1 5 GORCIUSIOfIS ocn eee aiii 28 References iia ri 29 Chapter 2 Genetic diversity and population structure of Rhizoctonia solani AG 3 in je npI Lo 36 2 1 The state of th GP sitet rias Adan 36 2 1 1 Sources of genetic variability in fungi oooococonnncccnnnnncccnnnnnacccnnnnnnncnnnnnnnerennnnenns 36 Paa AA TT 36 PE A NE 37 2 1 13 Gene a o ead et as ert ocean AE 37 2 1 1 4 Reproduction and matting system eese 38 21 10 Selecto casino m 39 2 1 2 Dynamics of diversity uidit iHa dE adi 39 2 1 3 Population genetics on plant pathogenic fungi essss 40 2 1 4 Genetic structure of populations eneeeeeeeeeeeeeeeeeneeeeen nennen nnns 40 2 1 5 Molecular markers and the study of population genetics
104. epartments were sampled 1 Nari o N 45 2 Cauca N79 3 Antioquia N 63 4 Cundinamarca N 171 5 Boyac N 110 6 Santander N 21 and 7 Norte de Santander N 14 N number of isolates obtained 1 2 2 Anastomosis group identification Mycelium for genomic DNA extraction from isolates collected was obtained from 5 day old cultures Each isolate was grown on plates containing PDA covered by a sterile cellophane sheet After incubation at room temperature mycelium from each isolate was harvested by scraping the mycelia from the cellophane membrane followed by freezing and lyophilization DNA was extracted with the QIAGEN DNeasy Plant Mini Kit The anastomosis group was determined by selective amplification of the ribosomal DNA rDNA region of the fungus using specific primers for R solani AG 3 Lees et al 2002 Polymerase chain reactions PCR were carried out in 10 ul volumes containing 10 50 ng of genomic DNA 10 mm of KCI 10 mm of NH4 SO 20mM of Tris HCI 2mM of MgSO 0 0196 of Triton X 100 pH 8 8 NEB 0 2 uM of each primer Microsynth 0 1 mM of each dNTP and 0 06 U of Taq polymerase NEB PCR conditions comprised initial denaturation of 20 2 min at 96 C followed by 35 cycles of denaturation for 30 s at 96 C annealing for 45 s at 57 C and elongation for 45 s at 72 C with a final extension step of 5 min at 72 C PCR products were visualized with UV on agarose gels In order to determine the AG from
105. es on teleomorphic and anamorphic stages in Rhizoctonia fungi Journal of General Plant Pathology72 400 403 Pringle A Taylor J W 2002 The fitness of filamentous fungi Trends in Microbiology 10 474 481 Pritchard J K Stephens M Donnelly P 2000 Inference of population structure using multilocus genotype data Genetics 155 945 959 Schlotterer C 2000 Evolutionary dynamics of microsatellite DNA Chromosoma 109 365 371 Simons S A Gilligan C A 1997 Relationships between stem canker stolon canker black scurf Rhizoctonia solani and yield of potato Solanum tuberosum under different agronomic conditions Plant Pathology 46 651 658 12 Stukenbrock E McDonald B A 2008 The Origins of Plant Pathogens in Agro Ecosystems Annual Review of Phytopathology 46 75 100 Tsror L 2010 Biology Epidemiology and Management of Rhizoctonia solani on Potato Journal of Phytopathology 158 649 658 Willi Y Frank A Heinzelmann R Kalin A Spalinger L Ceresini P 2011 The adaptive potential of a plant pathogenic fungus Rhizoctonia solani AG 3 under heat and fungicide stress Genetica 139 903 908 13 Chapter 1 Characterization of Anastomosis Groups associated to stem canker in potato crops in Colombia 1 1 The state of the art 1 1 1 Rhizoctonia solani Kuhn Rhizoctonia solani K hn anamorph teleomorph Thanatephorus cucumeris Frank Donk is the most studied species within gen
106. f 73 temperature For each plate the time to the formation of sclerotia was recorded The plates were scanned hp Scanjet 3570c and the colony sizes after 48 hours of growing were determined with the image analysis software ASSESS Lamari 2002 The data obtained with ASSES were transformed from pixels to cm An additional trial testing the in vitro response of 22 isolates to the fungicide thifluzamide at 3 and 14 2 ppm along the time was prepared The isolates were grown in presence of the fungicide for 96 hours and the growth of the colony was determined each 24 hours After 96 hours a plug of the micelial growth was transferred to a new plate with PDA 3 2 4 Pathogenicity tests Tubers of the cultivar Diacol capiro were used for the pathogenicity tests Each tuber was disinfested superficially first the sclerotia were removed manually and then the tubers were immersed in 1 of sodium hypochlorite for one minute and washed twice with distilled water Tubers were stored in paper bags in the dark to promote the development of sprouts 3 2 4 1 Pathogenicity on stems After a month of storage in the paper bags uniform sprouts 1 1 5 cm were took and transferred to pots with sterile quartz sand The sand was autoclaved twice at 15 PSI for 30 minutes in two consecutive days Pots were kept in a growing room for two weeks and then plants were inoculated For the inoculation fungal isolates were taken from long term storage plated
107. f Rhizoctonia solani Kuhn Annual Review of Phytopathology 25 125 143 Ogoshi A 1996 The genus Hhizoctonia In Sneh B Jabaji Hare S Neate S Dijst G Eds Rhizoctonia Species Taxonomy Molecular Biology Ecology Pathology and Disease Control Kluwer Academic Publishers Dordrecht The Netherlands pp 1 9 Olaya G Abawi G S 1994 Characteristics of Rhizoctonia solani and binucleate Rhizoctonia species causing foliar blight and root rot on table beets in New York state Pant Disease 78 800 804 Pal K van Diepeningen A D Varga J Hoekstra R F Dyer P S Debets A J M 2007 Sexual and vegetative compatibility genes in the aspergilli Studies in Mycology 59 19 30 Poromarto S H Nelson B D Freeman T P 1998 Association of binucleate Rhizoctonia with soybean and mechanism of biocontrol of Rhizoctonia solani Phytopathology 88 1056 1067 34 Priyatmojo A Yamauchi R Kageyama K Hyakumachi M 2002a Whole cell fatty acid composition to characterize and differentiate isolates of Rhizoctonia species associated with turfgrass diseases Jpn Journal of General Plant Pathology68 1 7 Priyatmojo A Yamauchi R Naito S Kageyama K Hyakumachi M 2002b Comparison of Whole Cell Fatty Acid Compositions of Isolates of Rhizoctonia solani AG 2 from Tobacco and Tulip AG 2 1 and AG BI Journal of Phytopathology 150 283 288 Reynolds M Weinhold A R Morris T J 1983 Comparison
108. f seed potatoes in field experiments Netherlands Journal of Plant Pathology 92 287 303 Escande A R Echandi E 1991 Protection of potato from Rhizoctonia canker with binucleate Rhizoctonia fungi Plant Pathology 40 197 202 Fox R 2006 Rhizoctonia stem and stolon canker of potato Mycologist 20 116 117 Frank J A Leach S S 1980 Comparison of tuber borne and soilborne inoculum in the Rhizoctonia disease of potato Phytopathology 70 51 53 Gonzalez D Cubeta M A Vilgalys R 2006 Phylogenetic utility of indels within ribosomal DNA and beta tubulin sequences from fungi in the Rhizoctonia solani species complex Molecular Phylogenetics and Evolution 40 459 470 Hall T A 1999 BioEdit a user friendly biological sequence alignment editor and analysis program for Windows 95 98 NT Nucleic Acids Symposium Series 41 95 98 Hamshou M Smagghe G VAn Damme E J M 2007 Analysisi of lectin Concentrations in different Rhizoctonia solani strains Communications in Agricultural and Applied Biological Sciences 72 639 644 Hartill W F T 1989 Some effects of Rhizoctonia solani on growth and yield potatoes Potato Research 32 283 292 Hide G A Hirst J M Stedman O J 1973 Effects of black scurf Rhizoctonia solani on potatoes Annals of Applied Biology 74 139 148 32 Hide G A Horrocks J K 1994 Influence of stem canker Rhizoctonia solani Kuhn on tuber yield tuber size reducing sugars
109. f the stem being larger in young plants Diseased plants are randomly distributed in the fields and aerial symptoms were not strong plants showed less height but purpling of the leaves was not commonly observed It was possible to find in a row one diseased plant followed by five or more asymptomatic ones Commonly plants with all the stems affected and the formation of roots above the point of infection were found Figure 1 2 The sexual state was observed on plants older than two months Figure 1 2 Symptoms of stem canker on potato plants Left formation of roots above the canker Right Plant with diseased and healthy stems The isolates were identified as Rhizoctonia based on morphological characteristics In young hyphae between eight to twelve nuclei were observed The young mycelium was white older isolates were brown Microscopically the ramification of mycelia was in right angle with a constriction in the base of hyphae Figure 1 3 23 Figure 1 3 Microscopic characteristics of Rhizoctonia solani isolates collected in this research Left multinucleated cells 1000X safranine O stain Right brown mycelia ramification of mycelia in right angle constriction in the hyphal base white arrows and septum formation near to the ramification Black arrows 400X lactophenol blue stain 1 3 1 Anastomosis group identification by PCR and sequencing of the ribosomal DNA A total of 433 isolates of R solani collected fr
110. for the subsequent analysis Each individual from each of the 18 populations of R solani AG 3 was genotyped using a specific set of polymorphic Simple Sequence Repeat SSR loci using fluorescent labeled forward primers Ferrucho et al 2009 Isolates from the states of Cundinamarca Boyac Antioquia and Nari o were processed in the plant pathology department of the Institute of Integrative Biology ETH Zurich Polymerase chain reaction PCR amplifications were performed in 96 well plates in a total volume of 15 ul containing 10 50 ng of total DNA 1 5 pl of 10X reaction buffer 10 mM KCI 10 mM NH4 2SO4 20 mM Tris HCl 2 mM MgSO 0 01 Triton X 100 pH 8 8 NEB 0 1 0 2 um of each labeled and non labeled primer NEB and Microsynth 0 1 mM of each dNTP and 0 06 uL of Taq DNA polymerase 5 U uL NEB Isolates collected on the states of Boyaca Santander Norte de Santander and Cauca were processed in the Institute of Genetics of the National University of Colombia following the conditions described previously but using Invitrogen supplies PCR conditions for all loci were initial denaturation at 96 C for 4 min 35 cycles of denaturation for 30 s at 96 C annealing for 30 s at specific temperature for each loci Ferrucho et al 2009 and elongation for 45 s at 72 C and a final extension cycle of 30 min Multiplex PCR reactions were conducted for 9 loci in three groups Group A TC_AG3_1 TC_AG3_7 and TC_AG3_ 14 Group B TC_AG3_6 T
111. g the contact of host and pathogen are two i inoculum density and ii growth rate which are critical in the disease dynamic Otten et al 2003 Otten et al 2005 Cunniffe and Gilligan 2009 Relationship among rate of radial growth of colonies of R solani and the biomass accumulation depend on the physical biological and chemical properties of the soil which are determinant for the dynamic of epidemics they modify the colonization efficiency as well as the growth of fungi changing their ability for the invasion Transmission of R solani is determined by its ability for growing on or through soil Exploration of mycelia from the inoculum source to the host occurs in a heterogeneous environment through a soil pore networks cracks aggregate surfaces and water films those factors increase the tortuosity and reduce the connectivity of the soil pores Gilligan and Bailey 1997 Otten et al 1999 Gill et al 2000 Otten et al 2001 71 The distribution of nutrient into the soil has strong effect upon the spatial organization of fungal mycelia Most resources are heterogeneously distributed in the soil and the invasion and persistence of parasites and saprophytes depends on its ability to colonize the substrate that is determined by the endogenous availability of nutrients and the ability of translocation into the colony the growth and the distance to susceptible hosts Ritz 2007 For a long time researchers were interested in th
112. gher than 5096 confirming the classification of isolates based on their ITS rDNA sequences Somatically incompatible isolates were no detected in the microscopic test However in the macroscopic somatic test the formation of tufts was common this is evidence of somatic incompatibility among AG 3 isolates The macroscopic interactions did not reproduce the categories of microscopic interactions observed In fact there was a continuing of somatic compatibility to incompatibility not detected in the microscopic test Figure 1 4 25 21D4p Vs 21A7p 18B4p Vs 15A1p 21A31p Vs 13A6p2 12D2p Vs 9C2p2 Figure 1 4 Macroscopic anastomosis reaction between Rhizoctonia solani AG 3 isolates a Strong tuft reaction between isolates collected in the same field b Strong tuft reaction between isolates collected in different field c Mild tuft reaction between isolates collected in different field and d merge reaction between isolates collected from different field Numbers underneath each figure are codes for isolate pairs 1 3 3 Pathogenicity tests Symptoms observed in the different hosts correspond to the typical symptoms of canker reported previously although severity varied among AGs Table 1 2 The isolation from asymptomatic tissue was successful showing that the absence of infection was not due to no viability on inoculum Table 1 2 Symptoms on different plant species caused by Rhizoctonia solani AG 2 1 an
113. h nuclear diversity low mitochondrial diversity regular recombination and gene flow Fungal Genetics and Biology 38 286 297 66 Annex 2 A Information content of eleven microsatellite loci used for multilocus genotyping of individuals of Rhizoctonia solani AG 3 from Colombian potato crops Microsatellite ace TC_AG3_0 TC_AG3_1 TC_AG3 6 TC AG3 7 TC AG3 8 TC_AG3 9 TC AG3 10 TC AG3 14 TC AG3 16 TC AG3 18 TC_AG3_19 Mour a om soda om om oei S nucleotide ada Snucleotide 3 nucleotide 3 nucleotide Total a size range 447 156 141 201 192 246 207 234 214 244 220 259 244 250 284 320 315 363 318 321 320 359 ae Repeat numbers 1 4 1 21 3 21 1 10 1 11 1 14 7 9 2 14 12 28 4 17 2 15 Subach1 N 28 2 6 4 6 2 6 2 3 5 5 6 47 Subach2 N 18 2 5 5 4 1 5 2 3 5 4 5 41 Cogua1 23 2 8 7 5 3 6 4 4 7 6 7 59 Cogua2 N 33 2 6 4 7 3 5 2 4 5 5 8 51 Sibate1 N 21 2 7 6 4 1 6 2 4 4 3 5 44 Sibate2 N 12 2 7 8 4 3 6 2 6 6 4 5 53 Pasto1 N 18 2 9 7 3 1 5 2 4 8 4 5 50 Pasto2 N 22 2 8 6 4 4 6 2 4 6 5 6 53 LaUnion1 N 17 2 7 4 6 2 5 3 3 6 4 5 47 LaUnion2 N 35 2 8 6 5 2 7 2 4 8 4 7 55 Soraca1 N 22 2 5 7 6 2 7 2 4 5 4 6 50 Soraca2 N 23 2 5 6 6 3 7 2 5 6 5 6 53 Ventaq1 N 20 2 5 8 5 3 5 2 6 6 4 6 52 Ventaq2 N 21 2 4 4 5 2 4 2 3 5 4 6 41 Carcasi1 N 10 2 5 7 4 2 6 3 5 5 4 6 49 Carcasi2 N 11 2 5 4 4 2 6 3 4 5 4 6 45 Chitaga N 14 2 6 4 3 3 5 3 5 3 3 6 43
114. hall et al 2007 India AG 3 and AG 4 Stems roots and soil Suresh and Mall 1982 Mexico AG 3 and AG 4 Stems stolons tubers Virgen Calleros et al 2000 Pakistan AG 3 AG 4 AG 5 Tubers Rauf et al 2007 Peru AG 3 AG 4 and non identified isolates Stems and tubers Anguiz and Martin 1989 South Africa AG 3 AG 5 AG 7 y AG 8 Stems stolons roots and soil Truter and Wehner 2004 USA AG 3 AG 5 and non identified isolates Stems Bandy et al 1988 USA and Alaska AG2 1 AG 3 and non identified isolates Stems Carling and Leiner 1986 Venezuela AG2 1 y AG 3 Stems stolons roots tubers Cede o et al 2001 The AG 2 1 was restricted to localities where weeds of Brassicaceae family are common Therefore the reduction of those weeds on potato fields may contribute to the reduction of inoculum and the consequent diminution the symptoms of stem canker and black scurf caused for this AG on stems of potato plants In Colombia potato is grown as the main crop in most geographical areas The most common rotation is with grasses which have not been reported as hosts of AG 3 and in the pathogenicity tests done in this study they were not affected by AG 3 or AG 2 1 In a previous work the use of ryegrass as crop rotation diminished the severity of stem canker on potato Talbot 2003 This makes to the grasses as the best crop rotation for control of the two AGs prevalent in Colombia especially co
115. he infection on stolons has great influence on the number and size of tubers Additionally severe infections cause deformation and cracking Sclerotia affect tuber quality reducing their marketability and are fundamental for the survival of the pathogen on the fields and they are the main source of inoculum for long distance dispersal Banville et a 1996 Jeger et al 1996 Tsror 2010 The life cycle of this fungus comprises two phases an asexual and a sexual The asexual phase is the most commonly observed in the fields Infection starts with hyphae growing on the plant surface from mycelia or sclerotia Then the fungus forms appressoria to penetrate plant cells Initial infection is followed by the release of enzymes that degrade cell walls kill the cells and promote the spread of hyphae in the dead cells Banville et al 1996 Tsror 2010 The sexual phase is characterized by formation of hymenia on the stem base Environmental factors such as air and soil temperature relative humidity wind velocity and concentration of O and CO can determine the occurrence of T cucumeris The Basidiospore dispersal occurs almost exclusively during the night but little is known about the relative importance of basidiospores as primary and secondary inoculum in the epidemiology of these diseases Naito 2006 Previous studies have employed various methods to examine the variation among isolates of R solani AG 3 Results of those studies have shown
116. hes the number of areal stems and in consequence the foliar area in the plants Cankers on stems alter the uptake and translocation of water and minerals to the leaves and the movement of photoassimilates to the tubers The losses caused by this pathogen are no well defined however it has been demonstrated that affects the size distribution of tubers Simons and Gilligan 1997b In Colombia the disease is not considered important in fact when we visited potato fields for sampling most farmers were not aware about the symptoms on stems With the exception of farmers that trade with specialized markets black scurf has no impact in the price of tubers for the internal market only for seed tuber and the production for industry In this research we generated important knowledge about the biology of the H solani AG 3 populations in Colombia With this work we demonstrate the importance of basic studies on the biology of this pathogen to understand the development of the disease and some epidemiological aspects In the next sections will present the summary of the research calling the attention to relevant aspects that should be considered for the control of the disease The ample sampling allowed to cover the main and representative localities along the country and gave us the possibility of drawing powerful conclusions about the populations of H solani AG 3 in Colombia as the first step to propose and reconsider the management measures of the disea
117. hyphae growing on surface of the plant later appresoria are formed previous to the penetration of the plant cells Initial infection is followed by the release of enzymes that degrade cell walls kill the cells and promote the spread of hyphae in dead cells Lehtonen 80 et al 2008b Mycelial growth is therefore an important fitness component for this species because the fungus grows and damages host plants with asexual hyphae The isolates showed high variation in the response to the two temperatures and to the fungicide evaluated The smaller average area of the colony was found with Thifluzamide at 14 2 ppm 0 53 cm then the growth at 15 C 2 23 cm At 25 C the largest size of the colonies was observed 20 16 cm These results are in agreement with previous reports about the ability of R solani to overcome the stress caused by fungicides and temperatures 24 and 27 C Willi et al 2011 The isolates tested in this research showed high variation in the response to the two temperatures and to the fungicide evaluated however the average among populations was similar This is evidence that although there is variation in the response among isolates into the fields the variation among them does not change This shows the ability of this pathogen to be adapted under different climate regimens as well as to different crop management in different geographical regions The optimal temperature for the growth in vitro of R so
118. ia esee Figure 1 2 Symptoms of stem canker on potato plants ssssssssee Figure 1 3 Microscopic characteristics of Rhizoctonia solani isolates collected in this research Figure 1 4 Macroscopic anastomosis reaction between Rhizoctonia solani AG 3 isolates Figure 2 1 Number of simulated populations Figure 2 2 STRUCTURE inferred membership coefficient for MLMGs Hhizoctonia solani AG 3 in Colombia esu iet ti Dust tne lote t enu Figure 3 1 Average of colony size Cm 48 hours after inoculation of petri dishes in isolates of R solani AG 3 collected on different municipalities and grown Figure 3 2 Proportion of growth of the isolates relative to the optimal temperature under in vitro A Figure 3 3 Average of colony size Cm in isolates collected on different municipalities and grown in presence of thifluzamide 14 2 ppm Figure 3 4 Proportion of growth of the isolates in presence of 14 2 ppm of fungicide relative to the growth without the fungicide Figure 3 5 Size lesion average on potato plants cv Capiro caused by isolates of H solani AG 3 collected on different municipalities in Colombia Figure 3 6 Percent of sclerotia on the tuber surface of the cv Capiro caused by isolates of H solani AG 3 collected on different municipalities Figure 3 7 Incidence of sclerotia on tubers of plants inoculated with isolates of H s
119. iados con papa en m rida Venezuela Interciencia 26 296 300 Ciampi M B Meyer M C Costa M N Jr Zala M McDonald B A Ceresini P C 2008 Genetic structure of populations of Rhizoctonia solani anastamosis group 1 IA from soybean in Brazil Phytopathology 98 932 941 FRAC 2011 FRAC Code List Fungicides sorted by mode of action including FRAC Code numbering Frank J A Leach S S 1980 Comparison of tuber borne and soilborne inoculum in the Rhizoctonia disease of potato Phytopathology 70 51 53 103 Hide G A Read P J Sandison J 1985 Stem canker Rhizoctonia solani of maincrop potatoes I Effects on growth and yield Annals of Applied Biology 106 423 437 Honeycutt C W Clapham W M Leach S S 1996 Crop rotation and N fertilization effects on growth yield and disease incidence in potato American Potato Journal 73 45 61 Jeger M J Hide G A Boogert P H J F Termorshuizen A J Baarlen P 1996 Pathology and control of soil borne fungal pathogens of potato Potato Research 39 437 469 Kassen R Rainey P B 2004 The ecology and genetics of microbial diversity Annual Review of Microbiology 58 207 231 Larkin R P Honeycutt C W 2006 Effects of different 3 year cropping systems on soil microbial communities and Rhizoctonia diseases of potato Phytopathology 96 68 79 Lehtonen M J Ahvenniemi P Wilson P S German Kinnari M Valkonen J P T 2008a
120. ide diminished the growth of the pathogen in all the doses evaluated At 100 ppm the growth was completely inhibited Annex 3 C For the response analysis a dose of 14 2 ppm was evaluated although this is a high dose this allows to detect variation in the response of the isolates and to detect differences among the individuals tested data not shown The colony sizes were independent of the volume of media in the Petri dish then for the temperature assays a fixed volume of PDA was not used 75 3 3 2 Temperature response The growth of the isolates was higher at 25 C for all the isolates tested in all the municipalities Figure 3 1 For both temperatures the growth was higher in the isolates from Chitaga and the lower in isolates from Silvia The difference among municipalities was not significant at both temperatures In each municipality there were isolates that grew well at 15 C and not at 25 C and others that had better response at 25 C E E o 8 mn gt 3 2 o o Subachoque Municipalities Ventaquemada Figure 3 1 Average of colony size Cm 48 hours after inoculation of petri dishes in isolates of H solani AG 3 collected on different municipalities and grown at 15 and 25 C The test for differences in the average growth of isolates was significant for isolates of Pasto and Silvia at 25 C at 15 C there were no differences among isolates into the municipalities The response of the isolates to low
121. indicates that potato is a suitable host for this AG and suggests that its presence in the field can enhance symptom expression of AG 3 the primary AG associated with potato The AGs 4 5 7 and 8 previously have been reported associated to stem canker on potato in different countries Anguiz and Martin 1989 Bains and Bisht 1995 Balali et al 1995 Campion et al 2003 Truter and Wehner 2004 Woodhall et a 2007 Cede o et al 2001 however in this study they were not identified The variability on the range of AGs present in potato fields in different countries could be related to the distribution of their hosts weeds and wild plants and the species used for crop rotation Binucleated isolates AG A AG E and AG I were present in stem canker lesions in potato Those Rhizoctonias have diverse ecological preferences some are non pathogenic and rather used for biological control of pathogenic Rhizoctonia because they cause a mild infection on the plants and may act as antagonists of pathogenic Rhizoctonia improving the defense responses of plants Carling and Leiner 1986 Poromarto et al 1998 Other binucleated Rhizoctonia can cause serious diseases like damping off on different species of plants Martin 1988 Escande and Echandi 1991 Demirci et al 2002 In this study pathogenicity tests with binucleated isolates indicated only mild symptoms restricted to stem surface and plants did not exhibit deep cankers These results
122. ine the somatic compatibility among isolates collected from different locations To determine the macroscopic vegetative reaction between pairs of isolates plates containing PDA were inoculated with disks of PDA colonized by each isolate and removed from the edge of an actively growing colony and located three cm apart each other As control each isolate tested was duplicated in the plate and then each plate contained four mycelia plugs from two different isolates The 21 isolates were tested in all the possible combinations Plates were incubated at 15 C and were evaluated after 21 days The macroscopic somatic reactions were defined as merge and tuft In the category merge the two cultures come together with little or no evidence of demarcation and in the category tuft there is an area of differentiation between the meeting of isolates in this area a band of hyphae raises above the level of mycelium on the agar surface in this reaction the color of the tuft mycelium is different from the color of the parent isolates MacNish et a 1997 1 2 4 Pathogenicity tests Three isolates of each anastomosis group were randomly chosen for the pathogenicity test Prior to inoculation each isolate was grown for 48 hours on PDA The following plants were tested as hosts i seedlings of carrot Daucus carota L bean Phaseolus vulgaris L corn Zea mays L tomato Solanum lycopersicum L lulo Solanum quitoense Lam pea Pisum sativum
123. ion2 and Chitaga with two loci the last populations presented one or no loci with heterocigote deficit Locus TCAG3 10 present heterocigote deficit in eight populations The test for gametic disequilibrium followed by Bonferroni correction for multiple tests was significant only for a few pairs of loci in some populations Populations with more than 1096 of locus pairs in disequilibrium were Subachoque1 16 3696 Cogua2 25 4596 and LaUnion2 14 55 Estimates of lA and 7 were significantly different from zero p value 0 001 for almost all populations however their values were low Table 2 6 Population Pasto1 presented departure from HWE proportions in 3 of 10 loci this population showed a significant value of Fis 0 2487 p value 0 0186 the same situation was present in the population Soraca2 this has 7 of 11 loci out of HWE Fis of 0 2764 p value 0 008 this is consistent with inbreeding The overall FIS value was 0 081 p value 0 0019 when populations Pasto1 and Soraca2 were removed from the analysis the overall Fis value was not significant different form zero 54 2 3 4 Admixture and hidden population structure The analysis was performed assuming both recent ancestry and permanent current gene flow admixture with correlated allele frequencies supported in the results of AMOVA and Rst The simulation of the best K using the information generated by STRUCTURE supports for the existence of two genetically distinct p
124. ippage and by proofreading errors during DNA replication Both mechanisms primarily change the number of repeats and thus the length of the repeat string Schl tterer 2000 Ellegren 2004 Karaoglu et al 2004 2 1 6 The soil borne plant pathogenic fungus Rhizoctonia solani Kuhn Rhizoctonia solani Kuhn teleomorph Thanatephorus cucumeris Frank Donk is a soil borne Basidiomycete fungus The anastomosis group three sub group PT AG 3PT is the causal organism of the Rhizoctonia disease complex in potato Wilson et al 2008 which results in two different symptoms stem canker and black scurf on the tubers Tsror 2010 On the last years the studies on genetic diversity of R solani have increased The most studied are the populations belonging to the AG 1 as pathogen of rice soybean and maize Analysis with codominant RFLP loci in a population of AG1 IA collected in Texas United States found evidence for clonal reproduction by finding the same genotype many times in the same and in separated fields however more than a half of the loci were in Hardy Weinberg Equilibrium and only a few of all possible pairs of loci were in linkage disequilibrium which indicates that recombination is also occurring Rosewich et al 1999 The same situation has been found on the subsequent studies on this AG on Brazil Ciampi et al 2008 China and India Bernardes de Asis et al 2009 using SSR markers the results suggest a reproductive mode varying
125. lani AG 3 was among 20 and 30 C In a previous research was found that the optimal temperature for mycelial growth production and germination of sclerotia is around 23 C Ritchie et al 2009 The soil temperature in the regions of potato production in Colombia is around 17 C The favorable response of the isolates to temperature above 15 C is an important concern respect to the response of the populations of this fungus in the climate warming context and is a demonstration that the fungus can be adapted rapidly to global climate change Willi et al 2011 On field conditions less incidence and severity is reported as the soil temperature increases however in the studies did not considered the change in different soil properties that can generate an unfavorable environment to the pathogen Simons and Gilligan 1997 The inhibition of the growth of R solani AG 3 below 10 C suggest that the pathogen have less colonization ability in cold environments and as consequence less disease intensity is expected However the pathogen survives and can be disseminated mainly as mycelia The quick response against the fungicide was remarkable In Colombia this is the single fungicide registered to the management of the disease however it is not widely used for instance the response of the isolates is not explained by the intensive use of the fungicide In fact there was a wide response among isolates This fungicide inhibits the succinate dehydrog
126. lation of potato with AG 13 led to formation of small stem canker lesions on shoots and roots Carling et al 2002 This indicates that in experimental situations some AGs are able to initiate limited necrosis although normally they do not infect potato Binucleated Rhizoctonia BNR isolates have been isolated from potato plants however they were mildly virulent or avirulent Carling and Leiner 1986 and have been proposed as an alternative to the disease control 1 1 4 Disease cycle 1 1 4 1 Infection of stems and stolons The infection process by R solani on stems and stolons of potato plants starts when mycelia or hyphae from a germinating sclerotia grow attracted by chemical exudates from the plants amino acids sugars organic acids and phenols Keijer 1996 Penetration may be mechanical or enzymatic Weinhold and Sinclair 1996 Mechanical penetration occurs when the fungus find a weak spot on the plant surface where it can break down the protecting layer When inside the host the fungus starts to grow inter and intracellularly degrading the tissue the result are necrotic lesions on epidermal tissue of shoots roots and stolons or damping off of the young seedlings Demirci and D ken 1998 In potato plants the disease potential varies over time as the tissues and organs mature show less susceptibility However a plant can escape to sprout pinch or stem canker and yet be severely affected by stolon infection Banville et a 1996
127. les of whole cell fatty acid composition Stevens Johnk and Jones 2001 Priyatmojo et al 2002a Priyatmojo et al 2002b and serology Adams and Butler 1979 have been used to classify groups in the Rhizoctonia complex and they support the anastomosis grouping These approaches have clarified the taxonomy genetic relationships and population structure of this complex pathogen Currently sequence analysis of ribosomal DNA rDNA is used as a simple and reliable methodology for the accurate designation of Rhizoctonia species and their subgroups Kuninaga et al 1997 Sharon et al 2006 Sharon et al 2008 13 AGs have been described they are designed as AG 1 through AG 13 Carling et al 2002 Truter and Wehner 2004 Sharon et al 2008 The first AGs described AG 1 2 3 and 4 produce the most destructive Rhizoctonia diseases around the world and those described later are considered less destructive pathogens and have more restricted geographical distribution Carling et a 2002 Binucleated Rhizoctonia BNR species are sometimes considered as an additional AG since they are able to anastomose with some of the existing AGs Kuninaga et al 1979 Sneh et al 1998 BNR species have been reported to be pathogenic Rinehart et a 2007 weakly pathogenic on different plant species Martin 1988 Olaya and Abawi 1994 or no pathogenic Individual isolates belonging to the same AG still can differ in host range virulence and in m
128. lone correction was performed to select only one individual of each MLMG to execute the subsequent tests 2 2 3 3 Gene diversity and population differentiation Allelic richness and the expected heterozygosity were calculated as indexes of gene diversity using the program FSTAT 2 9 3 2 Goudet 1995 Nei s unbiased gene diversity or expected heterozygosity was estimated as n n 1 x 1 Xip where p is the observed frequency of the ith allele and n is the sample size Nei 1978 Allelic richness was estimated as the mean number of alleles per locus El Mousadik and Petit 1996 for a standardized sample size of five individuals using rarefaction Hurlbert 1971 as described by Petit et al 1998 Finally differences in allelic richness and gene diversity among pairs of populations were tested using a bootstrapping approach based on 1 000 permutations for calculating p values 48 2 2 3 4 Population differentiation The distribution of gene diversity based on hierarchical analysis of molecular variance AMOVA was evaluated The fixation indices F statistics were calculated to quantify differentiation between pairs of populations and to assess the degree of population subdivision based on the sum of squared size differences for microsatellite loci Fisr Slatkin 1995 The null distribution of pairwise F statistics values under the hypothesis of no differentiation between two populations was obtained by permutating haplotypes
129. lvoire Phytopathology 89 414 420 Benali S Mohamed B Eddine H Neema C 2011 Advances of Molecular Markers Application in Plant Pathology Research European Journal of Scientific Research 50 110 123 Bernardes de Assis J Storari M Zala M Wang W Jiang W ShiDong L Jin M McDonald B A Ceresini P C 2009 Genetic structure of populations of the rice infecting pathogen Rhizoctonia solani AG 1 IA from China Phytopathology 99 1090 1099 Burdon J J Silk J 1997 Sources and Patterns of Diversity in Plant Pathogenic Fungi Phytopathology 87 664 669 Burnett J 2003 Fungal Populations and Species Oxford University Press USA 368 p 62 Campion C Chatot C Perraton B Andrivon D 2003 Anastomosis Groups Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France European Journal of Plant Pathology 109 983 992 Ceresini P C Shew H D Vilgalys R J Cubeta M A 2002a Genetic diversity of Rhizoctonia solani AG 3 from potato and tobacco in North Carolina Mycologia 94 437 449 Ceresini P C Shew H D Vilgalys R J Rosewich U L Cubeta M A 2002b Genetic Structure of Populations of Rhizoctonia solani AG 3 on Potato in Eastern North Carolina Mycologia 94 450 460 Ciampi M B Meyer M C Costa M N Jr Zala M McDonald B A Ceresini P C 2008 Genetic structure of populations of Rhizoctonia solani anasta
130. man welfare Science 316 1866 1869 Kassen R 2002 The experimental evolution of specialists generalists and the maintenance of diversity Journal of Evolutionary Biology 15 173 190 Kassen R Rainey P B 2004 The ecology and genetics of microbial diversity Annual Review of Microbiology 58 207 231 Kiffer E Morelet M 1999 The Deuteromycetes itosporic fungi Classification and generic keys Science Publishers INC USA 273 pp Kocher T D 2004 Adaptive evolution and explosive speciation The cichlid fish model Nature Reviews Genetics 5 288 298 Kolar C S Lodge D M 2001 Progress in invasion biology predicting invaders Trends in Ecology amp Evolution 16 199 204 Lamari L 2002 Assess Image analysis software for plant disease quantification APS Press The American Phytopathological Society St Paul Minnesota USA Leach J E Vera Cruz C Bai J Leung H 2001 Pathogen fitness penalty as a predictor of durability of disease resistance genes Annual Review of Phytopathology 39 187 224 Lees A K Cullen D W Sullivan L Nicolson M J 2002 Development of conventional and quantitative real time PCR assays for the detection and identification of Rhizoctonia solani AG 3 in potato and soil Plant Pathology 51 293 302 Leionen T O Hara R B Cano J M Merila J 2008 Comparative studies of quantitative trait and neutral marker divergence a meta analysis Journal of Evolutionar
131. mbining to a mixed system in which there are recombination events followed by clonal expansion during the growing season The evidences for those conclusions where the same genotype recovered many times in the same and in separated fields and in the opposite several loci in HWE and a few of all possible pairs of loci in linkage disequilibrium Rosewich et al 1999 Ciampi et al 2008 Bernardes de Assis et al 2009 There was no evidence for recent founder events or bottlenecks in the populations and the absence of structure does not allow inferring selection associated to geographical regions The SSR as neutral markers are not able to identify patterns of genetic variation associated to selection The variability in the response to temperatures the sensitivity to the fungicide and the aggressiveness on potato plants are important clues in the evolutionary history of this pathogen and additional studies are necessary to understand if those characteristics are adaptive evolutionary or phenotypic plasticity of the individuals into the populations of R solani AG 3 98 4 3 Variability of R solani AG 3 and the control of the diseases black scurf and stem canker The control strategies for the diseases caused by R solani AG 3 are varied but have low individual effect The genetic pathogenic and physiologic complexity of this pathogen shows that an integrate approach is necessary The knowledge here generated gives the possibility of propo
132. mosis group 1 IA from soybean in Brazil Phytopathology 98 932 941 Collard B C Y Jahufer M Z Z Brouwer J B E C K P 2005 An introduction to markers quantitative trait loci QTL mapping and marker assisted selection for crop improvement the basic concepts Euphytica 142 169 196 Cornuet J M Luikart G 1996 Description and Power Analysis of Two Tests for Detecting Recent Population Bottlenecks From Allele Frequency Data Genetics 144 2001 2014 Cubeta M A Vilgalys R 1997 Population biology of the Rhizoctonia solani complex Phytopathology 87 480 484 de Meeus T McCoy K D Prugnolle F Chevillon C Durand P Hurtrez Bouss s S Renaud F 2007 Population genetics and molecular epidemiology or how to d busquer la b te Infection Genetics and Evolution 7 308 322 El Mousadik A Petit R J 1996 High level of genetic differentiation for allelic richness among populations of the argan tree Argania spinosa L Skeels endemic to Morocco Theoretical and Applied Genetics 92 832 839 Ellegren H 2004 Microsatellites simple sequences with complex evolution Nature Reviews Genetics 5 435 445 Espinal C F Mart nez H Pinz n N Barrios C 2006 La cadena de la papa en Colombia Una mirada global de su estructura y din mica 1991 2005 In Agrocadenas Ed Evanno G Regnaut S Goudet J 2005 Detecting the number of clusters of individuals using the software STRUCTURE
133. n occurs suddenly a successful clone may increase in proportion this clone predominates for a time and finally can disappear as result of recombination This situation is detected when there is a single or a few multilocus genotypes showing wide distribution especially in epidemic areas The frequently recovering of the same genotype could be taken as evidence of clonality but when the clones are eliminated and the analysis is re run the populations behave as recombining it explains bias in the tests for recombination towards clonality and populations really have a random mating behavior Taylor et al 1999 Clonality produces a clear pattern into the populations for a series of polymorphic loci the same multilocus genotype is recovered over long distances or periods of time and loci are in GD In the other side if reproduction is sexual and mating is random multi locus genotypes are not repeatedly recovered and there is no association between alleles Anderson and Kohn 1995 Milgroom 1996 Formally the mating system of 7 cucumeris AG 3 is not known Is suggested that it is homotallic Cubeta and Vilgalys 1997 however there is no enough evidence for this supposition Our results suggest it is a heterothallic fungus Homothallic fungi present a kind of inbreeding which means that mating occurs among close relatives however our and previous results show the opposite situation high variability in pathogenicity morphology Campion et
134. ned with isolates collected from stem cankers they did not produce higher levels of cankers Annex 3 F Incidence of black scurf 0 0 l i l l E i Carcasi Chitaga Cogua La Union Pasto Sibate Silvia Soraca Subachoque Ventaquemada Control Municipalities Figure 3 7 Incidence of sclerotia on tubers of plants inoculated with isolates of R solani AG 3 collected on different municipalities in Colombia The cluster analysis showed what was found with the descriptive analysis There was no pattern of response associated with geographical location of the isolates but in each municipality there are isolates with differential responses to the stress factors evaluated 3 4 Discussion This was an exploratory research to study the variation on phenotypic and physiological traits in isolates of R solani AG 3 collected from potato infected plants in Colombia We were interested in determine if the pathogen is adaptable to stress conditions like fungicides and temperatures Mycelial growth was selected as variable because is linked to fitness in this pathogen Additionally differences in virulence and physiological responses were tested in the same isolates in order to identify individuals associated to geographical location or to physiological responses The life cycle of R solani AG 3 has an asexual and a sexual phase During the asexual phase the fungus produces mycelia and sclerotia as survival structures Infection starts with
135. not recommended for rotation The best options seem to be grasses P clandestinum and P pratense that were not affected when were inoculated with AG 3 isolates 4 3 3 Chemical control Fungicide application is the most common alternative for disease management Banville et al 1996 but this option is not sustainable because of the impact on non target soil microorganisms and the potential risk of resistance to fungicides In Colombia it is necessary to think on alternative options the only registered fungicide for stem canker and black scurf management showed short time effect in the inhibition of the fungus 24 hours at 3 ppm and 48 hours at 14 ppm although the manufacturer cite that this molecule is stable to hydrolysis and that the half life in soil ranges from 95 to 155 days The mode of action of thifluzamide and other carboxamides are thought to have a medium to high level of potential resistance FRAC 2011 Resistance to thifluzamide has been documented for specific fungi but is not a wide spread issue Our results would indicate that even though this fungicide is very stable in soil the low inhibition of micelial growth in time makes its use as the sole management strategy very unreliable 4 3 4 Resistant varieties Resistant cultivars are the desirable option to enhance the control of R solani disease in the field Different studies have reported potato germplasm with different responses to the pathogen Naz et al 2008 Ol
136. nsidering that AG 3 was able to infect several other species in controlled conditions 4 2 Genotypic and physiologic variability of R solani AG 3 R solani AG 3 has been reported variable in all the places where their populations have been evaluated The variability is manifest not only at the genetic level but in its physiologic and phenotypic characteristics Individual variation has great interest from an evolutionary perspective given that selection and evolution of traits arise from variable individuals present into the populations Bennett 1997 Individuals of the Colombian populations of R solani AG 3 are highly variable from a genetic perspective this pathogen has high evolutionary risk as has been proposed for pathogens with mixed reproduction and high and efficient gene flow McDonald and Linde 2002 Additionally results of this study show that this pathogen has the ability to grow well at temperatures above and under the optimal in fact the 97 temperature optimal in vitro is higher than expected considering that the temperatures on the regions were the samples were taken are mainly from 15 to 22 C The evolutionary potential of populations of organisms depends on the interaction of the evolutionary forces In the Colombian populations of R solani AG 3 the main evolutionary force acting was found to be gene flow an unexpected condition for a soil borne fungus condition that is explained for the movement of tuber see
137. o determine the evolutionary risk of plant pathogens depends on their biological ecological and genetic characteristics Table 2 1 The model considers that pathogens with mixed reproduction have the highest risk of evolution the sexual cycle generate new combinations of alleles genotypes The recombinant genotypes can migrate to different environments where they are tested The most fit combinations of alleles are held together and multiplied through asexual reproduction and selected clones may increase their frequency The clone s with highest fitness can become distributed over a wide area through genotype flow McDonald and Linde 2002 Table 2 1 Factors conditioning the evolutionary risk of plant pathogens Highest risk of evolution Lowest risk of evolution 1 High mutation rate Transposable elements active 1 Low mutation rate No transposons 2 Large effective population sizes Large overseasoning population Extinction of local populations rare No genetic drift no loss of alleles 2 Small effective population sizes No over seasoning propagules Extinction of local populations common Significant genetic drift loss of alleles 3 High gene genotype flow Sexual 3 Low gene genotype flow Asexual propagules dispersed by air over long propagules soil borne Quarantines effective distances 4 Mixed reproduction system Sexual 4 Asexual reproduction system Only outcrossing and asexu
138. o dextrose agar PDA Sclerotia from 25 day old cultures from each isolate growing on PDA plates with a sterile cellophane sheet were transferred to 1 8 ml cryotubes Nunc CryoLine System Denmark containing anhydrous silica gel for long term storage at 4 C Mycelia for genomic DNA extraction was obtained from 5 day old cultures on PDA containing a sterile cellophane membrane After incubation at room temperature mycelium from each isolate was harvested by scraping the culture from the cellophane membrane the mycelia was frozen and lyophilized DNA was extracted with the DNeasy Plant Mini Kit Qiagen The AG 3 isolates were determined by PCR with specific primers for the ITS 5 8S region of the ribosomal DNA Lees et al 2002 45 Table 2 2 Geographic origin of the populations of Rhizoctonia solani AG 3 used in this study State Municipality ta Geographical coordinates pui Potato cultivar po Cagua Cogual 5 04 32 N 73 58 33 W 2730 S tuberosum Gr phureja 2006 Cogua2 5 04 22 N 73 58 45 W 2722 S tuberosum Gr phureja 2006 Subachi 4 57 26 N 74 11 59 W 2931 S tuberosum R 12 2006 Cundinamarca Subachoque Subach2 4 57 IEN 749 12 56 W 3095 S tuberosum R 13 2006 abia Sibatel 4 25 26 N 74 15 08 W 3250 S tuberosum ICA Morita 2006 Sibate2 4 25 16 N 74 14 56 W 3296 S tuberosum Parda Pastusa 2006 Pasto1 01 14486N 77 34147W 3095 tuberosum Parda Pastusa 2007 Nari o Pasto Pasto2 01 137
139. of Anastomosis Groups of Rhizoctonia solani by Polyacrylamide Gel Electrophoresis of Soluble Proteins Phytopathology 73 903 906 Rinehart T A Copes W E Toda T Cubeta M A 2007 Genetic Characterization of Binucleate Rhizoctonia Species Causing Web Blight on Azalea in Mississippi and Alabama Plant Disease 91 616 623 Sharon M Kuninaga S Hyakumachi M Naito S Sneh B 2008 Classification of Rhizoctonia spp using rDNA ITS sequence analysis supports the genetic basis of the classical anastomosis grouping Mycoscience 49 93 114 Sharon M Kuninaga S Hyakumachi M Sneh B 2006 The advancing identification and classification of Rhizoctonia spp using molecular and biotechnological methods compared with the classical anastomosis grouping Mycoscience 47 299 316 Sneh B Burpee L Ogoshi A 1998 Identification of Rhizoctonia species APS press St Paul Minnesota USA pp 135 Stevens Johnk J Jones R K 2001 Differentiation of three homogeneous groups of Rhizoctonia solani anastomosis group 4 by analysis of fatty acids Phytopathology 91 821 830 Stodart B J Harvey P R Neate S M Melanson D L Scott E S 2007 Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia Mycological Research 111 891 900 Truter M Wehner F C 2004 Anastomosis Grouping of Rhizoctonia solani Associated with Black Scurf and Stem Canker of Potato in South
140. olani AG 3 collected on different municipalities in Colombia at 15 and 25 OC cis deste eie tuis tace sd in Colombia eeenn aa e eade 19 22 25 54 54 75 76 76 77 78 78 79 List of Annexes Annex 2 A Information content of eleven microsatellite loci used for multilocus genotyping of individuals of Rhizoctonia solani AG 3 from Colombian potato crops Annex 2 B Measures of differentiation among populations of Rhizoctonia solani AG 3 infecting potato in based on His ValUCS 6 cc cece eee eee e ee eee ee eens eee eeeeeaeeeeaeaeeees Annex 3 A Growth in vitro of isolates of Rhizoctonia solani AG 3 in response to four temperatures 5 egg dca RR a a AEn a a ERRARE RIA On EA Da AEn EA Ei ER MERE Annex 3 B Time to sclerotia formation in isolates of Rhizoctonia solani AG 3 in response to four temperatures 0 2 cece ccc eeee eee ee eee e eee cece ee eee e teens eaeneeneeeenaeaes Annex 3 C Growth of isolates of Rhizoctonia solani AG 3 in presence of different doses of the fungicide ThifluZamide cccececeee eee e ee eee ee ee eeeee eases eeeesnnieeees Annex 3 D Size of lesions on stems stolons and roots generated by the artificial inoculation with isolates of R solani AG 3 collected on different municipalities in Golomb ax sidshetin chins etc tenis wi A das pant Annex 3 E Test for differences among average values in the variables evalua
141. om symptomatic potato plants in Cundinamarca Nari o Antioquia Boyac Santander and Norte de Santander states were obtained Table 1 1 387 isolates were positive for the AG 3 with the specific primers Those samples amplified a 500 bp fragment according to the reported size The results of positive isolates were verified by sequencing of the ITS rDNA Four additional isolates that were negative with specific primers were classified as AG 3 based on their ITS rDNA sequence The sequence of the ITS rDNA of those four isolates showed a mutation point on nucleotide number nine into the forward primer this can explain their failure to amplify The ITS1 ITS2 regions of the rDNA from R solani AG 3 sequences from this study showed high sequence similarity 98 3 100 among them and with three AG 3 sequences from NCBI The sequence analysis allows the discrimination between isolates AG 2 1 from AG 3 Thus by the criteria of amplifying with specific primers plus sequencing of the rDNA 88 45 of the collection was identified as AG 3 Few other isolates were identified as AG 2 1 2 54 and binucleate Rhizoctonia AG A AG E and AG I 6 24 Around 3 of the isolates could not be assigned to any anastomosis group due to the poor sequence quality Table 1 1 probably the last group belong to isolates with large nucleotide differences in the primer region 24 Table 1 1 Number of isolates belonging to each anastomosis group of Rhizoctonia
142. opulation are interacting genotypes and whose driving mechanisms are the interactions among them These interactions may range from antagonistic to beneficial and the populations can themselves evolve sometimes fortuitous and unexpected Kassen and Rainey 2004 Epidemics of soil borne pathogens depend on the ability of the pathogen to contact the host The organisms used for biological control block the sites for infection acting against the pathogen infection Bailey and Gilligan 1997 In laboratory usually one strain of the pathogen is challenged with one strain of the biocontrol agent in this one by one interaction is easy to depict conclusions about the beneficial effect of the biocontrol against the disease However in the fields there are many different individuals in the populations of the pathogen that respond differentially under that situation only one strain of the biocontrol agent is fighting against a complex population of the pathogen When biological control of diseases is used in field conditions it is necessary to consider the complex environmental and the genetic diversity of the pathogen populations as well as the diversity of the interacting organisms all those factors contribute to the failure or success of biological control in the field The management of R solani AG 3 in Colombia requires an integrated approach The main objective must be to decrease its evolutionary potential which can be achieved reducing the popula
143. opulations from the 18 geographic locations Figure 2 1 and Figure 2 2 140 120 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 k Figure 2 1 Number of simulated populations Evanno et al 2005 1 00 0 80 0 60 0 40 0 20 0 00 Populations Figure 2 2 STRUCTURE inferred membership coefficient for MLMGs Rhizoctonia solani AG 3 in Colombia Each vertical bar represents one MLMG Each color represents the most likely ancestry of the cluster from which the genotype or partial genotype was derived Individuals with multiple colors are admixed genotypes The bar length indicates its membership coefficient Q to the distinctly colored populations Populations 1 Subachoque1 2 Subachoque2 3 Cogual 4 Cogua2 5 Sibate1 6 Sibate2 7 Silvia 8 Pasto1 9 Pasto2 10 Ventaquemada1 11 Soracai 12 Soraca2 13 Ventaquemada2 14 LaUnion1 15 LaUnion2 16 Carcasi1 17 Carcasi2 and 18 Chitaga Admixture was detected in the overall sample 20 67 N 61 of the evaluated genotypes have a mixed background of the two genetic populations The amount of admixture varied considerably among populations and all the populations present admixed MLMGs 55 Table 2 6 Tests for random association of alleles within each locus and between pairs of loci populations of Rhizoctonia solani AG 3 in Colombia lone Number of loci b b a Locus pairs at significant x C Cc A 0 State Location eae under HWE Fis p value l
144. orphological genetic and physiological features Intra specific groups ISG within AGs and vegetative compatible populations VCP comprising highly similar isolates of R solani have been identified by biochemical serological molecular and biological methods Ogoshi 1987 Kuninaga et al 1997 MacNish et al 1997 Isolates belonging to same VCP can be considered as clones representing identical fungal strains whereas ISGs within specific AGs are considered as subspecies Subgroups have been identified in AGs 1 2 3 4 6 8 and 9 Sharon et al 2008 The members each subgroup show hosts preferences this can be the result of particular ability to produce virulence factors Stodart et a 2007 Teleomorphs are originated under specific environmental conditions for each AG Hymenium is composed by short hyphal cell that branch frequently producing dense interwoven mats on which basidia are formed The sterigmata arise from the basidia ranging in number from one to seven The shape of basidiospores and basidia the number and size of basidia 15 basidiospores and sterigmata vary among species being important characters for classification of Rhizoctonia spp Sneh et al 1998 Naito 2006 1 1 3 Stem canker and black scurf on potato Solanum tuberosum L Black scurf and stem canker on potato caused by Rhizoctonia solani are economically important diseases causing both quantitative and qualitative damage in potato crops
145. ound in the fields may harbor AG 3 and although the amount of disease is low those plants may contribute to maintain and increase the inoculum of R solani AG 3 in soil Although host specific all AGs have proved to cause some level of damage in hosts different to their original host This is relevant considering that AG 3 live on alternate hosts Although the severity of the lesions is low the pathogen can on alternate hosts increasing the potential of inoculum in absence of its primary host potato Crop rotation is one of the main strategies of control of stem canker and black scurf and then it is necessary to select carefully the plant species for this practice to avoid the increase of inoculum by alternate hosts The absence of infection on grasses shows that they are a proper option for rotation AG 2 1 was exclusively found in samples from Boyaca and Cundinamarca Its occurrence on potato in these regions may be attributed to the abundance of two Brassicaceae hosts yellow R raphanistrum and purple mustard Brassica spp that are common weeds in potato fields Arrieta 2000 In fact this AG has been reported as a Brassicaceae pathogen Carling and Leiner 1986 28 The presence of individuals of different AGs on lesions does not necessarily indicate a pathogenic relationship between potato and those strains Carling and Leiner 1986 The ability of AG 2 1 isolates to cause cankers on potato stems under artificial inoculation
146. pion et al 2003 Lehtonen et al 2008 and genetic diversity Kuninaga et al 1997 Ceresini et al 2002a Ceresini et al 2002b Justesen et al 2003 Balali et al 2007 Woodhall et al 2007 This is an important insight in the population studies of this pathogen because genetically diverse populations can react easily and quickly to stress factors as those imposed to control their populations in the fields McDonald and Linde 2002 2 4 2 Population differentiation Several approaches have been used to estimate the amount of genetic differentiation into the populations Hedrick 2005 Wright developed an approach to partition the genetic variation in a subdivided population using three statistics in terms of correlation among alleles Nei 1977 showed that those coefficients can be expressed in terms of allele and genotype frequencies Thus Fs is a coefficient for evaluating population differentiation it ranges from 0 to 1 with cero indicating no differentiation among the populations evaluated and 1 showing that populations are totally different Hedrick 2005 Another way for testing population differentiation is AMOVA which estimates population differentiation making a hierarchical analysis of variance AMOVA make partition of the total variance in their components due to intra individual differences inter individual differences or inter population level differences it depends on the level tested Excoffier et al 1992 Th
147. raphical origin was not found and the wide variability in the response to the factors of stress evaluated Genetic and phenotypic diversity in the population of organisms is shaped by natural selection and adaptive evolution The process of adaptive evolution depends on how much heritable genetic variation exists for the traits that are exposed to selection Much of the variation observed in nature is neutral with respect to fitness being determined by stochastic processes choosing among ecologically equivalent types Kocher 2004 From an ecological perspective diversity is supported mainly by divergent natural selection which is plausible when there are free niches or the organism is highly competitive and displaces to its competitors The variety of niches available depends largely on the 82 physical structure of the environment Complex heterogeneous environments provide more niches and therefore maintain higher diversity than simpler environments In soil spatial variation is high with a huge richness of niches determined by the biotic and abiotic environment explaining the high biological diversity in this kind of environments The soil borne pathogen R solani is a complex specie it affects a wide range of hosts including dicots and monocots and has been adapted to different organs into the hosts underground and above ground In the sub specific groups AGs and subgroups the variability also is high showing the evolutionary poten
148. rs Conservation Biology 12 844 855 Piry S Luikart G Cornuet J M 1999 Computer note BOTTLENECK a computer program for detecting recent reductions in the effective size using allele frequency data Journal of Heredity 90 502 503 Pritchard J K Stephens M Donnelly P 2000 Inference of population structure using multilocus genotype data Genetics 155 945 959 Raymond M Rousset F 1995 GENEPOP Version 1 2 Population genetics software for exact tests and ecumenicism Journal of Heredity 86 248 249 65 Rice W R 1989 Analyzing tables of statistical tests Evolution 43 223 225 Rosewich U L Pettway R E McDonald B A Kistler H C 1999 High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG 1 IA Thanatephorus cucumeris from Texas Fungal Genetics and Biology 28 148 159 Rozen S Skaletsky H J 2000 Primer3 on the WWW for general users and for biologist programmers Bioinformatics Methods and Protocols Methods in Molecular Biology Humana Press Totowa New Jersey pp 365 386 Schlotterer C 2000 Evolutionary dynamics of microsatellite DNA Chromosoma 109 365 371 Schlotterer C 2004 Opinion The evolution of molecular markers just a matter of fashion Nature Reviews Genetics 5 63 69 Simons S A Gilligan C A 1997 Factors affecting the temporal progress of stem canker Rhizoctonia solani on potatoes Solanum tuberosum Plant Pathology 4
149. s that can be successful in the new environment ii Recombination between foreigner and native genotypes produce more novel genotypes with new combination of alleles and iii the continuous addition of resistance structures sclerotia to soil increases the potential of inoculum on the long run Additionally the variation in the response to stress factors of the isolates evaluated show that this pathogen has a high evolutionary potential therefore their populations react positively to differences in environments 4 3 2 Cultural practices Cultural practices together with host resistance generate an inadequate environment to the pathogen Practices that favor rapid emergence reduce the risk of root and stem cankers Jeger et al 1996 This occur when the colonization of the pathogen is diminished and the host advantaged Invasion of R solani occurs in a physical chemical and biological heterogeneous environment through a web of pores cracks and aggregates Otten et al 1999 Otten and Gilligan 2006 In that environment the pathogen interacts with soil particles water films organisms and with the host to cause the diseases Modification of the components of soil can diminish the effect of the soil diseases caused by R solani The main characteristic of the soil having an important effect on the development of epidemics is soil water high contents affect the pathogen Otten et al 1999 Ritz 2007 for what pre irrigation of dry soils b
150. s for a soil borne fungal plant pathogen Rhizoctonia solani Soil Biology and Biochemistry 31 1803 1810 Rauf C A Ahmad l Ashraf M 2007 Anastomosis groups of Rhizoctonia solani kuhn isolates from potato in Pakistan Pakistan Journal of Botany 39 1335 1340 Ritz K 2007 Spatial Organisation of Soil Fungi In Franklin R B Mills A L Eds The Spatial Distribution of Microbes in the Environment Springer 179 202 pp Rosewich U L Pettway R E McDonald B A Kistler H C 1999 High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG 1 IA Thanatephorus cucumeris from Texas Fungal Genetics and Biology 28 148 159 104 Scholte K 1989 Effects of soil borne Rhizoctonia solani Kuhn on yield and quality of ten potato cultivars Potato Research 32 367 376 Sharon M Kuninaga S Hyakumachi M Naito S Sneh B 2008 Classification of Rhizoctonia spp using rDNA ITS sequence analysis supports the genetic basis of the classical anastomosis grouping Mycoscience 49 93 114 Simons S A Gilligan C A 1997a Factors affecting the temporal progress of stem canker Rhizoctonia solani on potatoes Solanum tuberosum Plant Pathology 46 642 650 Simons S A Gilligan C A 1997b Relationships between stem canker stolon canker black scurf Rhizoctonia solani and yield of potato Solanum tuberosum under different agronomic conditions Plant Pathology 46 651 658 Suresh K
151. s was variable Table 3 1 For the pathogenicity test on stems half of the isolates tested for black scurf temperatures and fungicide sensitivity were used The isolates chosen were previously characterized with SSR markers 3 2 2 Preliminary tests Preliminary tests were made with 9 isolates randomly selected to choose the proper levels of the factors subject of evaluation First was checked if there were differences in the growth of the isolates using variable volumes of PDA Potato Dextrose Agar in the Petri plates 15 20 and 25 ml and the minimum number of repetitions required for the tests in PDA in vitro In a second test six doses of the fungicide thifluzamide 0 0 1 1 0 10 100 and 1000 ppm were evaluated to select one where the isolates show variability in their 72 growth The third test checked for the differential response to the temperatures growing the isolates at five temperatures 4 10 20 30 and 40 C The isolates were kept in different spaces for each temperature at 4 C in a cold room at 10 and 20 C in a fitotron Labline Biotronette without light at 30 C in an incubator WTBbinder and at 40 C in an incubator lab line instrumentals For all the tests in vitro the evaluation of the colony size was recorded at 24 48 and 72 hours after inoculation of the Petri dishes in order to define the accurate time to make the evaluation Table 3 1 Number of isolates used for each analysis
152. ses in Colombia 4 1 Anastomosis groups Plant pathogenic isolates of H solani are divided in intraspecific groups based on differences in pathogenicity cultural appearance morphology physiology ecology and DNA sequence Anderson and Stretton 1982 Ogoshi 1987 Andersen 1996 Sharon et al 2008 Those intraspecific groups are called Anastomosis Groups AG In different geographical regions where potato is produced research on AGs has shown that AG 3 is the most prevalent and the group that causes the most serious damage on plants and the higher infestation on tubers Additionally several AGs have been associated to symptoms and have been isolated from soils where potato is grown Table 4 1 In this research two AGs were associated with symptoms on plants and tubers AG 3 and AG 2 1 There was no relationship between the AG and the organ of the plant from where it was isolated 96 Table 4 1 Anastomosis Groups associated to Rhizoctonia diseases in potato crops around the world Country Source Reference Australia AG 3 AG 4 AG 5 y AG 8 Stems roots tubers and soil Balali et al 1995 Canada AG 3 AG 4 y AG 5 Stems stolons roots Bains and Bisht 1995 Chile AG 3 and non identified isolates Tubers Castro 2005 Finland AG2 1 AG 3 and AG 5 Stems stolons tubers Lehtonen et al 2008a France AG2 1 AG 3 y AG 5 Stems hymenia and tubers Campion et al 2003 Great Britain AG2 1 AG 3 y AG 5 Stems stolons roots tubers Wood
153. sing the proper approach for the management of this pathogen from a population view and considering its genetic and physiological variability Microbial communities are evolving entities that interact Only one individual rarely ever cause serious epidemics Disease symptoms typically manifest once the pathogen populations reach certain thresholds The severity of the disease is also influenced by evolution within the pathogen population that occurs within an ecological context defined by the host and the climatic conditions Kassen and Rainey 2004 Then it is important to have always in mind that in the fields the populations of pathogens interact with plants where pathogens are variable in time and space and plants have a narrow genetic base consequence of human selection process In the potato crops in Colombia R solani AG 3 maintain high genetic physiological and pathogenic variability and is able to be adapted to the fungicide thifluzamide and to react positively to the increase of temperature Those characteristics improve its adaptability The growth of the fungus is reduced in response to stress however it is not inhibited completely In those conditions the development of the disease will be slower allowing to the plants improve their defenses against the attack of the fungus The differences among isolates from one locality allows to infer that within fields the disease can be expressed in differential ways with isolates more inhibi
154. solani associated to symptoms of stem canker and black scurf on potatoes in Colombia Department Municipality Field AG 2 1 AG 3 AG A AG E AG I NI TOTAL 1 1 29 1 3 0 0 34 Subachoque 2 0 20 0 0 1 0 21 C 1 2 24 3 1 3 0 33 Cundinamarca ogua 2 1 36 1 0 0 0 38 ibale 1 1 22 0 0 1 0 24 2 3 15 1 0 1 h 2 o 1 0 18 0 2 1 1 22 Nari o Pasto 2 0 22 0 0 0 0 22 1 0 20 3 0 0 0 23 Antioquia La Union 2 0 35 1 0 0 2 38 1 2 24 0 0 0 0 26 Soraca 2 1 32 2 0 0 0 35 Boyaca 1 0 20 2 0 0 3 25 Ventaquemada 2 0 22 0 0 0 2 24 Santander Carcasi 1 9 H a 2 i i i 2 0 10 0 0 0 2 12 Norte de Santander Chitaga 1 0 14 0 0 0 0 14 Cauca Silvia 1 0 9 0 0 0 0 9 Number 11 383 14 6 7 12 433 2 54 88 45 3 23 1 39 1 62 2 77 NI Non identified From the total of 433 isolates analyzed 374 were isolated from cankers on the stems and 59 from sclerotia from the tubers seed that originated the plants collected 55 of the isolates from sclerotia were identified as AG 3 two as AG 2 1 one as AG A and one as AG E From the 374 isolates collected on stems 328 were identified as AG 3 nine as AG2 1 13 as AG A five as AG E and 7 as AG I 1 3 2 Hyphal interactions Microscopic somatic interactions between the AG 3 PT tester and the sample of 60 isolates of H solani AG 3 resulted in positive anastomosis reactions The frequency of reactions C1 C2 and C3 was hi
155. st way Abstract Rhizoctonia solani AG 3 Kuhn teleomorph Thanatephorus cucumeris Frank Donk is a main soil borne pathogen on potato crops around the world Actually this pathogen is recognized as important in Colombia however there is no information about the pathogen and the disease This research was conducted using biological and molecular tests to know the Anastomosis Groups AG associated with symptoms in the main producing potato areas in Colombia Additionally the genetic variability of the isolates and the distribution of variability along the main potato producing areas in Colombia as well as the response of the pathogen to temperatures and fungicides and its aggressiveness on potato plants were tested The samples were collected from symptomatic stems of potato on naturally infested fields and then were processed in laboratory The isolates were subject of different analysis The AG 3 of R solani was the most common associated to symptoms in fields The isolates from different geographical populations were highly variable and the variability was not structured geographically All the populations are genetically similar showing the effect of gene flow in the genetic structure of the populations The evaluation of the response of the pathogen to two temperatures the sensitivity to the fungicide thifluzamide and the aggressiveness on different organs of the potato plants showed that the isolates of all the geographical localities vary in
156. t bean lettuce maize onion sweet clover and sunflower were susceptible to AG 3 isolates Carling and Leiner 1986 The alternative hosts that can be either symptomatic or latently infected have to be considered in the integrated management of the disease In Colombia potato is grown in the Andean highlands 2500 3200 masl The departments with the largest production are Cundinamarca Boyac Nari o and Antioquia and they contribute with 70 of the national potato production Espinal et a 2006 Seed production is mainly domestic Although some potato growers use certified tuber seed it is impossible to guarantee tubers free of R solani and the pathogen is easily dispersed between regions on infested seed tubers In Colombia the knowledge about the relative importance of the distinct AGs on the etiology of Rhizoctonia diseases on potatoes and about the disease itself is still scarce Farmers are not aware of underground symptoms on stems and in consequence there are no estimations about the importance of this pathogen in quality and tuber yield The main goal of this research was to generate knowledge about the etiology of Rhizoctonia diseases on potatoes based on a large scale population sampling in Colombia The specific objectives were i to identify and characterize the relative importance of the distinct R solani AGs associated with potato stem canker and black scurf diseases in Colombia using a PCR based method and classical somatic
157. tances for attacking and manipulate the plant for their own benefit Strange and Scott 2005 Farmers are constantly challenging pathogens using practices to reduce plant diseases into the agro ecosystems causing strong directional selection on their populations 3 1 1 Genetic diversity and the evolutionary forces Molecular analyses have revealed high genetic diversity in the populations of plant pathogens Prevalence of diversity in populations depends on the fitness of the individuals that is determined by the combined ability of an organism to survive and reproduce Leach et al 2001 Kassen and Rainey 2004 it is measured as the ability of one genotype to leave offspring relative to others The mechanisms underlying changes in fitness are the interactions among genotypes and between genotypes and the environment For plant pathogens several traits such as reproductive rate growth infection efficiency and amount of disease aggressiveness have been used to measure fitness Leonard and Czochor 1980 69 Population genetics is focused mainly on genetic processes such as mutation genetic drift gene flow mating system and natural selection those are called evolutionary forces and are responsible for the extent of genetic variation in every population of living organisms McDonald and McDermott 1993 McDonald 2004 Although all of them are determinants for the genetic structure of the populations of organisms in this chapter we
158. te de tesis y el consejo examinador no seran responsables de las ideas emitidas por el autor Articulo 218 de los Estatutos de la Universidad Nacional de Colombia Esta tesis ha sido escrita en el formato establecido por la Universidad Nacional de Colombia para tesis de maestria y doctorado Resoluci n 001 de 2011 Por la cual se establecen los procedimientos para la publicaci n de las tesis de maestr a y doctorado de los estudiantes de la Universidad Nacional de Colombia en el Repositorio Institucional UN ACTA DE CALIFICACION TESIS DE DOCTORADO EN CIENCIAS AGROPECUARIAS AREA AGRARIA NFASIS EM FITOPATOLOG A He 481 Acta de Gones o de Fuceltad 47 dul 3 de Octubre de 2011 FECHA gt Nowlembre 21 de 2011 ESTUDIAMTE ROSA LILIA FERRUCHO DIRECTOR Calas Garcia Dorper TITLE CONSTRUCTION OF PATHOGENIC BIOLOGIC AND GENETIC BASES OF THE COLOMBIAN POPULATIONS OF Rh rocfonia sofa AG 3 NECESSARY FOR THE DEVELOPMENT OF MANAGEMENT STRATEGIES OF STEM CANKER AND BLACK SCURF DISEASES OF POTATO JURADO z Aprobade por al Comil cad mico Asecor da Poegrado y Conse da Facultzd del c a 3 de Octubra da 20141 Acta 017 PRESIDENTE OSCAR ARTURO OLIVEROS GARAY JURADO PAULO CEREGSIHI JURADO CELESTE LUNDE JURADO ALBA MARINA COTES El jurado calfcador en plero de acuerdo con lo establecida en el Articuln 35 par grados del 1 al 6 de la Resoluci n de Consejo de Facultad Ho 152 de 2005 sobre REGLAMENTO DE LOs SEMINARIOS DE IMWESTI
159. ted the plants do not get ill and in the places where are located the isolates best adapted the incidence and severity will be higher The possibility of diminishing R solani AG 3 in soils is remote The production of resistance structures allows it to survive in soils for long periods of time With rotation the population of the fungus diminishes but never will be close to zero Additionally the use of seed with some degree of sclerotia and mycelia always adds inoculums to the soil Therefore the control on the movement of tubers with visible structures must be considered as a main tool of management of the disease along with crop rotation 4 3 1 Legal control The use of pathogen free seed tubers is the most important tactic because in this way the inoculum potential is reduced Previous studies have shown that tuber borne as well as soil borne inoculum is important in the severity of the disease and the symptoms are more severe when both of them are present in the field Frank and Leach 1980 Carling et al 1989 Scholte 1989 Tsror and Peretz Alon 2005 The epidemics are strong when 99 the inoculum on tubers is higher than 10 of tuber surface covered by sclerotia Simons and Gilligan 1997 With low levels of inoculum on tubers the degree of the disease caused probably is not significant however there are three main consequences of the arrival of new genotypes i continuous introduction of new genotypes into the population
160. ted in pathogenicity tests of isolates of Rhizoctonia solani AG 3 cccccceceee eee eeeeee ees Annex 3 F Symptoms induced by Rhizoctonia solani AG 3 plants of the cv Diacol CADIFO Aen shies toc Ree oie ca ened dea A eae ow inte le eee A AN Annex 3 G Principal component analysis for the test in vitro ssuusuuuusus Annex 3 H Principal component analysis for the scores of the disease on stems roots and stolofisz sve ee E RN RENE RM DRIN A e EE RUE Annex 3 1 Principal component analysis for the scores of the disease on tubers 66 67 85 86 87 88 90 91 92 Introduction Rhizoctonia solani K hn Anastomosis Group 3 AG 3 teleomorph Thanatephorus cucumeris Frank Donk is an important soil borne pathogen of potato Banville et al 1996 R solani AG 3 affects plants on different growth stages Early in the season the fungus causes necrosis in emerging sprouts and the killing of the sprout tip later in the season dark brown cankers are formed at the base of the stems for that reason the disease is named stem canker Banville et al 1996 Jeger et al 1996 Simons and Gilligan 1997 At the end of the crop cycle sclerotia are formed on the tubers surface especially after vine death in this state the disease is called black scurf Cankers formed on the stem base cause growth delay as secondary symptom due to the diminishing of water and minerals uptake by the plant T
161. that populations of this pathogen are genetically variable Ceresini et al 2002a Ceresini et al 2002b Campion et al 2003 Justesen et al 2003 with differences in virulence among isolates Carling and Leiner 1990 Bains and Bisht 1995 Lehtonen et al 2008 and differences in their sensitivity to fungicides Campion et al 2003 Lehtonen et al 2008 Those characteristics are important when proposing control measures of the diseases because variable populations are more susceptible to overcome the control measures imposed in order to diminish their populations and to reduce the effect of the diseases in the fields The amount and distribution of genetic variation within and among populations is called genetic structure and is determined by the evolutionary history of populations then its study gives insights into the evolutionary processes that shaped a population structure McDonald and Linde 2002 and can be used to predict the evolutionary potential of the pathogens and to formulate control strategies of the diseases In Colombia potato is grown at the high lands in the Andean zone 2500 3200 masl The departments that have the highest production are Cundinamarca Boyac Nari o and Antioquia they contribute with 70 of the national potato production Seed production is mainly domestic with local production and informal exchange among farmers only a few of them use certified tuber seed and this has contributed to the sprea
162. the response to the factors evaluated The levels of black scurf and cankers were low and variable among isolates in the cultivar of potato evaluated Key words Soil borne fungi Biological diversity Population structure Evolution Adaptability Resumen Rhizoctonia solani AG 3 K hn teleomorph Thanatephorus cucumeris Frank Donk es un pat geno del suelo importante en cultivos de papa a nivel mundial Aunque este pat geno es importante en Colombia actualmente no existe informaci n acerca del pat geno y la enfermedad Esta investigaci n se realizo utilizando pruebas moleculares y biol gicas con el fin de conocer los Grupos de Anastomosis GA asociados a s ntomas en las principales regiones productoras de papa en Colombia Adicionalmente la variabilidad gen tica y su distribuci n geogr fica as como la respuesta del pat geno a dos temperaturas a un fungicida y su agresividad sobre plantas de papa fueron evaluadas Las muestras fueron colectadas en tallos de plantas sintom ticas de papa en campos naturalmente infestados posteriormente fueron procesadas en el laboratorio y los aislamientos obtenidos se sometieron a diferentes an lisis El GA 3 de R solani fue el com nmente asociado a los s ntomas en campo Los aislamientos colectados en diferentes regiones geogr ficas fueron variables y la variabilidad no est asociada a regiones geogr ficas las poblaciones son gen ticamente similares mostrando el efecto del flujo de genes
163. throughout the world Banville et al 1996 Jeger et al 1996b Banville and Carling 2001 Tsror 2010 Quantitative losses occur due to infection of the stems stolons and roots that affects size and number of tubers Carling et al 1989 Qualitative losses occur mainly through the production of misshapen tubers and the development of sclerotia on the tuber surface downgrading their quality James and McKenzie 1972 Hide et al 1973 Frank and Leach 1980 Anderson and Stretton 1982 Carling et al 1989 Tsror and Peretz Alon 2005 The severity of the symptoms of Rhizoctonia disease in potato depends on inoculum potential on tubers and in soil along with local climatic conditions AG 3 is most virulent in cool growing conditions Carling and Leiner 1990 Bains and Bisht 1995 Campion et al 2003 Justesen et al 2003 Economic impact of black scurf depend on the market of destine of the tubers The highest impact occur on the tuber seed market and depends on the norms of each country for the internal trade as well for the importation from external countries Banville et al 1996 The effect of Rhizoctonia disease on the number size distribution and tuber quality leads to yield losses ranging from 10 to 30 on marketable size tubers Carling et al 1989 For a long time R solani AG 3 was documented as host specific and the main R solani group infecting potato Carling and Leiner 1986 Carling and Leiner 1990 Bains and Bisht
164. tial of this specie which is adapted to a wide range of ecological conditions being able to occupy a wide range of niches in different agriculture ecosystems 3 5 Conclusion Populations with high genetic variation have shown to have better potential to adapt to changing environments Populations of Rhizoctonia solani AG 3 in Colombia are highly diverse in genetic and phenotypic traits Isolates in each population showed a different response to the temperatures fungicides and presented differences in aggressiveness on potato stems and tubers this show that those populations have high evolutionary potential with consequences for the management of the diseases caused by the pathogen References Belotte D Curien J B Maclean G Bell G 2003 An experimental test of local adaptation in soil bacteria Evolution 57 27 36 Burnett J 2003 Fungal Populations and Species Oxford University Press USA 368 p Carling D E Leiner R H 1990 Virulence of isolates of Rhizoctonia solani AG 3 collected from potato plant organs and soil Plant Disease 74 901 903 Cunniffe N J Gilligan C A 2009 Scaling from mycelial growth to infection dynamics a reaction diffusion approach Fungal Ecology 1 133 142 de Meeus T McCoy K D Prugnolle F Chevillon C Durand P Hurtrez Bouss s S Renaud F 2007 Population genetics and molecular epidemiology or how to d busquer la b te Infection Genetics and Evolution 7 308 3
165. tion and recolonization events McDermott and McDonald 1993 Mutation will generate new alleles if the pathogen is random mating those alleles will be mixed generating novel combinations Selection chooses the best combination of alleles in a particular environment Finally under clonal reproduction the new allele combination will be multiplied and the migration moves the genotypes to new areas If the environment is favorable they can be established in the new geographical areas in the new fields farmers cause bottlenecks by using different measures to reduce their effect on the crops McDonald and Linde 2002 Viewing pathogens in a metapopulation context reinforces the importance of the ecological scenary to explain the generation and maintenance of diversity in pathogen populations The interplay of fungal life history and host population size induces asynchrony in pathogen behavior among demes and affects the probability of local drift extinction and recolonization in time and space Drift and migration are opposing forces acting to reduce and increase 40 within population genotype diversity respectively and simultaneously increasing and decreasing the variability between populations Their combined effect leads to a dynamic ever changing patchwork of distinct individual pathogen demes which may be strengthened by differences in the local selective biotic and abiotic environment Burdon and Silk 1997 2 1 3 Population genetics on
166. tion size into the fields and limiting the movement of inoculum among localities References Andersen T F 1996 A comparative taxonomic study of Rhizoctonia sensu lato employing morphological ultrastructural and molecular methods Mycological Research 100 1117 1128 Anderson N A Stretton H M 1982 The Genetics and Pathology of Rhizoctonia solani Annual Review of Phytopathology 20 329 347 102 Anguiz R Martin C 1989 Anastomosis groups pathogenicity and other characteristics of Rhizoctonia solani isolated from potato in Peru Plant Disease 199 201 Bailey D J Gilligan C A 1997 Biological control of pathozone behaviour and disease dynamics of Rhizoctonia solani by Trichoderma viride New Phytologist 136 359 367 Bains P S Bisht V S 1995 Anastomosis group identity and virulence of Rhizoctonia solani isolates collected from potato plants in Alberta Canada Plant Disease 79 241 242 Balali G R Neate S M Scott E S Whisson D L Wicks T J 1995 Anastomosis group and pathogenicity of isolates of Rhizoctonia solani from potato crops in South Australia Plant Pathology 44 1050 1057 Bandy B P Leach S S Tavantzis S M 1988 Anastomosis group 3 is the major cause of Rhizoctonia disease of potato in Maine Plant Disease 72 596 598 Banville G J Carling D E Ostysko B E 1996 Rhizoctonia disease on potato Rhizoctonia species taxonomy molecular biology ecology pathology and
167. tions from avirulence to virulence are not common and by themselves would not cause the breaking of resistance but if a mutation is coupled to efficient directional selection virulent strains can increase their frequency causing the loss of effectiveness of resistance genes McDonald and Linde 2002 2 1 1 2 Genetic drift Genetic drift occurs when a small and randomly subset of individuals from a population survive to catastrophic events reducing population size and when a small random subset individuals colonizes new host populations founder event Allele frequencies between original and the surviving or founding populations will be different over short periods of time genetic drift lead to unpredictable changes on the allele frequencies in plant pathogen populations McDonald and Linde 2002 McDonald 2004 In plant pathogens founder events occur when a pathogen is introduced into a new area this usually happens by the movement of contaminated propagation material as result of the breaking of quarantines Control of diseases into the fields generates bottlenecks due to the reduction on population size of the pathogen after control the populations arise from few isolates reducing the genetic background of the population McDonald and Linde 2002 The probability of generation of new alleles is proportional to the size of the population in consequence large populations will have more mutants than small ones thus large populations likely
168. uction is the asexual at least at the spatial scales sampled Recombination although sporadic has significant effect into the populations This is an important insight in the population studies of pathogens meiotic recombination contributes to the genetic and genotypic diversity generating individuals with new allele combinations that can be advantageous This research brings evidence for no preference among isolates infecting stems and causing black scurf however it is necessary to propose a deep study to evaluate the inoculum source of initial infections on stems and tubers Association of MLMGs by cultivar was not found tests on susceptibility of cultivars to R solani AG 3 show that susceptibility in the host depends on the isolate used and that cultivars do no select for genotypes of the fungus References Agapow P M Burt A 2001 Indices of multilocus linkage disequilibrium Molecular Ecology Notes 1 101 102 Anderson J B Kohn L M 1995 Clonality in Soilborne Plant Pathogenic Fungi Annual Review of Phytopathology 33 369 391 Balali G R Neate S M Kasalkheh A M Stodart B J Melanson D L Scott E S 2007 Intraspecific variation of Rhizoctonia solani AG 3 isolates recovered from potato fields in Central Iran and South Australia Mycopathologia 163 105 115 Banniza S Bridge P D Simons S A Holderness M 1999 Characterisation of populations of Rhizoctonia solani in paddy rice fields in C te d
169. umber and frequencies of alleles at individual loci in a population it increases as the number of alleles increases and the relative frequencies of those alleles become more similar McDonald and Linde 2002 Genotype diversity refers to the number and frequencies of multilocus genotypes or genetically distinct individuals in a population Anderson and Kohn 1995 Taylor et al 1999 McDonald and Linde 2002 Halkett et al 2005 The genetic structure of pathogen populations offers insights in their evolutionary potential Understanding the genetic structure of pathogen populations is useful to infer the life histories and the evolutionary processes that shape the populations in agroecosystems McDonald and Linde 2002 The knowledge on diversity its geographical distribution and their relation to environmental issues could be useful to optimize the breeding programs and also to predict fungicide resistance McDonald and Linde 2002 Knowledge on the distribution of genetic diversity within and among populations can be used to identify migration patterns and to reveal cryptic recombination Ceresini et al 2002b The degree of similarity between geographically separated populations provides evidence of gene flow The gene flow has 41 important effect on the effectiveness of control strategies due to the movement of novel virulence genes or genotypes well adapted to the new cropping areas McDonald and Linde 2002 A model proposed t
170. us Rhizoctonia The main characteristics of this specie were defined as follows i Some shade brown hyphal pigmentation ii Branching near to the distal septum of cell in young vegetative hyphae iii Constriction of hyphae and formation of septa at short distance from the point of origin of hyphal branches Carling and Leiner 1990 iv The presence of dolipore septa and v multinucleate cells in young vegetative hyphae Characters never existing include presence of clamp connections conidia rhizomorphs and sclerotia differentiated on ring and medulla Sneh et al 1998 Morphology of teleomorphs also has been used as criteria for classification The teleomorph of Rhizoctonia belongs to the subdivision Basidiomycotina class Hymenomycetes and almost all Rhizoctonias belong to the Subclass Holobasidiomycetidae In nature R solani mainly exists as vegetative hyphae and sclerotia Sclerotia are composed of compact masses of cells generating tight hyphal clump that protects and preserves the fungus over non optimal times Sneh et a 1998 The fungus is dispersed via mycelia or sclerotia present on tuber seeds in contaminated plant debris or in soil Keijer 1996 The host range of R solani is wide it causes diseases on important crop plants including species in the Solanaceae Fabaceae Asteraceae Poaceae and Brassicaceae as well as ornamental plants and forest trees Ogoshi 1996 Disease symptoms include leaf blights leaf spots damping
171. val of a population in the changing environment The high genetic and genotypic diversity the gene flow and the evidence of a mixed reproductive mode within Colombian populations of R solani AG 3 are characteristics for pathogens with high evolutionary potential McDonald and Linde 2002 Pathogens with high evolutionary potential require special attention when generating control measures such as fungicide applications and resistance genes Additionally the gene flow through contaminated tuber seed must be minimized in order to avoid dissemination of virulence genes and the generation of advantageous genotypes 2 5 Conclusions The Colombian population of Rhizoctonia solani AG 3 is not geographically structured These results support observations about dispersal of this pathogen in contaminated tuber seed which is the most efficient mechanism of dispersion of this fungus between geographical distant populations The analysis with structure showed two genetic pools associated to the geographical populations of R solani AG 3 en Colombia Frequent human mediated dispersal of asexual propagules mycelia and sclerotia among populations would lead to the widespread distribution of clones among populations Long 61 distance dispersal of sclerotia and mycelia is suggested by the presence of the same multilocus genotypes in different counties Although clones are important for dispersion it does not seem that the predominant mode of reprod
172. versity and to detect groups associated to ecological parameters The multilocus genotype of an individual helps to compute its probability of belonging to a given subpopulation This is useful to determine the proportion of individuals that are immigrants and to identify the geographical origin of the individuals de Meeus et al 2007 Additionally different approaches based on Bayesian statistics and Markov Chain 10 Monte Carlo simulations have been developed to find hide population structure due to unknown factors that contributes to shape the genetic structure of the populations Huelsenbeck et al 2011 Pritchard et al 2000 By analyzing genetic and genotypic data is possible to find evidences of random mating inbreeding or asexuality into the populations This allows inferring the predominant reproductive mode into the populations Testing for linkage between pairs of loci Hardy Weinberg Equilibrium and calculating the inbreeding coefficient Fis value is possible to determine deviation of panmixia Reproduction and mating systems have effect on the way that gene and genotypic diversity is distributed within and among individuals into the population Recombining populations of pathogens can put together new combinations particularly those related to virulence or resistance to fungicides generate more successful genotypes in agro ecosystems Milgroom 1996 McDonald and Linde 2002 Gr nwald et al 2006 de Meeus et al 2007
173. were not evenly distributed for the loci in all the populations As a trend each locus had two or three alleles with frequencies higher than 20 and they corresponded to sizes differing in one repetition In locus TC_AG3_8 alleles with frequencies higher than 85 were observed Frequencies of the most common alleles varied among populations but no consistent patterns among loci were observed A total of 18 private alleles were found in whole population Nine populations had private alleles La Union2 Cogua1 Sibate1 Cogua2 Soraca2 pasto1 pasto2 LaUnion1 and Chitaga 295 MLMGs were found into the sample of 355 isolates analyzed clonal fraction 15 Table 2 3 The MLMGs were evenly distributed into the populations evenness 0 89 Low clonal fractions were observed for almost all populations Subachoque2 and Ventaquemada2 showed the higher values of clonal fraction 0 33 and 0 28 respectively and the lowest clonal fraction was found in Carcasi2 and Silvia with no repeated clones Clones of a particular MLMG were commonly found within the same field 58 MLMGs were shared among populations and there was one MLMG repeated 11 times into the whole population Subachoque1 shared the highest number of MLMGs 8 shared MLMGs from 23 isolates The populations Sibate1 and Pasto1 did not share MLMGs with any population The mean genotypic diversity estimated using Stoddart and Taylor s index was 14 5 and scaled to the sample size this value was 84 9
174. y Biology 21 1 17 Leonard K J Czochor R J 1980 Theory of genetic interactions among populations of plants and their pathogens Annual Review of Phytopathology 18 237 258 McDonald B A 2004 Population Genetics of Plant Pathogens The Plant Health Instructor aps online http www apsnet org edcenter advanced topics PopGenetics Pages default aspx McDonald B A Linde C 2002 Pathogen population genetics evolutionary potential and durable resistance Annual Review of Phytopathology 40 349 379 84 McDonald B A McDermott J M 1993 Population genetics of plant pathogenic fungi Bioscience 43 311 319 Milgroom M G Peever T L 2003 Population Biology of Plant Pathogens The Synthesis of Plant Disease Epidemiology and Population Genetics Plant Disease 87 608 617 Murashige T Skoog F 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiologia Plantarum 15 473 497 Ostfeld R S Glass G E Keesing F 2005 Spatial epidemiology an emerging or re emerging discipline Trends in Ecology amp Evolution 20 328 336 Otten W Filipe J A N Bailey D J Gilligan C A 2003 Quantification and analysis of transmission rates for soil borne epidemics Ecology 84 3232 3239 Otten W Filipe J A N Gilligan C A 2005 Damping off epidemics contact structure and disease transmission in mixed species populations Ecology 86 1948 1957 Otten W Gilligan C

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