Articulo 9 - Use of soil microorganisms as strategy.indd
Contents
1. Murphy and Riley 1962 in a spectrophotometer Genesys 20 at 890 nm Rev Fac Nal Agr Medell n 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological Monitoring soil microorganisms in avocado plantations Seventy rhizosphere samples of three to four years old avocado plants were collected from commercial plantations located at the towns of San Pedro Entrerrios and Santa Rosa Northern Antioquia and Rionegro El Retiro La Ceja El Carmen de Viboral and San Vicente Eastern Antioquia In each farm fine roots and rhizosphere soil 200 g of avocado plants were collected at 0 10 cm of depth packed into plastic bags and later transported in polystyrene boxes to the Laboratory of Ecology and Environmental Conservation LECA at the Universidad Nacional de Colombia Soil pH 1 2 water and soluble P in 0 01 M CaCl Olsen and Sommers 1982 were also measured in these samples and already reported by Serna et al 2012 Mycorrhizal colonization Samples of fine roots were cut in 1 cm fragments rinsed with tap water and transferred into 50 mL plastic vials 20 mL of KOH 10 solution were added to clear root contents Phillips and Hayman 1970 and removed 24 h later After that root fragments were washed with tap water and then maintained in H O 35 solution for 30 min and washed again with tap water Then 20 mL of HCI 10 solution were added and 5 min later removed without water rinsing 20 mL of t2. Jaramillo and Osorio 2009 Gonz lez and Osorio 2008 Currently the presence of effective PSM in the rhizosphere of avocado plants has not been confirmed The aim of this study was to evaluate the use of AMF and PSM in avocado plants at the Northern and Eastern Antioquia Colombia and to detect potential soil and crop factors that limit their effectiveness in enhancing plant P uptake and growth MATERIALS AND METHODS Soil phosphate sorption Soil surface samples A horizon 0 20 cm were collected from two places Santa Rosa and El Pe ol which are representative of the two productive areas of avocado at Northern and Eastern Antioquia respectively To quantify the soil P fixation capacity the method developed by Fox and Kamprath 1970 was used Briefly this consisted of the use of 3 g soil dry basis sieved at lt 2 mm that were transferred into plastic centrifuge tubes volume of 50 mL which received grading amounts 0 2 000 mg kg of H PO SOLUFOS dissolved in 30 mL of 0 01 M CaCl Two drops of toluene were added into each tube to impair microbial activity Then the tubes were shaken in a reciprocal shaker at 100 rpm 30 min each 12 h during six days at room temperature 25 27 C After the incubation period the tube and their contents were centrifuged for 15 min at 2 500 x g and the uppernatant was passed through a Whatman No 1 filter paper Soluble P concentration was measured by the molybdate phosphate method
3. mostly Andisols that reduce the amount of P available for plant roots IGAC 1988 The ion phosphate H PO is strongly adsorbed onto the surface of soil minerals allophane goethite kaolinite in such a way that most of the applied P fertilizers gt 90 became 1 Associated Professor Universidad Nacional de Colombia Sede Medellin Faculty of Sciences School of Biosciences A A 3840 Medellin Colombia lt nwosorio unal edu co gt 2 Agronomic Engineer Student in Geomorphology and Soils Universidad Nacional de Colombia Sede Medellin Faculty of Sciences A A 3840 Medellin Colombia lt sandraluz571 gmail com gt 3 Agronomic Engineer M Sc in Geomorphology and Soils Universidad Nacional de Colombia Sede Medellin Faculty of Sciences A A 3840 Medellin Colombia lt bemontoy gmail com gt Received October 07 2011 Accepted October 18 2012 Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 non available Havlin et al 1999 Miyasaka and Habte 2001 Besides free ions of aluminum and iron can also precipitate phosphate into insoluble compounds Barber 1995 As a consequence high doses of soluble P fertilizers are regularly applied in avocado plantations Serna et al 2012 however this increases the production costs causes plant nutrient imbalances micronutrient deficiencies and increases the risk of pollution of surface water Miyasaka and Habte 2001 Montoya 2007 In addition
4. the variation of the international price of soluble P fertilizer in Colombia all P fertilizer is imported is a challenge to the food production as occurred in 2007 and 2008 when these increased by 355 Hylton 2008 For all these reasons it is desirable increase the efficiency of soluble P fertilizers by using soil microorganisms Osorio 2012 Given the role of arbuscular mycorrhizal fungi AMF and phosphate solubilizing microorganisms PSM Thies and Grossman 2006 these can be used as an attractive biotechnological strategy in avocado plantations In some countries such as Mexico Chile Brazil and United States California the use of AMF during the nursery stage of avocado has been successful in enhancing water and nutrient uptake particularly P and reduce the pos transplant stress Menge et al 1978 Hernandez 2001 In Colombia recently some authors has been investigating the effect of AMF inoculation in avocado cv Villagorgona Montoya 2007 cv Hass Monta ez 2009 and cvs Santana Lorena and Com n Melo 2011 whose findings indicate that mycorrhizal inoculation with different AMF in each case significantly promoted plant growth at the nursery On the other hand Bola os and Saenz 2010 found that in addition to the plant growth promotion the mycorrhizal inoculation also reduced significantly the avocado root rot caused by some phytopathogens Fusarium sp and Phytohphtora cinnamomi the reduction w
5. P fertilizer in these soils may limit the benefits of these fungi on avocado plants Currently effective PSM of avocado rhizosphere have not been detected In some cases these microorganisms are absent in the avocado rhizosphere and in other cases their low capacity to solubilize P discourage their use in the crop Key words Arbuscular mycorrhiza phosphate solubilizing microorgansims Andisols fixation of phosphorus Resumen En los ltimos a os el rea cultivada con aguacate en Colombia ha crecido notoriamente y se espera que esta tendencia contin e en los pr ximos a os Sin embargo muchos de los suelos de la zona andina utilizados para su siembra exhiben fuerte acidez y niveles deficientes de varios nutrientes particularmente f sforo P Para corregir este problema se hacen aplicaciones de cales y fertilizantes fosf ricos solubles en mucho casos las dosis aplicadas son altas Esto eleva innecesariamente los costos de producci n crea desbalance nutricional en la planta y genera riesgos de contaminaci n ambiental Una alternativa biotecnol gica es el uso de microorganismos solubilizadores de fosfato PSM y hongos formadores de micorriza arbuscular AMF En este trabajo se describe el aislamiento caracterizaci n y evaluaci n de la efectividad de AMF y PSM para mejorar la absorci n de P por las plantas de aguacate La asociaci n micorrizal ha probado ser una alternativa factible de implementar sin embargo el excesivo
6. based only on the effects on plant nutrition and growth it is possible that other benefits on water uptake and reduction on some plant diseases incidence Allen 1996 Smith and Read 1997 must be considered in relation to the convenience of AMF inoculation under these conditions On the other hand consistently with the treatments only those plants grown in inoculated soils developed the mycorrhizal association The value of this variable fluctuated between 57 to 77 and it was not significantly affected by the level of soluble P However at the two lowest levels 0 002 and 0 2 mg L there was abundance of vesicles and arbuscules in the roots while at the highest level of soil soluble P 0 2 mg L the roots were exclusively colonized by intraradical hyphae without vesicles and arbuscules This should be a matter for further research Monitoring soil microorganisms in avocado plantations Mycorrhizal colonization of avocado plant roots In avocado plantations the values of mycorrhizal colonization were variable these fluctuated between 0 to 90 Figures 5a and 6 The significance of this should be considered in relation to the level of soil P available For instance at low soil soluble P concentration lt 0 05 mg L the mycorrhizal association promotes plant growth B Figure 5 A Roots of avocado plants colonized by AMF B Petri dishes containing colonies of potential PSM capable of rock phosphate dissolution Note t
7. management Prentice Hall Upper Saddle River NJ 499 p Hernandez C 2001 Efecto del hongo micorrizal Glomus intraradices Schenk and Smith en el crecimiento del portainjerto Mexicola Persea americana Mill cultivado bajo 5 tratamientos de fertilizaci n Facultad de Agronom a Universidad Cat lica de Valpara so Chile 95 p Hue N V and Fox R 2010 Predicting plant phosphorus requirements for Hawaii soils using a combination of phosphorus sorption isotherms and chemical extraction methods Communications in Soil Science and Plant Analysis 41 133 143 Hylton K 2008 Visi n global del mercado de fertilizantes pp 7 18 En Jim nez F ed Actualizaci n en fertilizaci n de cultivos y uso de fertilizantes Sociedad Colombiana de la Ciencia del Suelo Bogot 236 p INFOAGRO 2006 El cultivo del aguacate En http www infoagro cons frutas frutas tropicales aguacate htm 11 p consulta septiembre 2007 Instituto Geogr fico Agustin Codazzi IGAC 1988 Suelos y bosques de Colombia Subdirecci n Agrol gica Bogot 127 p Jaramillo S and N W Osorio 2009 Mycorrhizal dependency of coffee seedlings at three levels of soil solution phosphorus Suelos Ecuatoriales 39 1 143 147 Jaramillo S P J M Silva y N W Osorio 2004 Potencial simbi tico y efectividad de hongos micorrizo 6655 arbusculares de tres suelos sometidos a diferentes usos Revista Facultad Nacional de Agronom a Medell n 57 1 22
8. mg kg potassium nitrate 380 mg kg and magnesium sulfate 3 280 mg kg Then KH PO was applied in order to have three concentrations of soluble P in the soil solution 0 002 0 02 y 0 2 mg L The amount of P added was determined through a soil P sorption isotherm Fox and Kamprath 1970 Afterward the soil was inoculated with 75 g of the crude inoculun of G fasciculatum which contained 43 infective propagules most probable number Porter 1979 The uninoculated soil received 75 g of autoclaved inoculum and 20 mL of washings of the crude inoculum after removing AMF propagules by filtration with Whatman No 1 filter paper Two months old seedlings of avocado cv Villagorgona were obtained from Profrutales a commercial nursery at Palmira Colombia and transplanted into the treated soils according to treatments Seedlings were grown for 150 days under greenhouse conditions and watered if required to maintain 50 60 of the maximal water hold capacity Every two weeks 25 mL of the P free Hoagland solution were added per pot The experimental design was completely randomized treatments had a factorial arrangement 3x2 three levels of soil soluble P and two level of mycorrhizal inoculation inoculated and uninoculated Each treatment had five replicates Response variables were shoot dry weight oven dried for 96 h at 60 C foliar P concentration measured in the foliar ashes 500 C 3 h by the molybdate phosphate m
9. mg kg Juo and Fox 1977 These levels of soil P sorption are associated with volcanic ash soils Andisols and are comparable with other studies conducted in this area Osorio 2008 This very high P fixation is responsible for the high requirements of P fertilizers in these soils which is desirable to reduce by introducing other practices for instance i moderate liming to increase soil pH up to a range 5 5 6 0 ii use of organic amendments to reduce P sorption iii use of AMF and PSM to reduce the soil soluble P requirement iv improve placement of P fertilizers and v use of rock phosphates Havlin et al 1999 Effectiveness of AMF associated to avocado plants The AMF population in the rhizosphere had low diversity because this was dominated by three species Glomus fasciculatum present in 35 45 of the samples G fistulosum 20 30 and E colombiana 40 50 others morphotypes likely Glomus were less frequent and were not identified The origin of these AMF native or introduced was unclear however their presence has been registered Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological 2500 o El Pe ol Santa Rosa y 492 42In x 2244 4 2 20004 r 0 97 P o 15004 g 10004 2 O g y 554 23In x 1990 lt 500 r 0 99 0 T T 1 0 0 0 1 0 2 0 3 0 4 Soluble Pi mg L Figure 1 Isotherm of P sorption of two soils from the No
10. respective fungus Plants were grown during 45 days under greenhouse conditions and watered if required to maintain 50 60 of the maximal water hold capacity Each week 25 mL of the P free Hoagland solution were added The experimental design was completely randomized treatments consisted of the three inoculations and an uninoculated control Each treatment had three replicates Response variables were shoot dry weight oven dried for 96 h at 60 C shoot P content measured by the molybdate phosphate method developed by Murphy and Riley 1962 after obtain ashes from the shoot tissues 500 C 3 h and mycorrhizal colonization as explained above but in this case H O was not used and fucsin acid 0 15 was used for staining roots 6647 Data were subjected to analysis of variance and mean separation through the multiple range test of Duncan in both cases a significant P lt 0 05 was used Statistical analyses were conducted in the software Statgraphics Centurion version XV Effect of mycorrhizal inoculation on avocado plants To this experiment an Andisol volcanic ash soil from the Experimental Forestry Station Piedras Blancas Santa Elena Medell n was used The soil was air dried sieved lt 4 mm autoclaved 120 C 0 1 MPa 2 h and then transferred into plastic bags 5 6 kg per bag Soil pH was adjusted to 6 0 with calcium oxide CaO additionally the following compounds were added ammonium nitrate 173
11. 03 2214 Joergensen R G and F Wichern 2008 Quantitative assessment of the fungal contribution to microbial tissue in soil Soil Biology and Biochemistry 40 12 2977 2991 Juo A S R and R L Fox 1977 Phosphate sorption characteristics of some bench mark soils of West Africa Soil Science 124 6 370 376 Melo Y P 2011 Respuesta de la inoculaci n de micorrizas en pl ntulas de aguacate Persea americana Mill variedad Hass en diferentes sustratos Tesis Maestr a Universidad Nacional de Colombia Palmira 136 p Menge J R Davis E Johnson and G Zentmyer 1978 Mycorrhizal fungi increase growth and reduce transplant injury in avocado California Agriculture 32 4 6 7 Miyasaka S and M Habte 2001 Plant mechanisms and mycorrhizal symbioses to increase phosphorus uptake efficiency Communications in Soil Science and Plant Analysis 32 7 8 1101 1147 Monta ez B I 2009 Efecto de la micorrizaci n en plantas de aguacate Persea americana L durante la fase de vivero en suelos provenientes de los Llanos Orientales Tesis Maestr a Universidad Nacional de Colombia Bogot 145 p Montoya B 2007 Determinaci n de la dependencia micorrizal del aguacate Tesis Magister en Geomorfolog a y Suelos Universidad Nacional de Colombia Medell n Msolla M M J M R Semoka and O K Borggaard 2005 Hard Minjingu phosphate rock An alternative P source for maize production on acid soils in Tanzania Nutrient Cyc
12. Fertility of Soils In http link springer com article 10 1007 2Fs00374 012 0737 7 15 p consulta noviembre 2012 Bernal J y C Diaz 2008 Tecnologia para el cultivo del aguacate Manual T cnico No 5 CORPOICA Rionegro 241 p Bolan N 1991 A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants Plant and Soil 134 189 293 Bola os M M y E P S enz 2010 Efecto de hongos ben ficos sobre la nutrici n y sanidad de aguacate http www corpoica org co http www corpoica org co sitioweb Archivos oferta ArticuloEfectoHongosbenficosnutaguacate pdf 10 p consulta noviembre 2012 Brundett M N Bougher B Dell T Grove and N Malajczuk 1996 Working with mycorrhizas in forestry and agriculture Australian Centre for International Agricultural Research Monograph No 32 374 p Cabrales E M y R Campo 2010 Hongos micorrizo arbusculares asociados a los principales cultivos de los suelos sulfatados cidos de C rdoba Colombia Suelos Ecuatoriales 40 1 57 61 Cancio M A G L Madrid R O Campo y E M Combatt 2005 Efecto del encalamiento en la diversidad de micorrizas vesiculo arbusculares y nem todos asociados al ma z en suelos sulfatados cidos en el departamento de C rdoba Colombia Suelos Ecuatoriales 35 2 24 27 Cano C A 1996 Manejo de un banco de germoplasma de hongos formadores de micorriza arbuscular MA pp 125 143 En Guerrero E ed Micor
13. Use of Soil Microorganisms as a Biotechnological Strategy to Enhance Avocado Persea americana Plant Phosphate Uptake and Growth Uso de Microorganismos del Suelo como Estrategia Biotecnol gica para Mejorar la Absorci n de F sforo y el Crecimiento de Plantas de Aguacate Persea americana Nelson Walter Osorio Vega Sandra Luz Serna G mez and Beatriz Elena Montoya Restrepo Abstract In recent years the area cultivated with avocado has increased significantly in Colombia and this trend is expected to continue in the coming years However most of the soils in the highlands of the Colombian Andean that have been used to grow avocado are strongly acidic and nutrient deficient particularly phosphorus P To alleviate these problems high rates of lime and P fertilizers are frequently applied in many cases the applied dose are excessive This increases production costs produces plant nutrient imbalance in the plant and increases the risk of polluting surface waters A biotechnological alternative is the combined use of phosphate solubilizing microorganisms PSM and arbuscular mycorhizal fungi AMF This work describes a series of experiments carried out to isolate characterize and evaluate the effectiveness of AMF and PSM in increasing plant growth and P uptake of avocado plants The results indicate that the mycorrhizal association is an effective alternative that can easily be established in the crops however the excessive use of
14. as higher when some biocontrol agents such as Trichoderma Paecilomyces or Pseudomonas fluorescent were co inoculated with the AMF The use of PSM can enhance the soil P availability by dissolving native or applied P compounds particularly rock phosphates RP Omar 1998 Osorio and Habte 2009 The advantage of using RP is that they are locally available in many countries including Colombia and are cheaper than P soluble fertilizers unfortunately their low solubility discourage their use but this can corrected by effective PSM Msolla 6646 Osorio N W Serna S L Montoya B E et al 2005 Ojo et a 2007 This strategy has been successfully proved by several authors in many plant species and soils Sreenivasa and Krishnaraj 1992 Whitelaw 2000 Osorio and Habte 2001 Barea et al 2005a 2005b but in Andisols this may have a limited efficiency because the P released can be retained by soil minerals Bolan 1991 To correct this limitation the co inoculation with PSM and AMF seems to be desirable because the mycorrhizal hyphae may absorb the released P by PSM and in this way improve plant P uptake Osorio and Habte 2009 However the excessive use of P fertilizers and their residual effects on soil P availability Serna et al 2012 may impair the benefits of AMF as reported in other crops e g coffee Coffee arabica lulo Solanum quitoense tomato Solanum lycopersicum Gonz lez 2006 Montoya 2007
15. ate value of PSM population at the rhizosphere Second the presence of these microorganisms is not a guarantee to improve soil P availability since either competition antagonism among microbes and or the soil P sorption capacity may limit the effectiveness of them to enhance plant P uptake Osorio 2011 Then their presence in the rhizosphere only indicates that their potential role in enhancing soil P availability may occur on the other hand their absence certainly indicates that the microbial solubilization of P will 6652 not happen Even so we should not forget that the counts conducted only detect those rhizospheric PSM cultivable and all discussion just made here is irrelevant Thus in 39 of the soil samples studied where PSM were not detected the mechanism of microbial P solubilization perhaps does not occur In the rest of the samples 61 PSM were found and likely they contribute to enhance soil P availability to plant roots Some other factors might help to PSM effectiveness such as the mycorrhizal association because the fungal hyphae would absorb the P released Rev Fac Nal Agr Medell n 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological by PSM In addition the presence of NH favors the dissolution of RP while NO is inhibitory Osorio 2008 In vitro test for PSM efficiency The in vitro test allow us to detect that some microorganisms produced acidity in the medium and thus red
16. bia Medell n 158 p Gonz lez O y N W Osorio 2005 Evaluaci n de la efectividad de in culos micorrizales Suelos Ecuatoriales 35 2 19 23 Gonz lez O A y N W Osorio 2008 Determinaci n de la dependencia micorrizal del lulo Acta Biol gica Colombiana 13 2 63 174 Guerrero E 1996 Micorriza Fundamentos y estado de arte pp 3 46 En Guerrero E ed Micorrizas Recurso Biol gico del Suelo Fondo FEN Bogot 208 p Guerrero E C Rivillas y E L Rivera 1996 Perspectivas de manejo de la micorriza arbuscular en ecosistemas tropicales pp 181 206 En Guerrero E ed Micorrizas Recurso Biol gico del Suelo Fondo FEN Bogot 208 p Habte M and A Manjunath 1987 Soil solution phosphorus status and mycorrhizal dependency in Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 Leucaena leucocephala Applied and Environmental Microbiology 53 797 801 Habte M and N W Osorio 2001 Arbuscular Mycorrhizas Producing and applying Arbuscular mycorrhizal inoculum University of Hawaii Honolulu 47 p Hamdali H A Smirnov C Esnault Y Ouhdouch and M J Virolle 2010 Physiological studies and comparative analysis of rock phosphate solubilization abilities of actinomycetales originating from Moroccan phosphate mines and of Streptomyces lividans Applied Soil Ecology 44 1 24 31 Havlin J J Beaton S L Tisdale and W Nelson 1999 Soil fertility and fertilizers An introduction to nutrient
17. e vivero Suelos Ecuatoriales 35 5 11 Serna S L B Montoya y N W Osorio 2012 Monitoreo del pH y f sforo soluble en la rizosfera de rboles de aguacate en el Norte y Oriente Antioque o Suelos Ecuatoriales 42 2 93 97 Sierra J A 2006 Dependencia micorrizal de cuatro especies forestales y efectividad micorrizal de Andisoles en funci n de su uso y manejo Tesis Magister en Bosques y Conservaci n Ambiental Universidad Nacional de Colombia Medell n 84 p Phillips J M and D S Hayman 1970 Improved procedures for clearing and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection Transactions of the British Mycological Society 55 158 161 Porter W 1979 The Most Probable Number method for ennumerating infective propagules of vesicular arbuscular micorrizal fungi in soil Australian Journal of Soil Research 17 515 519 Smith S and D Read 1997 Mycorrhizal Symbiosis Academic Press London UK 605 p Smith S E FA Smith and I Jakobsen 2003 Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses Plant Physiology 133 1 16 20 Smith S E I Jakobsen M Gr nlund and F A Smith 2011 Roles of arbuscular mycorrhizas in plant Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 phosphorus nutrition interactions between pathways of phosphorus uptake in arbuscular mycorrhizal roots have important implications for under
18. ethod Murphy and Riley 1962 and mycorrhizal colonization measured as explained above Data were subjected to analysis of variance and mean separation through the LSD test in both cases a 6648 Osorio N W Serna S L Montoya B E significant P lt 0 05 was used Statistical analyses were conducted in the software Statgraphics Centurion version XV In vitro test for PSM efficiency Selected colonies of PSM were purified with a sterile loop and aseptically transferred by duplicates into a 250 mL Erlenmeyer flask that contained the medium for PSM agar free Osorio and Habte 2001 The flasks were continuously shaken in a reciprocal shaker at 150 rpm 25 C for 7 days for comparison an uninoculated control was included At the end of the incubation period the medium pH was measured by introducing directly a Ht selective electrode Also the concentration of P in the medium was measured by the molybdate phosphate method for this purpose the solution was removed by centrifugation for 15 min at 2 500 x g and the uppernatant was filtered by a Whatman No 43 filter paper 2 um of aperture size RESULTS Soil P sorption isotherm The value of P amount of P mg kg required to achieve soil solution P concentration of 0 2 mg Lt Hue and Fox 2010 was 1 098 and 1 452 mg kg for Santa Rosa and El Pe ol soils respectively Figure 1 These results allow classify the soil P sorption capacity as very high P0 2 gt 1000
19. ght was significantly reduced by 26 These results probed that AMF inoculation is highly promissory to enhance plant performance on avocado concentration of P in the soil solution For instance at plantations in soils with high P fixation as those 75 a AMF 2 S a D 2 45 a v 5 z E 15 0 0 001 0 01 0 1 1 Soil Soluble Pi mg L Figure 3 Shoot dry weight of avocado plants 150 days after inoculation with G fasciculatum AMF in comparison to uninoculated plants AMF at three levels of soil solution P concentration Bars indicate the value of LSD P lt 0 05 AMF 0 002 AMF 0 002 AMF 0 02 Figure 4 General view of uninoculated AMF and inoculated plants with G fasciculatum AMF grown at three levels of soil solution P concentrations 0 002 0 02 and 0 2 mg L 150 days after transplanting Note the increase in plant growth with the mycorrhizal inoculation at the lowest and medium level of soil P and the decrease at the highest level 6650 Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological considered in this study e g Santa Rosa and El Pe ol The use of these fungi may help to reduce the amount of P fertilizers and in this way reduce the production costs The combination of AMF and RP application seems to be very attractive to evaluate in avocado plants since positive results have been observed in other soils a
20. gt 0 2 mg Lt This increases in production costs raises environmental concerns and creates plant nutrient imbalance A valid strategy is the use of arbuscular mycorrizal fungi AMF in this 6653 study the fungus Glomus fasciculatum was capable of increasing avocado plant growth at low levels of soil soluble P 0 002 y 0 02 mg L but the effect became negative at high levels 0 2 mg L The use of phosphate solubilizing microorganisms PSM is quite attractive given its potential to dissolve rock phosphate and native soil P compounds However the excessive levels of soil soluble P detected in avocado plantations threaten the proper functioning of the mycorrhizal association in many cases this association was absent of the plant system The counts of PSM in the rhizosphere of avocado were quite variable in some cases they were not detected and those isolated were ineffective in dissolving RP ACKNOWLEDGEMENT This work was financially supported by the Direction of Research of the Universidad Nacional de Colombia We want to thank the criticism of the reviewers whose contributions improved the document We also thank Claudia Alvarez for reading the final version BIBLIOGRAPHY Allen M 1996 The ecology of arbuscular mycorrhizas a look back into the 20 th century and a peek into the 21st Mycological Research 100 7 769 782 ASOHOFRUCOL FNFH 2005 Convocatoria para el proyecto Programa de desarrollo de proveedores Im
21. he presence of yellow halos around the colonies that indicate the acidity production Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 6651 but at higher concentrations it decreases Montoya 2007 Recently in the same samples studied here Serna et al 2012 found that more than 60 of the soil samples had soil soluble P gt 0 2 mg L Under such conditions the presence of AMF in the roots may have negative effects on plants This indicates that to determine the convenience of the mycorrhizal Osorio N W Serna S L Montoya B E inoculation in avocado plantations it is necessary to monitor the levels of soil P availability as well as the presence of infective mycorrhizal propagules in the soil Sierra 2006 PSM in the avocado rhizosphere The interpretation of PSM plate counts from the rhizosphere has some 50 4 40 gt 305 oO 5 O oO l j il 25 50 50 75 gt 75 oe of mycorrhizal colonization 50 gt 40 38 6 B 32 9 S 30 Q o 3 20 11 4 10 8 6 i 1 4 0 El o Ln Oo LN o Ln Koj N Ln tS Oo N N wo N 00 Ln m 4 T T o p Y wn Oo Ww o LN N wn Oo N wo N co wn H a rc N m Range for PSM x 10 UFC g Figure 6 Histograms of frequency for root mycorrhizal colonization above and counts of potential PSM in the rhizosphere below of avocado at the North and East of Antioquia Colombia n 70 limitations First there is no certainty about which is the adequ
22. l their use Frequency histograms were prepared for both variables mycorrhizal colonization and rhizosphere PSM Identification of AMF spores Spores of AMF were isolated from the 20 rhizosphere samples following the method described by Habte and Osorio 2001 Briefly this consisted of wet sieving aliquots 5 g dry basis of rhizosphere samples in nested series of sieves 500 250 100 and 50 um of aperture pore The AMF spores were collected from the last two sieves and observed under the microscopy for their morphological identification following the guides of INVAM www invam caf wvu edu Test for the effectiveness of AMF Crude inocula of these AMF species were taken from the collection of the LECA and multiplied separately during four months in a sterile substrate soil sand 2 1 proportion autoclaved 120 C 0 1 MPa 2 h with corn Zea mays and brachiaria grass Brachiaria decumbens as host plants To evaluate the effectiveness of the inocula the method developed by Habte and Manjunath 1987 was used in this case the host plant Leucaena leucocephala was used as indicator plant given its very high mycorrhizal dependency and fast growth These plants were grown in plastic pots 900 mL that contained soil pH 6 0 and soluble P concentration of 0 02 mg Lt and were separately inoculated with each crude inoculum spores hyphae infected roots in a soil sand substrate with 25 g pot each gram had 250 270 spores of the
23. ling in Agroecosystems 72 299 308 Murphy J and J P Riley 1962 A modified single solution method for the determination of phosphate in natural waters Analytica Chimica Acta 27 31 36 Oberson A E K Bunemann D K Friesen I M Rao and P C Smithson 2006 Improving phosphorus fertility in tropical soils through biological interventions pp 531 546 In Uphoff N ed Biological approaches to sustainable soil systems CRC Press Boca Raton Fl 764 p 6656 Osorio N W Serna S L Montoya B E Ojo O D A A Kintomo E A Akinride and M O Akoroda 2007 Comparative effect of phosphorus sources for grain amaranth production Communications in Soil Science and Plant Analysis 38 35 55 Olsen S R and L E Sommers 1982 Phosphorus pp 403 430 In A L Page and D R Keeney eds Methods of soil analysis Part 2 Chemical and Microbiological Properties American Society of Agronomy and Soil Science Society of America Madison WI 1159 p Omar S A 1998 The role of rock phosphate solubilizing fungi and vesicular arbuscular mycorrhiza VAM in growth of wheat plants fertilized with rock phosphate World Journal of Microbiology and Biotechnology 14 211 218 Osorio N W and M Habte 2009 Strategies for utilizing arbuscular mycorrhizal fungi and phosphate solubilizing microorganisms for enhanced phosphate uptake and growth of plants in the soils of the tropics pp 325 351 In Khan M S A Zaidi and J Mu
24. nd crops Sreenivasa and Krishnaraj 1992 Omar 1998 Barea et al 2005a 2005b Osorio 2008 Although AMF do not dissolve RP they can absorb more efficiently the P release from the RP than the non mycorrhizal roots Bolan 1991 Smith et al 2003 Smith et al 2011 When AMF take P up reduce the concentration of Pi in the soil solution favors the RP dissolution In addition the mycorrhizal association may promote the activity ofthe rhizosphere microorganisms capable of P dissolution PSM Barea et a 2005a 2005b and enhance plant P uptake by plant roots and or mycorrhizal hypha Whitelaw 2000 Osorio and Habte 2009 Since the soil P availability can control the effects from positive to negative of AMF inoculation Figure 3 it is worrisome that the avocado growers apply high rates of P fertilizers with the consequent effect on soil soluble P levels Serna et al 2012 found in the same soil samples used here that the soil soluble P ranged from 0 01 to 1 78 mg L 47 of the rhizospheric soil samples exhibited levels of soluble P excessively high 0 25 1 69 mg L According to our results it is logic to expect that native or applied AMF would reduce plant growth under these conditions In order to avoid these problems it is recommendable to monitor the level of soluble P and mycorrhizal colonization of plant roots before making decisions on AMF inoculation It must be clarified that the consideration made here are
25. plementaci n y certificaci n de buenas pr cticas agr colas en fincas productoras de frutales de clima fr o exportables en el departamento de Cundinamarca Bogot Mayo 2005 Barber S 1995 Soil nutrient bioavailability a mechanistic approach Willey Interscience New York 398 p Barea J M R Azc n and C Azc n 2005a Interaction between mycorrhizal fungi and bacteria to improve plant nutrient cycling and soil structure pp 195 212 In Buscot F and A Varma eds Microorganisms in soils roles in genesis and functions Springer Verlag Heidelberg 419 p Barea J M D Werner C Azc n and R Azc n 2005b Interaction of arbuscular mycorrhiza and nitrogen fixing simbiosis in sustainable agricultura In Werner D and W E Newton eds Nitrogen Fixation in 6654 Osorio N W Serna S L Montoya B E Agriculture forestry Ecology and the Environment Kluwer Academic Publishers Netherlands 349 p Bashan Y and G Holguin 1998 Proposal for the division of plant growth promoting rhizobacteria into two classifications biocontrol PGPB plant growth promoting bacteria and PGPB Soil Biology and Biochemistry 30 9 1225 1228 Bashan Y A A Kamnev and de Bashan L E 2012 Tricalcium phosphate is inappropriate as a universal selection factor for isolating and testing phosphate solubilizing bacteria that enhance plant growth a proposal for an alternative procedure Biology and
26. rizas Recurso Biol gico del Suelo Fondo FEN Bogot 208 p Rev Fac Nal Agr Medell n 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological Daza P C y N W Osorio 2011 Promoci n de crecimiento y absorci n de f sforo de pl ntulas de Leucaena por un hongo micorrizal en un suelo degradado por miner a de aluvi n Suelos Ecuatoriales 41 2 144 149 D az D C O L Hern ndez L M Z rate y J F Garc a 2011 Efecto de la aplicaci n de diferentes fertilizantes sobre la actividad micorrizal en gulupa Suelos Ecuatoriales 41 2 115 121 FONTAGRO 2006 M todos no destructivos y marcadores moleculares para la determinaci n de fechas apropiadas de cosecha de cinco variedades de aguacate Fondo Regional de Tecnolog a Agropecuaria Proyecto FTG 24 01 En http www fontagro org en convocatorias convocatoria 2001 propuestas formales mY C3 A9todos no destructivos y marcadores moleculares Consulta abril 2010 Fox R and E Kamprath 1970 Phosphate sorption isotherms for evaluating the phosphate requirements of soils Soil Science Society of America Journal 34 6 902 907 Giovannetti M and B Mosse 1980 An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots New Phytologist 84 489 500 Gonz lez O 2006 Determinaci n de la dependencia micorrizal de frutales de clima frio Tesis Magister en Geomorfolog a y Suelos Universidad Nacional de Colom
27. rth Santa Rosa an East El Pe ol Antioquia Colombia Santa Rosa P 1 098 mg kg El Pe ol P 1 452 mg kg in other soils and crops of Colombia Guerrero 1996 On the other hand the evaluation of these AMF from inocula Guerrero et al 1996 Cano 1996 Cancio et al 2005 allows us to separate them according to their effectiveness for Salamanca and Cano 2005 Jaramillo and Osorio enhancing plant performance Figure 2 The results showed 2009 Cabrales and Campo 2010 Daza and Osorio that the inoculum of G fasciculatum was more effective in 2011 D az et al 2011 Furthermore these AMF enhancing Leucaena plant P uptake and growth than the other species are commonly used in commercial mycorrhizal AMF inocula studied The increase in shoot dry weight with G inocula frequently applied at the Northern and Eastern fasciculatum inoculation respect to uninoculated plants was Antioquia regions for avocado plantations of 75 while the shoot P concentration was 3 75 fold higher 3 0 2 5 Shoot dry weight g plant Shoot P content mg plant in Control G fasciculatum G fistulosum E colombiana Control G fasciculatum G fistulosum E colombiana Figure 2 Mycorrhizal effectiveness of three inocula measured through the effects on Leucaena shoot dry weight SDW mg plant and shoot P content mg plant in comparison to the uninoculated plants control Different letters over the columns indicate significant differences Duncan te
28. rypan blue 0 15 dissolved in glycerol and lactic acid were added and the roots were left at room temperature for 48 h for staining Brundrett et a 1996 Next the excess of trypan blue was removed by glycerol and lactic acid for 48 h Root colonization was measured by the grid line intercept method Giovanneti and Mosse 1980 Rhizosphere PSM Once in the laboratory rhizosphere samples were hand dispersed in order to destroy soil aggregates Serial dilutions 107 10 were prepared with a 0 01 M CaCl solution Aliquots of 100 uL were aseptically transferred into Petri dishes that contained a sterile 120 C 0 1 MPa 40 min selective medium for PSM Osorio and Habte 2001 The medium contained per liter 1 0 g of NaCl 0 2 g of CaCl 2H O 0 4 g of MgSO 7H O 1 0 g of NH NO 10 g of glucose 15 g of Agar 3 5 g of Huila rock phosphate and 5 mL of bromothymol blue as a pH indicator Petri dishes were incubated at 28 C for three to five days Colony forming units were counted and some morphotypes were preliminary selected for further studies based on the presence of yellow halo around the colony abundance and growth After that an in vitro test Osorio 2011 was conducted to quantify the capacity of the isolates to dissolve Huila Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 RP as explain above Selected microorganisms were transferred to Petri dishes with the same medium for purification and conservation at 4 C unti
29. sarrat ed Microbial strategies for crop improvement Springer Verlag Berlin 358 p Osorio N W 2008 Effectiveness of microbial solubilization of phosphate in enhancing plant phosphate uptake in tropical soils and assessment of the mechanisms of solubilization Thesis Ph D University of Hawaii Manoa 391 p Osorio N W 2011 Effectiveness of phosphate solubilizing microorganism in increasing plant phosphate uptake and growth in tropical soils pp 65 80 In Maheshwari D K ed Bacteria in agrobiology Plant nutrient management Springer Verlag Berlin 345 p Osorio N W 2012 Manejo de Nutrientes en Suelos del Tr pico Universidad Nacional de Colombia Medellin 339 p Osorio N W and M Habte 2001 Synergistic influence of an arbuscular mycorrhizal fungus and P solubilizing fungus on growth and plant P uptake of Leucaena leucocephala in an Oxisol Arid Land Research and Management 15 263 274 Posada R H M S nchez E Sieverding K Aguilar y G P Heredia 2012 Relaciones entre los hongos filamentosos y solubilizadores de fosfatos con algunas variables ed ficas y el manejo de cafetales Revista de Biolog a Tropical 60 3 1075 1096 Rev Fac Nal Agr Medell n 65 2 6645 6657 2012 Use of soil microorganisms as a biotechnological Salamanca C R y C A Cano 2005 Efecto de las micorrizas y el sustrato en el crecimiento vegetativo y nutrici n de cuatro especies frutales y una forestal en fase d
30. st P lt 0 05 and the shoot P content was 6 8 fold higher Although the The technique used was very simple and useful to benefits on plant performance with G fistulosum inoculation evaluate inoculum quality as illustrated before by were lower than with G fasciculatum the shoot dry weight Gonz lez and Osorio 2005 as well as to determine P concentration and P content were 72 3 fold and 5 32 the mycorrhizal effectiveness of native AMF in soils fold significantly higher than in the uninoculated plants By Jaramillo et al 2004 Sierra 2006 However the contrast the inoculum of colombiana was ineffective to results should be associate to the quality of a particular enhance plant performance inoculum AMF spores number and viability and other Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 6649 infective propagules and not to the AMF species As a conclusion the crude inoculum of G fasciculatum was the most effective and therefore used in further studies Effectiveness of G fasciculatum on avocado plants The results showed that the inoculation with this fungus significantly P lt 0 05 increased avocado plant growth Figures 3 and 4 However this effect depended on the Osorio N W Serna S L Montoya B E the level 0 002 mg L the increased on plant growth was by 48 at 0 02 mg L this was by 35 By contrast at the level of 0 2 mg L the plant response was negative to the inoculation because the shoot dry wei
31. standing and manipulating plant phosphorus acquisition Plant Physiology 156 3 1050 1057 Sreenivasa M and P U Krishnaraj 1992 Synergistic interaction between VA mycorrhizal fungi and a phosphate solubilizing bacterium in chili Capsicum annuum Zentralblatt f r Mikrobiologie 147 1 2 126 130 Thies J and J M Grossman 2006 The soil habitat and soil ecology pp 59 78 In Uphoff N ed Biological approaches to sustainable soil systems CRC Press Boca Raton FL 764 p Welch S A E Taunton and J F Banfiled 2002 Effect of microorganisms and microbial metabolites on apatite dissolution Geomicrobiology Journal 19 343 367 Whitelaw M A 2000 Growth promotion of plants inoculated with phosphate solubilizing fungi Advances in Agronomy 69 99 151 Yusdar H A R Anuar M M Hanafi and H Azizah 2007 Analysis of phosphate rock dissolution determining factors using principal component analysis in some acid Indonesian soils Communication in Soil Science and Plant Analysis 38 1 2 273 282 Zaidi A M S Khan M Ahemad M Oves and P A Wani 2009 Recent advances in plant growth promotion by phosphate solubilizing microbes pp 23 50 In Khan M S A Zaidi and J Musarrat ed Microbial strategies for crop improvement Springer Verlag Berlin 358 p 6657
32. ucing pH down to 3 1 Figure 7 which is required to dissolve RP Welch et al 2002 Oberson et al 2006 Yusdar et al 2007 Hamdali et al 2010 Bashan et al 2012 Despite that the concentration of P in solution was relatively low if compared with other effective PSM s Aspergillus niger and Mortierella sp used with the same test Osorio and Habte 2009 Zaidi et al 2009 Bashan et al 2012 at pH of 3 1 a value of soluble P around 15 mg L is 3 5 3 0 2 5 ul Pi in solution mg L o ul expected Osorio 2012 however the value found was only 3 2 mg L although this level was significantly higher than that in the uninoculated control clearly it suggests a low effectiveness Osorio 2008 Likely the microbes did solubilize RP but most of it was immobilize in their cells This transformation of P from insoluble RP toward microbial P is not necessarily unfavorable for plants because this pool is later subjected to decomposition by other microorganisms leaving P available for root uptake Joergensen and Wichern 2008 Posada et al 2012 The lack of effective PSM s in the rhizosphere of avocado plants lead us to consider the use of effective PSM obtained from other plant rhizosphere 2 0 1 I 0 0 d i 22 41 i 45 I 31 26 Control Isolates Figure 7 Concentration of soluble P in a culture medium as a function of the inoculation with rhizosphere microorganisms in comparison with a
33. uninoculated control The number of the isolate is an internal code This approach is valid because the microbial solubilization of P compunds does not require a specific relationship between plant microbe as other interactions e g legume rhizobio In this way the relationship plant PSM is quite similar to that observed between plant PGPR Bashan and Holguin 1998 It has been proposed that an effective PSM must be i a good competitor for resources in the rhizosphere especially carbonaceous compounds ii efficient colonizer of the rhizosphere and or the rhizoplane and iii an active producer of organic acids di and tri carboxylic acids such as oxalic acid and citric acid Osorio 2011 Bashan et al 2012 Also the PSM effectiveness will be higher in the presence of mycorrhizal plants particularly if they grow in soils with high P sorption capacity as those considered in this study Rev Fac Nal Agr Medellin 65 2 6645 6657 2012 CONCLUSIONS In general the soils from the North and East Antioquia exhibited a very high P sorption capacity P0 2 gt 1 000 mg kg which is responsible of the low soil P availability observed in these soils under natural conditions soil soluble P lt 0 05 mg L limiting the plant productivity To prevent plant P deficiency avocado growers apply high rates of soluble P fertilizers in many cases the amount applied is excessive and thus soil soluble P increase at levels unnecessarily high
34. uso de fertilizantes fosf ricos puede limitar los beneficios de estos hongos en las plantas de aguacate Hasta ahora no se han detectado PSM efectivos en la rizosfera de aguacate En algunos casos estos microorganismos est n ausentes en la rizosfera del aguacate y en otras circunstancias la baja capacidad para solubilizar P desestimula su uso en el cultivo Palabras clave Micorriza arbuscular microorganismos solubilizadores de fosfato Andisoles fijaci n de f sforo Currently the cultivation of avocado Persea americana in Colombia has received much attention due to the high demand for this fruit in national and international markets Bernal y Diaz 2008 The Colombian government has planned to increase the area with avocado plantations from the current 16 220 ha to 29 600 ha in the year 2020 Also the mean yield is expected to increase from 11 2 t ha to 13 54 t ha although the potential yield estimated is 30 t ha Bernal and Diaz 2008 In order to reach international markets good agricultural practices must be adopted FONTAGRO 2006 However the intensive use of agrochemicals is a serious limit to satisfy this objective ASOHOFRUCOL FNFH 2005 INFOAGRO 2006 A constraint for agricultural production in the Andean region where avocado is intensively cultivated is the low soil phosphate P availability IGAC 1988 Gonzalez and Osorio 2008 This occurs due to the high P sorption capacity of these soils
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