The introduction of VAMF into agricultural systems benefits the growth and the health of the crops, especially in abandoned areas with poor soils. As a pre-requisite for the application of the symbiots, the fungi must be isolate and selected with respect to their efficiency for the plants and their adaptability to the environmental conditions in the field. Different methods of isolation and the selection procedure are described. The mass production of the selected fungi can be carried out in the greenhouse or in the field. The inoculation of the plants with the VAMF is possible in the nurseries as well as in the field. Two methods of application - inoculation of the plants with a homogenous mixture of the inoculum in the substrate and with a concentrated inoculum layer respectively - are presented

FELDMANN, F.

IDCZAK, E.

MACEDO, J. L. V. de

MULLER, I.

WERITZ, J.

English

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~I~A ~~~o·'dó~I~~MANAGEMENT AND REHABILITATION OFDEGRADED LANDS AND SECONDARY FORESTS.IN AMAZONIA:EMBR t:PA - CPAABibliot&caAn Intemational Symposium/WorkshopApril18-22, 1993Hotel TropicalSantarém, Pará - BrazilOrganized by:Intemational Institute of Tropical ForestryUSDA - Forest ServiceSouthem Forest Experiment StationRio Piedras, Puerto Rico, USAEMBRAPA/CPATUCentro de Pesquisa Agroflorestal da Amazônia OrientalBelém, Pará, BrasilIn Collaboration with:Instituto Brasileiro do Meio Ambiente edos Recursos Naturais Renováveis - IBAMASantarém, Pará, BrasilWith Support from:Tropical Forestry ProgramIntemational ForestryU.S. Department of Agriculture - Forest ServiceWashington, DC, USAandMan and the Biosphere Programme (MAB)UNESCOParis, FranceISOLATION, SELECTION AND PRODUCTION OF VESICULAR-ARBUSCULARMYCORRHIZAL FUNGI (VAMF) AND THEIR APPLICATION IN MIXEDCROPPING SYSTEMSF. Feldmannl, I. MüIler1, J. Weritz2, J.L.V. Macedo-, E. IdczakllUniversitat Hamburg, Institut for Angewandte Botanik, Hamburg, Germany2Biologische Bundesanstalt, Institut for Pflanzenschutz im Gartenbau, Braunschweig, Germany3Centro de Pesquisa Agroflorestal da Amazônia Ocidental, CPAA/EMBRAPA, AM, BrazilSUMMARYThe introduction of vesicular-arbuscular mycorrhizal fungi (VAMF)into agricultural systems benefits thegrowth and the health of the crops, especially in abandoned areas with poor soils. As a prerequisite for theapplication of the symbionts, the fungi must be isolated and selected with respect to their efficiency for theplants and their adaptability to the environmental conditions in the field. Different methods of isolation andthe selection procedure are described. The mass production of the selected fungi can be carried out in thegreenhouse or in the field. The inoculation of the plants with the VAMFis possible in the nurseries as wellas in the field. Two methods of application - inoculation of the plants with a homogeneous mixture of theinoculum in the substrate and with a concentrated inoculum layer respectively - are presented.INTRODUCTIONIn tropical habitats the benefits of vesicular-arbuscular mycorrhizal fungi (VAMF)to their hostplants are of special importance. By means of im-proved nutrient supply the fungi cause significantgrowth enhancement of the plants in the poor soilsof the tropics (5ieverding 1987;Lin 1986). Further-more, the colonization of roots by mycorrhizal fungiincreases the resistance of the host plants against rootpathogens (Zambolim 1986)and reduces the sever-ity of foliar disease (Feldmann 1991). The toleranceof mycorrhizal plants against stress (e.g. extremetemperatures, high humidity or drought and trans-planting) is generally higher compared to non-myc-orrhizal plants (Schonbeck 1987;Müller 1991). Inagricultural systems a high deficiency of thesymbiotical fungi has been observed (Feldmann1991). To regain the "vitality of the soil" with re-spect to VAMF,the application of the fungi at thetime of sowing or planting or even earlier in thenurseries is necessary. In Manaus, Amazonas 5tate,a mixed cropping system of perennial plants hasbeen installed in which the management takes ac-count of the requirements of the fungi (less fertiliza-tion, less pesticides, more secondary vegetation).Theplantation has been established in an abandonedarea (former rubber plantation). To improve the"ecological fitness" of the plants, they have beeninoculated with VAMFin the nursery or at the timeof planting into the field.50 far the use of VAMFin plant production sys-tems and agriculture is not a common practice. Thisis mainly due to the difficulties involved in the iso-lation, selection, multiplication and applicationof the VAMF. The purpose of this paper is todemonstrate the methods from the first step of iso-lation of the mycorrhizal fungi to the final step ofapplication in the nurseries and in the field.ISOLATION OF VESICULAR-ARBUSCULARMYCORRHIZAL FUNGI (VAMF)VAMFcan be used in the form of:pure isolates derived from single or multiplespore cultures of one fungal speciesa mixture of defined pure isolatesa mixture of unknown VAMFspecies withproven stability of the population and itseffects.The establishment of pure isolates (1) and iso-lates of an unknown mixture of VAMF(2) is shownin Figure 1. In both cases the origin of the fungi isfield soil. Besides the living VAMFspores, field soiloften contains large numbers of dead spores of622! ISDlatiDn Df VAMF IOulllval8 hOSI planla for ""MFIn lha 8011( •. g. cor n) ,,,,,,,,, 2,,,,,,,•..,I1 i;ORemov. lha planla Irom Ih. eeuaflal 4-8 w•• k. end wuh lhaloota Iholoughly1 ,tTlansplanl Ih. myeorrhlzal planlsInlO coru atner a ftl18d wllh aporous sub'llsl.~llvala lha planls fOI 8 waaks1 ~ '22Lal Ih. planla dlY slowly In 2 E.Ilsel lha "'MF apol's 110mlha sube rr ere ................•.............• Ih. '011 by alavlng andL- -' cantrllugallonL- ~I Selection I1. + 2Inoculum ProductionFIGURE 1. Procedure of isolation of VAMP.(1) pure isolate, (2) isolate of an unknown mixture of VAME1 !Exllsel lhe "'MF sporea tromlhe substraleRapall lha a'apa d8serlb.dabove wlth • Ilngle apor.as lhe Inoculum SOUIC.mycorrhizal fungi. As it would be time consumingto establish single spore isolates directly from theoriginal soil sample, we prefer to enhance the num-ber of living spores by cultivation of a host plant(e.g. com, onion, bell pepper) in the soil. This willcause the propagation of the fungi in the substrate.A disadvantage of this method at increasing thenumber of living VAMF spores is the possibility ofpre-selection of those fungi which are more adapted.'""to the given growth conditions. To minimize thispre-selection, the use of different plant species andgrowth conditions is recommended. Four to eightweeks after sowing or planting, the plant roots willbe colonized by the VAMF. For the establishment ofpure isolates, the plants are taken from the substrateand the roots are rinsed thoroughly. To avoid con-tamination with root pathogens a surface steriliza-tion (e.g. with ethanol) of the roots can be an63advantage. The plants are then transplanted intoplastic sacks or other containers such as pots or boxescontaining a porous substrate. They are irrigatedmoderately and fertilized once a week with a lowphosphate fertilizer « 60ppm P). After a growthperiod of eight weeks, the plants are not wateredfurther, thus inducing the spore production of theVAMF. The spores can be removed from the sub-strate by the method of wet sieving and centrifuga-tion (Daniels and Skipper 1984; Feldmann 1991). Toestablish pure cultures, the procedure described isrepeated with a single spore as the inoculum source(single spore isolate) or with several spores of thesame fungal species (multiple spore isolate), whichis often problematic as it is hard to determinewhether similar spores belong to the same species.For the production of a VAMF-isolatecontain-ing an unknown mixture of VAMFspecies (2), sev-eral steps of the procedure described above can beomitted (see Figure 1). Furthermore, it is possible toextract the VAMFspores directly from the field soiland use them as the primary inoculum. Thismethodwill diminish the contamination with pathogenswhich rnight be present in the soil.Before the established VAMF-isolates can beused in nurseries or on an agricultural scale, theymust be selected with respect to their effectiveness.SELECTION OF EFFECTIVE VAMF-ISOLATESAlthough the VAMFare distributed worldwideand most of the plant species live in symbiosis withthese fungi, a specificity in the interaction betweenfungus and host plant exists (Estaun et al. 1987, Grawet aI. 1979, Idczak 1992). Specificity is revealed withrespect tothe host plant (plant species/ plant cultivar)the nature of the effect (which also dependson the environmental conditions).Therefore the selection of effective VAMFisolates isnecessary.THE SELECTION PROCEDUREList:* the nature of the effect(s) desired (e.g.plant growth enhancement, increase ofresistance against pathogens ...),* the given abiotic factors during the se-lection which should be similar to thefinal conditions under which the fungiwill be used (substrate, temperature,pH ...).Ensure the infectivity of the fungal inocu-lum which will be tested (check whether apotential host plant, e.g. com, is colonizedby the fungi).Determine whether the plant for which thefungal isolate is to be selected is mycotroph(Janos 1987; Feldmann 1991), i.e. testwhether the VAMFare able to colonize theroots and whether the mycorrhizal plantsbenefit from the symbiosis.If the plant is mycotroph, analyse the de-gree of mycorrhizal dependency (obligateor facultative dependency; Feldmann 1991).Determine again the abioticselectionfactors,considering the degree of dependency, theconditions in the area where the fungi willbe used and the kind of management.Compare the effectiveness of the isolateswith respect to the desired effect.In addition to the described steps in the selec-tion of effective VAMFisolates, there are other fac-tors which should be taken into account during theselection process, such as the competitive ability andpersistence of the fungi under natural conditions(Abbot et al. 1992). Once a VAMFisolate has beenchosen, inoculum production on a large scale is apre-requisite for the application in nurseries or inthe field.INOCULUM PRODUCTIONA detailed description of the production ofVAMFinoculum for use in the tropics has been pre-sented by Feldmann and Idczak (1992). Thereforeonly the main aspects of inoculum production aredescribed here.The inoculum production can be differentiatedinto an initial phase (Figure 2) and mass production(Figure 3).64A BcFIGURE 2. Initial phase of inoculum production(Feldmann & Idczak 1992). See text fordetails.BAFIGURE 3. Mass production of inoculum(Feldmann & Idczak 1992). See text for details.Initial phase:Fill two thirds of a pot (volume about 500ml) with quartz sand and layer the start in-oculum (soil containing the VAMF,infectedroot pieces or funga I spores) on topoSurface sterilize the seeds of a host plant (e.g.com or onion), rinse them with water after-wards to remove all pesticides, sow theminto the pots and cover them with sand.Water the pots moderately (the humidity ofthe substrate should be a little below fieldcapacity) and fertilize the plants with a lowphosphate fertilizer (P <60ppm, details seeFeldmann and Idczak 1992). Avoid the useof pesticides.After a growth period of three to four weeks,the roots of the host plants should be colo-nized by the VAME Control the root colo-nization with a microscope. In the event oflow infectivity of the start inoculum the cul-ture period can be extended by one to twoweeks.When the roots of the host plant are wellcolonized, separate the plants from eachother and wash the roots. Sometimes itmight be necessary to surface sterilizethe roots (70% ethanol, two minutes) toeliminate pathogenic microorganisms.The plants treated in this way are used forthe mass production of VAMFinoculum.Mass production:The mycorrhizal plants are transplanted intolarger pots (volume about 51)or comparablecontainers, which might be even larger, butstill allow the roots to explore the wholevolume (A). The use of expanded clay asthe substrate (method described by Dehneand Backhaus 1986)has various advantagessuch as its low weight, easy application andthe fact that contamination by pathogenscan be largely avoided and subsequent de-contamination is aIso possible. But othersubstrates like sterilized sandy soils can beused as well.65Irrigation and fertilization should be carriedout as described above (initial phase).After three to four months of plant growth,the roots should fill the whole volume of thecontainer. From this moment on, the plantsare no longer irrigated. The drought stressstimulates the spore production of theVAMEne week after termination of irrigation, theplants are cut off and the pots with the rootsare kept dry for another two weeks (B). Af-ter this period the roots are removed fromthe substrate.The substrate is spread out ín a thin layerand air-dried rapidly (C). The dry inocu-lum can be stored under cool and dryconditions for more than a year.Altematively to the procedure of inoculum pro-duction described above, the multiplication of theVAMFcan be carried out directly in the field as well(in-situ production). Feldmann (1991)showed thatthe inoculation of host plants (e.g. com) with VAMFin the field at the time of sowing or planting resultsin an increase of the number of VAMFspores from 4to 57 spores per gram of soil after two months,APPLICATION OF VAMF INOCULUMThe inoculation ofplants with mycorrhizal fungican be performed in different ways:The fungal inoculum can be mixed homo-geneously with the substrate in which theplants are grown.A concentrated layer of the inoculum canbe placed below the seeds or the plant roots.The method of inoculation and the amount ofthe applied inoculum (O;0.2; 1; 5 and 10% VoI.)determine the development of the mycorrhizalassociation (Figure 4).FIGURE4. Root colonization by VAMFin dependency of the method of application and the amountof the inoculum applied.homogeneou8 mlxtureroot colonlutlon l'fol110..------------ --'---,day. after Inoculatlon~ 0.0" Inoo. -+- o •••• l/tOo. ~ 1.0" Inoe.-R- 11.0" Inoc. -><- 10,0" Inoo.concentrated appllcatlonroot colonlutlon I,..'40.--------------·-····--··/./30 -.-- .... -"OL---~---J---~-_----~I----ro ~ ~ ~ ~ ~daya after Inoculat1on~ 0.0" Inoo. -+- 0.2" Inoc. 4".- 1,0" Inoc.-- 11.0" l/tOo. -><- 10.0" Inoc.66The highest degree of root colonization of rub-ber tree seedlings (Hevea sp.) is achieved by use ofmore than 1% (vIv) of inoculum in the substrate.The homogeneous distribution of the inoculum fi-nally leads to a higher degree of root colonizationcompared to the method of "concentrated applica-tion", although a higher percentage of root coloni-zation is achieved with the latter method during theearly stages of the development of the symbiosis.This is probably due to the fact that in the case of a"concentrated application", the roots of a seedlinghave contact to a large number of infection unitswhen growing through the inoculum layer. At thismoment a heavy infection of the root system occurrs.After the roots have passed the inoculum layer nofurther infection units of VAMF are present. Thefungus has to spread over the whole root systemfrom the site of the primary penetration points. Incase of a homogeneous distribution of the fungalinoculum, new infections of the roots by the fungusare possible even after a longer time of the develop-ment of the root system. For this reason we recom-mend applying the inoculum in the form of ahomogeneous mixture, but the "concentratedapplication" has also been proved to be successfulin the field.THE USE OF VAMF IN MIXED CROPPINGSYSTEMSThe introduction of VAMF in agricultura 1systems as a management factor is simple. If theisolation, selection and the production of the startinoculum are carried out by officialinstitutes or spe-cialized companies, the farmer has to produce theinoculum on a large scale,which isbest done in con-tainers with sand or sterilized porous soil. Theproduced inoculum is then used for the inoculationofplants in the nursery by mixing it homogeneouslywith the substrate. In this way the plants willalready be mycorrhizal when they are transplantedinto the field. This will be of benefit to the plantsduring the first critical phase of adaptation to thenew environmental conditions.If a farmer wants to inoculate non-mycorrhizalperennial plants which are already planted into thefield, he can do 50 by use of "nurse plants". Thismeans that he should sow or plant crops which areoften used in inoculum production (e.g. com) doseto the perennial plants and inoculate them withVAME The "nurse plants" will multiply the VAMFinoculum in the soil and the roots of these mycor-rhizal plants will grow close to the roots of theperennial plants and thus inoculate them. If the man-agement in the plantation takes into account the re-quirements of the VAMF(less fertilizer, less pesti-cides, more secondary vegetation), the fungi canestablish themselves in the field.REFERENCESAbbot, L.K.; Robson, A.D. and Cazey, C. 1992. Selectionof inoculant vesicular-arbuscular mycorrhizal fungi.In: Norris, J.R.; Read, D.J. and Varrna, A.K. (eds.):Methods in Microbiology, Volume 24,Academic Press,London, pp. 1-21.Daniels, B.A. and Skipper, H.D. 1984. Methods for therecovery and quantitative estimation of prop~ulesfrorn soi!. In: Schenck, N.C. (ed.): Methods and prin-cipies of mycorrhizal research. Am. Phytopath. Soc.,St. Paul, MI, USA, pp. 29-35.Dehne, H.W. and Backhaus, C.S. 1986. The use of VAMin plant production 1. Inoculum production. Z.Pflanzenkrankheiten und Pflanzenschutz 93:415-424.Estaun, Y.;Calvet, C. and Hayman, D.5. 1987. Influenceof plant genotype on mycorrhizal infection: responseof three pea clutivars. Plant and Soi! 103:295-298.Graw, D.; Moawad, M. and Rehm, S. 1979. Host specific-ity and the effectiveness of VA-mycorrhiza. Z. furAcker- und Pflanzenbaur 148 (2):85-98.Feldmann, F. 1991. Die Mykorrhiza desKautschukbaumes Hevea spec. Muell. Arg.:Vorkommen am Naturstandort und in Plantagen,Wirkung auf das Resistenzverhalten, Nutzung imPlantagenbau. Dissertation, Technische UniversitatBraunschweig, 148 pp.Feldmann, F. and Idczak, E. 1992. Inoculum productionof vesiculararbuscular mycorrhizal fungi for use intropical nurseries. In: Norris, J.R.; Read, D.J. andVarrna, A.K. (eds.): Methods in Microbiology,Volume 24, Academic Press, London, pp 339-357.Idcz ak, E. 1992. Wechselwirkung zwischen VA-Mykorrhizapilzen und Nutzpflanzen: Isolat- undSortenspezifitat, Biochemie und Physiologie derWurzel, Pathogenabwehr. Dissertation, UniversitatHamburg, 142 pp.[anos, D.P. 1987. VA mycorrhizas in humid tropical eco-systems. In: Safir, G.R. (ed.): Ecophysiology of VAmycorrhizal plants, CRC Press, Boca Raton, Florida,USA,107-134.Lin, M.T. 1986. Uso de micorrhizas em fruticultura. Rev.Bras. Frutic. (Cruz das Almas, BA) 8(3):47-55.Sieverding, E. 1987. On farm production of VAM inocu-lum. In: Sylvia, D.M.; Hung, L.L. and Graham, J.H.:Proceedings of the 7th North American Conferenceon Mycorrhizae (NACOM), Gainesville, Florida, P:284.67

Isolation, selection and production of vesicular-arbuscular mycorrhizal fungi (VAMF) and their application in mixed cropping systems.

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Isolation, selection and production of vesicular-arbuscular mycorrhizal fungi (VAMF) and their application in mixed cropping systems.

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