About TERI The Biotechnology and Management of Bioresources...

About TERIA dynamic and flexible organization with a global vision and a local focus,TERI, The Energy and Resources Institute, was established in 1974 as theTata Energy Research Institute. While initially, TERI’s focus was mainly ondocumentation and information dissemination, research in the fields of energy,environment, and sustainable development was initiated towards the end of 1982.The genesis of these activities lay in TERI’s firm belief that efficient utilizationof energy, sustainable use of natural resources, large-scale adoption of renewableenergy technologies, and reduction of all forms of waste would move the processof development towards the goal of sustainability.

The Biotechnology and Management of Bioresources DivisionFocusing on ecological, environmental, and food security issues, the division’sactivities include working with a wide variety of living organisms, sophisticatedgenetic engineering techniques, and, at the grass-roots level, with villagecommunities. The division functions through two areas—the Centre forMycorrhizal Research, and Plant Tissue Culture and Molecular Biology.It is actively engaged in mycorrhizal research. The Mycorrhiza Network hasspecifically been created to help scientists across the globe in carrying outresearch on mycorrhiza.

The Mycorrhiza Network and the Centre for Mycorrhizal CultureCollectionEstablished in April 1988 at TERI, New Delhi, the Mycorrhiza Network firstset up the MIC (Mycorrhiza Information Centre) in the same year and theCMCC (Centre for Mycorrhizal Culture Collection) – a national germplasmbank of mycorrhizal fungi – in 1993. The general objectives of the MycorrhizaNetwork are to strengthen research, encourage participation, promoteinformation exchange, and publish the quarterly newsletter Mycorrhiza News.

The MIC has been primarily responsible for establishing an informationnetwork, which facilitates sharing of information among the network membersand makes the growing literature on mycorrhiza available to researchers.Comprehensive database on Asian mycorrhizologists and mycorrhizal literature(RIZA) allow information retrieval and supply documents on request.

The main objectives of the CMCC are to procure strains of both ecto andVA mycorrhizal fungi from India and abroad; multiply and maintain these fungiin pure culture; screen, isolate, identify, multiply, and maintain nativemycorrhizal fungi; develop a database on cultures maintained; and providestarter cultures on request. Cultures are available on an exchange basis or onspecific requests at nominal costs for spore extraction or handling.

Vol. 17 No. 1April 2005

2 Mycorrhiza News 17(1) • April 2005

The extent of VAM infection in plant roots is usuallymeasured, in terms of infected root length as apercentage of total root length, made visible by non-vital stains like trypan blue or chlorozol black.These stains, however, do not differentiate betweenactive and inactive or dead cells of the fungalhyphae. In order to have a clear understanding of thedynamics of VAM infection and the development offungal hyphae in roots and in soil, use of vital stainsis necessary. A vital stain stains only the living cellsso that metabolically active VAM fungal hyphae inroots as well as in soil can be detected andquantified. Such stains determine the activity ofdifferent fungal enzymes: NBT (Nitrobluetetrazolium) is used to determine the activity ofsuccinate dehydrogenase found in mycorrhizal fungi.FDA (Fluorescein diacetate) is used to determinethe activity of esterases in VAM fungal hyphae. Vitalstaining methods have also been used to study theprogress of infection and the effects of fungicides,light and other environmental factors, and soilphosphorus levels on the development of VAMinfection (Sukarno and Dickson 1992).

Methods of staining and extraction ofenzymes from rootsIn studies on enzyme staining conducted at the SoilScience Department, Waite Agricultural ResearchInstitute, University of Adelaide, Glen Osmond,Australia, NBT was used to determine the activity ofvarious dehydrogenase enzymes found in mycorrhizalfungi, particularly SDH (succinate dehydrogenase).NBT is a yellow tetrazolium salt, which is reducedto purple formazan. Other tetrazolium salts have also

been used in various investigations. FDA was used todetermine the activity of mycorrhizal fungi. FDApassively enters the cells down a concentrationgradient and is hydrolysed by esterases to release freefluorescin, which fluoresces at a wavelength of 490nm. The usefulness of these vital stains differsdepending on the situation: NBT is particularlyuseful in staining root segments or sections anddistinguishes fungal structures, the phloem in thevascular system, and passage cells in the hypodermis.Thick (120 µm) sections of the roots can be cutusing a freezing microtome. This slicing does notappear to kill the fungus, which has the ability toreseal the cut ends. Cut cells of the root are killedand do not take up the stain, thus allowing the fungalcells to be differentiated from the plant cells. FDA isunsuitable for use on root sections because ofautofluorescence in both plant and fungal walls,which makes it difficult to demarcate the areaoccupied by an active fungus. FDA is useful instaining external hyphae. A soil sample is dilutedwith distilled water, which is stirred rather thanblended to avoid possible damage to hyphae.Aliquotes of this solution are taken using a Milliporefilter set-up which produces a thin layer of residueon the filter. Purple staining with NBT gave poorcontrast between the hyphae and the dark backgroundof soil particles; FDA worked better and produced astrong fluorescence that contrasted clearly with thebackground (Sukarno and Dickson 1992).

In studies conducted at the Department of LandResource Science, University of Guelph, Ontario,Canada, a modified vital staining technique, used inintraradical VAM colonization, was applied toextraradical VAM hyphae in order to understand how

Assessment of a metabolically active VAM infection by vital stainingmethodsSujan Singh*T E R I, Darbari Seth Block, I H C Complex, Lodhi Road, New Delhi – 110 003, India

ContentsAssessment of a metabolically active VAM infection byvital staining methodsSujan Singh 2

Research findingsEffect of dual inoculation (AM fungi and Rhizobium)

on chlorophyll content of Vigna unguiculata (L).Walp. var. Pusa151 10

Germination response of mango stones to differentarbuscular mycorrhiza fungi 11

New approachesA new approach for characterization of arbuscular

mycorrhiza fungi 15

Centre for Mycorrhizal Culture CollectionUser-friendly digital herbaria of arbuscular mycorrhizal

fungi for facilitating identification 15

Improved variety rapeseed mustard: T ER I cultivationUttam–Jawahar 17

Biography of Dr Andre Fortin 18

Recent references 19

Forthcoming events 24

Mycorrhiza News 17(1) • April 2005 3

VAM fungal mycelia respond to environmentalstresses such as freezing and disturbances such astillage. In this method, hyphae are extracted fromsoil, collected on nylon mesh filters, and incubatedin NBT succinate solution (NBT succinate).Succinate dehydrogenase, which is present in viablefungal hyphae, reacts with NBT and is readilyreduced to a dark blue or purple formazancompound. The hyphae are then fixed, cleared inKOH, and counterstained with AF (acid fuchsin).This counterstaining not only increases the contrastbetween viable and non-viable hyphal segments butalso allows total hyphal length to be measured fromthe same slide used for measuring hyphal viability.Both formazan and AF will persist for extendedperiods if samples are stored in the dark, whereasfluorescent stains fade quickly, which limits their useto immediate observations of samples. This vitalstaining technique also has the advantage of beingrelatively inexpensive and simple to use. However,the stains do not distinguish between mycorrhizaland non-mycorrhizal fungi. Such differentiation canbe obtained on the basis of morphologicalcharacteristics (Heather, Shaffer, and Miller 1993).

In further studies conducted at the Department ofBotany of the University of Guelph, the NBTsuccinate method was further modified for theassessment of SDH activity in leek, maize, andpigmented soybean colonized by VAM fungi. Rootstreated with NBT succinate, cleared in (w/v) KOHin a drying oven (55 °C) for 24 hours or longer, andobserved as whole mounts revealed signs ofintraradical VAM fungus colonization more clearlythan the roots cleared by the standard method ofboiling in 20% (w/v) chloral hydrate. A combinationof the two techniques by clearing roots in boilingchloral hydrate followed by clearing in 5 KOH forprolonged periods also improved the visibility ofintraradical fungal structures. Bleaching the rootstreated with NBT succinate using the standardNH

3-H

2O

2 method removed pigmentation from roots

and did not affect the viability indicator, namelyformazan. Roots of pigmented soybean collectedfrom the field were successfully cleared and bleachedto reveal the signs of viable and non-viableintraradical fungal structures. Counterstaining theroots with AF made both viable and non-viableintraradical fungal structures clearly visible. TheNBT-succinate solution infiltrated all intraradicalfungal structures after 24 hours. Formazan productswere observed at similar concentrations in viablestructures after 24, 36, and 48 hours (Schaffer andPeterson 1993).

Extraction of hyphae from VAM rootsIn studies conducted at the National GrasslandResearch Institute, Nishinasuno, Tochigi, Japan, onisolation of metabolically active internal hyphae fromarbuscular mycorrhiza in onion, a method wasdescribed wherein roots of onion, colonized by

Gigaspora margarita were digested for one hour at 30°C with solution containing cellulase and pectinaseand then homogenized with a blender at a low speed.The internal hyphae were collected from thehomogenate by centrifugation on a discontinuousgradient of Percoll and then purified further byfiltration. Enzyme histochemical staining showedthat the collected internal hyphae had active SDHand alkaline and acid phosphatases (phosphoricmonoester hydrolases). Specific activities (proteinbasis) of several enzymes in a crude extract of theinternal hyphae were compared with those extractedfrom axenically germinated spores of Gi. margarita.The specific activities of hexokinase and of theenzymes involved in phosphate metabolismalkalinease acid and phosphatases and in the TCAcycle (SDH and malate dehydrogenase) were muchgreater in the internal hyphae than in the germinatedspores. The specific activity of phosphofructokinasewas similar in both. By contrast, the specific activityof glucose-6-phosphate dehydrogenase was greater inthe germinated spores (Saito 1995).

In studies conducted at the Department ofAgricultural Biochemistry, Waite AgriculturalResearch Institute, Glen Osmond, Australia, enzymicactivity of hyphae and vesicles of the VAM fungusGlomus intraradices in the roots of Allium porrum wasmeasured before and after enzymic digestion of thefungus. Using NBT to detect the activity of SDHand the fluorescence of acridin orange, theresearchers observed a decline in the activity ofenzymes of the mycorrhizal fungus during enzymicdigestion. No enzymic treatment was found thatpermitted removal of the fungus from roots withoutplasmolysis of vesicles and a reduction in enzymeactivity (McGee and Smith 1990).

Quantification of active VAM infectionMost research on the interactions betweenmycorrhizal fungi and plants requires thedetermination of the extent to which the fungicolonize the root systems. It is, however, importantto obtain data that give a better estimate of theintensity (in terms of root volume or mass infected)and the quality of infection (for example, arbusculesor vesicle production) and the proportion ofpropagules that remain viable over different duration(Smith and Dickson 1990).

In another study by the same researchers, imageanalysis was used to quantify different parameters ofinfective propagules which include numbers of cellscontaining arbuscules relative to the total number ofinfected cells in the root cortex, the proportion ofthe root volume colonized by arbuscular infection,and the activity of arbuscules as shown by theintensity of staining of SDH with NBT. The resultsindicated that over a period of nine weeks, thenumber of cortical cells containing arbuscules andthe activity of SDH in individual arbuscules declinedappreciably (Smith and Dickson 1990).


Yet another study by the same team describes themethods for objective measurement of VAMinfection in the roots of Trifolium subterraneum, peas,leeks, and cucumber based on vital staining oftransverse sections of fresh roots, followed byevaluation of the infection and determination of thenumber of living arbuscules and hyphae in eachtransverse section. The most useful method involvedfreeze-sectioning of roots embeded in gelatincontaining glycerol and DMSO, followed by stainingthe sections with NBT overnight to reveal SDHactivity. Two systems of computer-aided imageanalysis were evaluated for the determination of thenumber, perimeter, and cross-sectional area of theintercellular hyphae and arbuscules. This approach,which is clearly objective, is an advance over theearlier methods based on ranking sections orsegments of root based on the intensity ofdevelopment of arbuscules. The data were used tocalculate the area of interface between the plant andthe fungus in infected roots. The results showed thecontribution of arbuscules to the total infection.Also, the area of interface declined markedly withage of the plants and the infection (Smith andDickson 1991).

In further studies, Smith and Dickson (1992)refined the method of detection of active VAMinfection by vital enzyme staining. The fungus wasquantified by observing transverse sections of rootsusing the chromatic colour image analysis system(L R Jarvis, Leading Edge). Automaticdiscrimination was achieved by selecting the averagecolour intensity of the fungal structures within thesections. This segment of the image is then digitizedand used for calculating fungal areas, perimeters,and the numbers of different structures includinghyphae, vesicles, and arbuscules. For soil samples,the length of active hyphae was determined by usinga grid intersect placed in the eyepiece of afluorescence microscope.

In studies conducted in the Soil MicrobiologyDepartment, Estacion Experimental del Zaidin,Granada, Spain, the percentage of infected rootlength and SDH activity were assessed in maize,cucumber, and wheat inoculated with Glomusmosseae. The results indicated that although thehistochemical assay of SDH detects the metabolicfunction of the fungus, quantifying this activity as apercentage of mycorrhizal root length along whichthe activity was observed was not indicative of theefficiency of the fungus in promoting plant growth(Vierheilig and Ocampo 1990).

Use of enzyme staining in studying thedynamics of VAM infectionVital staining methods have been used in trackingthe course of VAM infection in time and space andto study the effects of fungicides, phosphates,environmental factors, and interactions with othermicroorganisms in VAM infections.

The course of VAM infectionIn studies conducted at the Instituto ColtivazioniArboree dell’ Universita, Via Giuria 15, Turin,Italy, activities of acid and alkaline phosphatase,esterase, and succinate dehydrogenase were assessedin extraradical mycelium of Glomus clarum byhistochemical methods. Staining was observed for allenzymes except acid phosphatase. The amount ofactive mycelium decreased after the initial infection.In older hyphae, stained deposits were irregularlydistributed inside the hyphae. It is possible that insome hyphal tracts the enzymatic activity wasreduced following the formation of vacuoles(Schubert and Mazitelli 1990).

In studies conducted in the Plant ScienceDepartment of Macdonald College, McGillUniversity, Ste-Anne de Bellevue, Quebec, Canada,the extraradical hyphae of G. intraradices associatedwith a mixture of Saranac lucerne and bromegrass(Bromus inermis cv Tempo) were tested for metabolicactivity by three enzyme-staining procedures.Mycorrhizal plants were grown for 6, 9, and 12weeks and the vital staining methods were evaluatedand compared. The SDH activity was assessed by thereduction of NBT and NADH diaphorase activity bythe reduction of INT (indonitro tetrazolium). TheFDA (fluorescin diacetate) hydrolysis was used toassess the activity of esterases in the extraradicalhyphae. Percentage of intraradical hyphae active inlucerne and bromegrass were determined afterstaining root samples with NBT and chlorozol blackE. The percentage of live intraradical infectiondeclined as the symbiosis matured, whereas that ofextraradical hyphae did not (the NBT, however, didnot show this contrast) suggesting a differentturnover for extra- and intraradical hyphae. TheFDA gave the best estimates of enzyme activity,followed by INT and NBT in that order (Hamel,Fyles, and Smith 1990).

In studies conducted in INRA, CNRS,Phytoparasitol laboratory, Genet & AmelioratPlantes, Dijon, Cedex, France, a histochemicalprocedure was developed to estimate the proportionof AM infections showing fungal alkalinephosphatase activity in the total amount of fungaltissue (indicated by trypan blue staining) and in theliving mycelium (indicated by SDH activity). Inroots of leek and Platanus acerifolia, only a smallproportion of living intraradical mycelium showedalkaline phosphatase activity during early infectionby Glomus spp. but this increased greatly just beforethe mycorrhizal growth response of the host plant.Infection revealed by all three stains reached amaximum at six weeks after inoculation after whichthe level of trypan-blue-stained infection remainedconstant whereas that showing SDH and alkalinephosphatase activity declined. Alkaline phosphataseactivity was absent from virtually all abortive entry-point hyphae formed on the roots of a resistant myc(–), nod (–) mutant of pea, although SDH activity


was detected. The results indicate that alkalinephosphatase activity is induced by colonization ofhost roots and that this fungal enzyme could be auseful marker for analysing the symbiotic efficiencyof AM infection (Tisserant, Gianinazzi-Pearson,Gianinazzi, et al. 1993).

Further studies were conducted in the samelaboratory to define the relationships between rootorder dynamics, AM development, and thephysiological state of symbiotic Glomus fasciculatumin a woody plant species, Platanus acerifolia, usingSDH and alkaline phosphatase to detect activemycelium. Arbuscular mycorrhiza influenced rootsystem development in P. acerifolia and was closelyrelated to the appearance of different root orders.The most active mycelium, characterized by fungalSDH and alkaline phosphatase activities, was mainlylocalized in the newly formed lateral roots. Nineweeks after inoculation with G. fasciculatum, theproportion of mycelium showing the activity ofalkaline phosphatase strongly decreased in all rootorders, probably because of increased phosphoruscontent of the host plant (Tisserant, Gianinazzi, andGianinazzi-Pearson 1996).

In studies conducted at the Department of PlantPathology, Kansas State University, Manhattan,Kansas, USA, the effect of burning on thedevelopment of mycorrhizal symbiosis as determinedby enzyme staining was determined in big bluestemAndropogon gerardii. Burning is an integral part oftall grass prairie maintenance and regularlystimulates plant growth and productivity. Eight daysafter burning, no differences were found in per centroot colonization between burned and unburned plotsas determined by staining with trypan blue.However, in SDH assays, burning increased thepercentage of root tissues colonized by metabolicallyactive mycorrhizal fungi. This increase inmycorrhizal activity may contribute to the burst ofplant growth following burning (Bentivenga andHetrick 1990).

In studies conducted at the Soil ScienceDepartment, AFRC Institute of Arable CropsResearch, Rothamsted Experimental Station,Harpenden, Herts, UK, the SDH activity in hyphaeof the VAM fungus G. mosseae in symbioticassociation with leek (Allium porrum) wasinvestigated by histochemical staining in situ. Leekseedlings were transplanted to sand culture andinoculated with spores of G. mosseae placed justbelow the base of the stem. At 14, 25, 35, and60 days after transplanting, the growth medium(sand) was flooded with NBT chloride solution,thereby displacing the nutrient solution. Thisallowed the sites of SDH activity in external andinternal fungal structures to be stained withoutphysically disturbing the symbiotic system. Aftercounterstaining the harvested roots and myceliumwith AF, it was possible to differentiate clearlybetween the metabolically active and inactive regionsof the fungus. The lengths of both external hyphae

and infected roots increased exponentially and werein constant proportion (1.4 m hyphae per cm ofinfected root) for up to 60 days. The percentagelength of external hyphae with SDH activityremained almost constant (80%). In each infectedlength of root, there was a gradation of SDH activityfrom the inactive distal (older) hyphae to uniformlyactive proximal (younger) hyphae (Saito, Stribley,and Hepper 1993).

Studies were conducted at the Department ofBotany, Mansoura University, Mansoura, Egypt, toinvestigate the response of soybean (Glycine max) toinoculation with VAM fungus G. mosseae atflowering and maturity stages of plant growth.Activity of fungal SDH and alkaline phosphataseindicated that the response of soybean to mycorrhizalinfection was higher at flowering at maturity. Thelevel of total mycorrhizal infection was not generallyrelated to plant growth and phosphorus content(AbdelFattah 1997).

Studies conducted in the College of Life Scienceand Technology, Huazhong Agricultural University,Wuhan, Hubei, China, compared two VAM fungi,G. mosseae and G. intraradices, for abundance ofintraradical and soil-borne hyphae in associationwith Astragalus sinicum, a legume with small seeds,and Glycine max by enzyme staining. A. sinicum wasmore responsive than G. max to mycorrhizalformation, especially in early growth stages. Biomassallocation was greater in roots than shoots formycorrhizal A. sinicum while the opposite was truefor G. max. Hyphal development in root and soilcompartments was estimated by trypan blue stainingand SDH and alkaline phosphatase activity. Totalfungal abundance increased steadily in roots and soilwith time, leading a maximum eight weeks aftersowing. Activity of SDH and alkaline phosphatase inAM hyphae increased in roots during plant growthbut decreased in soil at later harvests. Mycorrhizalroot mass in A. sinicum and G. max increased about14-fold and 2.5-fold, respectively, but total length ofsoil hyphae produced per plant differed little so thatthe pattern of abundance of the two fungi in soil andin roots varied considerably with the host plant(Zhao, Trouvelot, Gianinazzi, et al. 1997).

In studies conducted at the University of Pisa,CNR, Centre Studio Microbiol Suolo, Departmentoand Biotecnol Agrarie, Via Borghetto 80, Pisa, Italy,enzyme staining was used to find out protoplasmiccontinuity in hyphal fusions of VAM fungi. Studiesconducted on G. mosseae colonizing roots ofT. vulgaris and P. cerasifera showed that themycelium spreading from the colonized roots ofT. vulgaris and P. cerasifera showed hyphal fusions tothe extent of 64% and 78%, respectively, of the totalcontacts occurring between the hyphae amountingto 0.66 and 0.51 anastmoses per mm of the hyphae,respectively. Histochemical localization of theSDH activity in hyphal bridges demonstratedprotoplasmic continuity, while the detection ofnuclei in the hyphal bridges confirmed the viability


of anastomosed hyphae. The ability of extraradicalmycelium to form anastomoses and to exchangenuclei suggests that beyond nutritional flow, flow ofinformation might also be active in the network(Giovannetti, Fortuna, Citernesi, et al. 2001).

In studies conducted at the Department ofAgricultural Biochemistry, Waite AgriculturalResearch Institute, on the effect of photon irradianceon phosphorus uptake and growth of onioninoculated with G. mosseae, NBT chloride was usedas a vital stain for SDH activity. During the6-week-long experiment, all the infections revealedby trypan blue staining were physiologically active.Branched arbuscules always had SDH and alkalinephosphatase activity. Thus, in young plants,observing of arbuscules gives a good indication ofthe amount of active infection (Smith andGianinazzi-Pearson 1990).

Effect of phosphorus on metabolic activityof VAM as determined by enzyme stainingIn studies conducted at the Faculty of Agriculture,Yamagata University, Tsuruoka, Yamagata-Ken,Japan, on the effect of phosphate application on thematabolic activity of hyphae in VAM root, onionseedlings were transplanted in 200-ml pots filledwith autoclaved sand and inoculated withGi. margarita. The seedlings were grown in growthchambers and phosphate-free nutrient solution wasapplied every two days. (At 40 days aftertransplanting, three treatments were given: injectionof 0.1% P solution to shoots, (2) application of8 mg P/L nutrient solution, and (3) application ofP-free nutrient solution). As before, the solutionswere applied every two days. At 40 and 80 days afterthe treatment, metabolic activity of the internalhyphae was evaluated with SDH. After the enzymaticstaining, the hyphae were counterstained with AF.The percentage of root length infected, number ofappressoria per unit root length, and shoot weightwere measured. Shoot growth was increased a littlein P-free treatment. VAM infection was decreased inplants that received P. The number of appressoriaper unit length of infected root decreased with timeand was not affected by the treatments. There was asignificant positive correlation between the numberof appressoria per plant and the length of infectedroots per plant. At 40 days after treatment, thepercentage of hyphae showing SDH activity did notdiffer among the treatments. The results suggest thatmetabolic activity of internal hyphae is little affectedby phosphate application for a short period and thatthe inhibition of infection by phosphate applicationoccurs at the stage of hyphal penetration of roots.(Tawaraya, Saito, Morioka, et al. 1992 and 1994)

Studies conducted at the Laboratore dePhytoparasitology, INRA-CNRA, Station deGenetique et d’ Amelioration des Plantes, Dijon,Cedex France, on the effects of phosphateapplication fertilization on the physiological activity

of VAM infection in soybean and pineapple usedfungal SDH and alkaline phosphatase. Soybean andpineapple were used because of their different growthrates and response to phosphate. Total mycorrhizalinfection as estimated by trypan blue staining wasfound to be reduced by phosphate application soilswith adequate levels of P. In P-deficient soils, fungalinfection in pineapple roots was not affected byfertilizer application. The level of total mycorrhizalinfection was not related to plant growth. Fungalalkaline phosphatase staining and to a lesser extent,SDH activity were related to plant growth. Thisprocedure is thus potentially useful in estimatingendomycorrhizal infection as a measure of efficiencyof the symbiosis (Guillemen, Orozco, Gianinazzi-Pearson, et al. 1995).

Effect of fungicides on metabolic activityof VAM as determined by enzyme stainingStudies conducted at the Station d’ Amelioration desPlantes, INRA, Dijon, Cedex, France, showed thatapplication of either benomyl or captan to soilsignificantly decreased the growth VAM-inoculatedonion four weeks after the treatment but not that ofnon-VAM plants. Fungal colonization of onion asindicated by non-vital staining with chlorozol BlackE was depressed two weeks after fungicideapplication. However decrease in metabolicallyactive VAM fungal tissue revealed by SDH assaycould be detected as early as three days after thetreatment. There was little difference between thefungicides in their effect on VAM fungi used.SDH assay is thus useful in studying the effectsof fungicides (Kough, Gianinazzi-Pearson, andGianinazzi 1987).

Studies conducted at the Department of PlantPathology, J N Agricultural University, IndoreCampus, India, on the effect of fosetyl Al(tris-o-ethyl phosphorate), a systemic anti-oomycetes, symplast fungicide, used at threeconcentrations for seed and foliar application oncolonization of soybean roots by G. mosseae andG. fasciculatum showed that the fungicide treatmentincreased colonization, intramatrical vesicles,arbuscules, and extramatrical spores significantly.SDH assay showed greater colonization andincreased growth and yield of soybean in plantstreated with the fungicide (Vyas 1990).

In experiments conducted at the Department ofMicrobiology, Swedish University of AgriculturalSciences Uppsala, Sweden, the influence of twosystemic fungicides, propiconazol, and carbendazim(bavistin), at field application rates on the functionsof three VAM fungi was studied. Short-terms32 P-transport and SDH activity in external hyphaeof G. intraradices, G. claroideum, andG. invermanium in symbiosis with pea (Pisumsativum) were measured. In the experimental system,the hyphae grew into two root-free hyphalcompartments. The fungicides were applied to both


compartments 24 days after sowing and 32P wasadded to only one in each pot. Four days later, theeffect of the fungicide on fungal P transport wasmeasured as the difference in 32 P content in treatedand untreated plants. SDH activity in fungal hyphaewas determined in hyphal compartments with no 32 P.Carbendazim severely inhibited 32 P transport andSDH activity in external hyphae at an applicationrate of 0.5 µg per litre of soil. The ergosterolinhibitor, propiconazol, affected none of theseparameters. The fungicides had similar effects on allthe three fungal species although P-transportefficiency and SDH activity differed markedly amongthe species (Kling and Jakobsen 1997).

Effect of interactions with other soilmicroorganisms on metabolic activity ofVAM as determined by enzyme stainingStudies conducted at CSIS, Estacion, Experimentaldel, Zaidin, Department of Microbiology, Suelo andSistemas Simbiont, Granada, Spain, compared theeffect of two Rhizobium mililoti strains, the wild type(WT) and its GM (genetically modified) derivate, onphysiological activity of the VAM fungus, G. mosseae,during the colonization of alfalfa (Medicago sativa)roots as affected by the water status in the growingmedium. Activities of SDH and alkaline phosphatasewere used as a measure of the effect. The VAMplants did not differ significantly in their growthresponse to the two strains at any water level. Thislack of response did not adversely affect thedevelopment of AM symbiosis. In well-wateredplants, SDH activity indicated that about 80% of theAM mycelium in plants inoculated with GMRhizobium were alive throughout the experimentcompared to only 10%–20% in those inoculated withthe WT strain. Both the rhizobial strains behavedsimilarly under water-limiting conditions in regardto AM development (Vazquez, Bejarano, Azcon,et al. 2000).

In another study at the same department, SDHwas also used to study the effect of two soil fungi,Alternaria alternata and Fusarium equiseti, onmetabolic activity of extraradical hyphae of G. mosseae.Both the saprophytes decreased plant dry weight andmycorrhization when introduced into the rhizospherebefore inoculation with G. mosseae but they did notaffect the percentage of root length colonized by theAM endophyte when inoculated two weeks afterG. mosseae. However, these saprophytes did affect themetabolic activity of G. mosseae as assessed by theSDH activity. The results suggest that thesaprophytic fungi may affect the mycorrhizal fungidirectly in the extramatrical phase of the latter butless so after the AM fungus is established affectedless (McAlister, Garcia Garrido, et al. 1997).

Studies conducted at the Department of CienciesBiologicas, Universidad de Buenos Aires, 1428Buenos Aires, Argentina, on the interaction betweenAspergillus niger and G. mosseae showed that

percentage germination and the length of hyphae ofgerminated spores of G. mosseae decreasedsignificantly in the presence of A. niger on bothwater agar and malt extract agar. Shoot dry weightand percentage of AM colonization of roots of maizeand lettuce decreased when A. niger was inoculatedat the same time or two weeks before G. mosseae butunaffected when A. niger was inoculated two weeksafter G. mosseae. However, metabolic activityresulting from AM colonization, measured as apercentage of mycelium showing SDH activitydecreased in all the treatments (McAllister,Garcia-Romera, Martin, et al. 1995).

Further studies conducted at the CSIC,Department Microbiology, Estacion ExperimentalaZaidan, Granada, Spain, on the interaction betweenG. mosseae and Trichoderma koningii or Fusariumsolani showed that T. koningii decreased plant dryweight and AM colonization on roots of maize andlettuce when inoculated into the rhizosphere beforeor at the same time as G. mosseae but had no effectwhen inoculated two weeks later. The effect ofF. solani on the AM colonization of lettuce roots wassimilar to that of T. Koningii although F. solani didnot affect AM colonization of maize roots.T. koningii adversely affected the metabolic activityof AM mycelium as assessed by SDH activity in allthe treatments (McAllister, Garcia-Romera,Godeas, et al. 1994).

Studies conducted at the University NacionalAgraria la Selva, Faculty Agronomy, Tingo Maria,Huanaco, Peru, on single or dual inoculation ofalfalfa (Medicago sativa cv. Aragon) with Rhizobiummeliloti strain 10 F28 and G. fasciculatum showedthat dual-inocualted plants had higher shoot dryweight, the lowest root-to-shoot ratio, and higheroxygen uptake and nodule nitrogenase activity thanthose inoculated with either of the two organisms.However, dry weight of the roots of plants inoculatedwith the VAM alone was higher than those fromother treatments. These differences were notcorrelated with SDH activity, which was at a similarlevel in all the treatments. The data suggest thatRhizobium may affect metabolism and that the effectis not achieved via the tricarboxylic acid pathway(Piccini, Ocampo, and Bedmar 1988).

ReferencesAbdelFattah G M. 1997Functional activity of VA-mycorrhiza (Glomusmosseae) in the growth and productivity ofsoybean plants grown in sterilized soilFolia Microbiologica 42(5): 495–502

Bentivenga S P and Hetrick B A D. 1990Mycorrhizal contribution to tallgrass prairie. pp 21[In Proceedings of the Eighth North American Conference onMycorrhiza Innovation and Hierarchical Integration,Jackson, Wyoming, 5–8 September 1990]Wyoming: University of Wyoming. 324 pp.


Giovannetti M, Fortuna P, Citernesi A S, Morini S,Nuti M P. 2001The occurrence of anastomosis formation andnuclear exchange in intact arbuscular mycorrhizalnetworksNew Phytologist 151(3): 717–724

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Kough J L, Gianinazzi-Pearson V, and Gianinazzi S. 1987Depressed metabolic activity of vesicular-arbuscular mycorrhizal fungi after fungicideapplicationsNew Phytologist 106: 707–715

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McAllister C B, Garcia-Romera I, Martin J, Godeas A,Ocampo J A. 1995Interaction between Asperpgillus niger van Tiegh.and Glomus mosseae (Nicol. and Gerd.) Gerd. andTrappeNew Phytologist 129(2): 309–316

McGee P A and Smith S E. 1990Activity of succinate dehydrogenase in vesicular-arbuscular mycorrhizal fungi after enzymicdigestion from roots of Allium porrumMycological Research 94(3): 305–308

Piccini D, Ocampo J A, and Bedmar E J. 1988Possible influence of Rhizobium on VA mycorrhizametabolic activity in double symbiosis of alfalfaplants (Medicago sativa L) grown in a potexperimentBiology and Fertility of Soils 6(1): 65–67

Saito M. 1995Enzyme activities of the internal hyphae andgerminated spores of an arbuscular mycorrhizalfungus, Gigaspora margarita Becker HallNew Phytologist 129(3): 425–431

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Schaffer G F and Peterson R L. 1993Modifications to clearing methods used incombination with vital staining of roots colonizedwith vesicular-arbuscular mycorrhizal fungiMycorrhiza 4(1): 29–35

Schubert A and Mazitelli M. 1990Enzymatic activities of VAM extraradicalmycelium at different stages of developmentAgriculture, Ecosystems and Environment 29(1–4):349–353

Smith S E and Gianinazzi-Pearson V. 1990Phosphate uptake and arbuscular activity inmycorrhizal Allium cepa L.: effects of photonirradiance and phosphate nutritionAustralian Journal of Plant Physiology 17(2): 177–188

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Smith S E and Dickson S. 1991Quantification of active vesicular-arbuscularmycorrhizal infection using image analysis andother techniquesAustralian Journal of Plant Physiology 18(6): 637–648

Sukarno N and Dickson S. 1992Vital staining of vesicular-arbuscular mycorrhizalfungi, pp. 46–47In Proceedings of the International Symposium onManagement of Mycorrhizas in Agriculture, Horticulture andForestry, p. 86, edited by D JasperAustralian Institute of Agricultural Sciences. 125 pp.[28 September–2 October 1992, University of WesternAustralia, Nedlands, Australia]

Tawaraya K, Saito M, Morioka M, Wagatsuma T. 1992Effect of phosphate on succinate dehydrogenaseactivity of hyphae of Gigaspora margaritaIn Proceedings of the International Symposium onManagement of Mycorrhizas in Agriculture, Horticulture andForestry, p. 69, edited by D JasperAustralian Institute of Agricultural Sciences. 125 pp.[28 September–2 October 1992, University of WesternAustralia, Nedlands, Australia]

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Tisserant B, Gianinazzi-Pearson V, Gianinazzi S,Gollette A. 1993In planta histochemical staining of fungal alkalinephosphatase activity for analysis of efficientarbuscular mycorrhizal infectionsMycological Research 97(2): 245–250

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Vazquez M M, Bejarano C, Azcon R, Barea J M. 2000The effect of a genetically modified Rhizobiummeliloti inoculant on fungal alkaline phosphataseand succinate dehydrogenase activities inmycorrhizal alfalfa plants as affected by the waterstatus in soilSymbiosis 29(1): 49–58

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*This paper has been compiled from TERI records in RIZA.


The symbiotic relationship between VAM (vesiculararbuscular mycorrhiza) fungi and the host plants hasbeen studied traditionally in terms of benefits to theindividual plant and fungi (Smith and Smith 1996;Francis and Read 1995). VAM association can affectthe host plants in terms of stomatal movement andphotosynthesis of leaves and has been shown to increasechlorophyll content and the rate of transpiration andphotosynthesis (Panwar 1991; Bethlenfalvay, Brown,and Franson 1988). Mycorrhizal infection is ofparticular value to legumes because it can increase theP (phosphorus) update: nodulation and symbioticnitrogen fixation by rhizobia require adequate supply ofP, and restricted root system leads to poor competitionfor soil P (Carling, Richle, and Johnson 1978).

Cowpea – Vigna unguiculata (L). Walp – is one ofthe principle pulses of India, grown as a vegetable inmost parts of the country. Fully mature seeds ofcowpea are boiled and eaten; they are also split andused as dal. The seed contains 24% protein and is arich source of calcium and iron (CSIR 1967). In lightof these facts, the present investigation was undertakento study the effect of dual inoculation (VAM fungiand Rhizobium) on the chlorophyll content ofV. unguiculata leaves.

Material and methodsSeeds of V. unguiculata (L.) Walp. Var. Pusa 151were obtained from the Agricultural ResearchStation, Vamban, Pudukkottai district, Tamil Nadu.Seeds free from visible defects were selected for thestudy. Seedlings were raised in pots containing amixture of sand and soil in equal proportion. Seedswere inoculated either with Rhizobium or withGlomus mosseae (applied as a layer on soil surface) orwith both together. One pot of seedlings was left

without inoculation as control. The leaves werecollected for analyses after 15, 25, and 40 days ofinoculation and the chlorophyll content was estimatedusing the method of Arnon (1949). All the experimentswere carried out in triplicate and the results wereexpressed as the average of the three replications.

Results and discussionPlants inoculated with VAM fungus alone or incombination with Rhizobium showed significantchanges in chlorophyll a and b and total chlorophyllcontent. The total chlorophyll content was thehighest in dual-inoculated plants, particularly infive-day-old leaves (see table below). Such anincrease might be due to an increase in the rate ofphotosynthesis and transpiration or increased growth(Sampathkumar and Ganeshkumar 2003; Hayman1983) or due to the presence of a large number ofchloroplasts in the bundle sheath of inoculated leaves(Krishna and Bhagyaraj 1984). Several studiesreported indicate that chlorophyll content is higherin the leaves of resistant varieties than those of thesusceptible ones as biochemical characters likephenols, proteins, and chlorophyll may play a vitalrole in making plants resistant to pathogens(Bhavani, Venkatasubbaiah , Sudhakar Rao, et al.1998; Charitha Devi and Reddy 2001). Thus thepresent study clearly reveals that there is an increasein the total chlorophyll content, because theassociation of VAM, Rhizobium, and the host isbelieved to have a possible effect on host defencemechanism and photosynthesis.

It is concluded that dual inoculation withmycorrhizal fungi and rhizobium species is effectivein increasing the chlorophyll content, leading toenhanced growth in legumes.

Effect of dual inoculation (AM fungi and Rhizobium) on chlorophyllcontent of Vigna unguiculata (L). Walp. var. Pusa 151S Rajasekaran and S M NagarajanDepartment of Botany, A V C College, Mannampandal – 609 305, India

Research findings

Effect of arbuscular mycorrhiza fungi and Rhizobium on chlorophyll content of cowpea

Chlorophyll A (mg/g) Chlorophyll B (mg/g) Total chlorophyll (mg/g)

Treatment Days after inoculation Mean Days after inoculation Mean Days after inoculation Mean

15 25 40 15 25 40 15 25 40Control 0.61 0.73 0.82 0.72 1.04 1.12 1.18 1.11 2.06 2.15 2.24 2.15Rhizobium 0.71 0.76 0.97 0.79 1.10 1.21 1.32 1.21 2.14 2.31 2.43 2.29Glomus mosseae 0.63 0.81 0.88 0.77 1.12 1.26 1.21 1.16 2.16 2.35 2.28 2.26Rhizobium and G. mosseae 0.77 0.92 1.07 0.92 1.22 1.25 1.64 1.37 2.27 2.35 2.73 2.45Mean 0.68 0.80 0.93 — 1.12 1.21 1.33 — 2.15 2.27 2.42 —


ReferencesArnon D I. 1949Copper enzymes in isolated chloroplasts.Polyphenol oxidase in Beta vulgarisPlant Physiology 24: 1–5

Bhavani U, Venkatasubbaiah K , Sudhakar Rao A,Saigopal D V R. 1998Studies on mosaic disease of sunflower:biochemical changes and growth parametersIndian Phytopathology 51: 357–358

Charitha Devi M and Reddy M N. 2001Growth response of groundnut to VAM fungus andRhizobium inocualtionPlant Pathology Bulletin 10: 71–78

CSIR (Council of Scientific Industrial Research). 1967Wealth of India: a dictionary of Indian rawmaterials and industrial products (Vol 10)New Delhi: CSIR. pp. 497–510

Hayman D S. 1983The physiology of vesicular arbuscularendomycorrhizal symbiosisCanadian Journal of Botany 61: 944–963

Krishna K R and Bagyaraj D J. 1984Growth and nutrient uptake of peanut inoculatedwith mycorrhizal fungus Glomus fasciculatumcompared with uninoculated onesPlant and Soil 77: 405–408

Sampathkumar G and Ganeshkumar A. 2003Effect of AM fungi and Rhizobium on growth andnutrition of Vigna mungo L. and Vignaunguiculata L.Mycorrhiza News 14(4): 15–18

Other readingsBethlenfalvay G J, Brown M S, and Franson R L. 1988Glycine–Glomus Bradyrhizobium symbiosisPlant Physiology 94: 723–728

Carling D E, Richle N E, and Johnson D R. 1978Effect of VAM on nitrate reductase and nitrogenaseactivity in nodulation and non-nodulating soybeanPhytopathology 68: 1590–1596

Francis R and Read D J. 1995Mutualism and antagonism in the mycorrhizalsymbiosis with special reference to impacts onplant community structureCanadian Journal of Botany 73: 1301–1309

Panwar J D S. 1991Effect of VAM and Azospirillum brasilense onphotosynthesis, nitrogen metabolism and grainyield in wheatIndian Journal of Plant Physiology 34: 357–361

Smith F A and Smith S E. 1996Mutualism and parasitism: diversity in functionand structure in the arbuscular (VA) mycorrhizalsymbiosisAdvanced Botany Research 22: 1–43

Because mango stones being recalcitrant, rapidlylose viability (Ruehle and Ledin 1955), they shouldbe collected and sown within a week of collection,especially in the high-latitude tropics and arid andsemi-arid regions where high evaporative demandprevails during the period when stones are available.In these areas, mango is being propagated onnondescript monoembryonic rootstocks followingex situ propagation (container-grown, detachedgrafting) with 30% to 40% germination. Good andearly germination of mango stones (seeds) is themain prerequisite to successful graftins.

Though Ram (1997) achieved good germinationby soaking the stones in or spraying them withgibberellins, the seedlings were unsatisfactory.According to Reddy (1991), mango responds well to

the AM fungal inoculation, in the form of increasedgirth, height, and leaf area. Earlier workers havereported that the AM fungi secrete plant growthregulators like gibberellins (Miller 1971; Crafts andMillar 1974; Slankis 1975), which are known topromote seed germination in mango and other crops.In order to use these benefits and to find out themost efficient AM fungi for germination and growthin mango, the present experiment was undertaken.

Materials and methodsAn investigation on the effect of the AM fungi ongermination in mango stone was carried out in theDepartment of Pomology, KRC College ofHorticulture, Arabhavi, during 2003/04. The

Germination response of mango stones to different arbuscularmycorrhiza fungi

Santosh C P Patil and P B PatilKittur Rani Channamma College of Horticulture, Arabhavi – 591 310, India


experiment was laid out in completely randomizedblock design with ten treatments (nine AM fungi andone uninoculated control) and four replications. Twohundred seed stones were sown per treatment perreplication in 18 x 12 cm polybags filled withpotting mixture (soil, sand, and FYM in the ratio of2:1:2). Inoculation with the AM fungal was carriedout by placing 5 g of inoculum uniformly, whichconsisted on an average of 19 chlamydospores pergram at a depth of 5 cm. After putting a thin layer ofsoil on the inoculum, stones from Alphonso mangoesobtained from a single lot of uniform size and shapefrom a processing unit were placed and covered withsoil. Days required for emergence of the plumule(germination), days taken for 50% germination, daystaken for completion of germination, germinationpercentage, germination vigour index, the number ofchlamydospores, and per cent root colonizationwere recorded.

The GVI (germination index) was computedusing the formula

X1

X2

X3

Xn

GVI = —— + —— + —— + ..............+ ——D

1D

2D

3D

n

where, X1, X

2, X

3 … X

n were the number of seeds

germinated and D1,

D2,

D3 … D

n were the days taken

for germination.The number of extrametrical chlamydospores

produced by the AM fungi were determined by usingwet sieving and decanting method as described byGerdemann and Nicolson (1963). The per cent rootcolonization was determined using the method asdescribed by Phillips and Hayman (1970). The datarecorded on various parameters were subjected toFisher’s method of variance and interpretation of datawas done as described by Panse and Sukhatme (1967).

Results and discussionDays required for emergence of plumuleMango stones inoculated with Glomus leptotichum,Acaulospora laevis, Glomus intraradices, and Gigasporamargarita required the least number of days (7.00) forinitiation of germination (emergence of plumule).However, these values were statistically at par with thedays taken for germination of seeds inoculated withGlomus fasciculatum and Glomus monosporum (7.50days) and control (8.00 days). Inoculation with Glomusbagyaraji (13.00 days), Sclerocystis dussii (10.75 days),and Glomus mosseae (10.75 days) led to significantlymore days for initiation of germination than thoserecorded with control (8.00 days) or with othertreatments (see table below).

Days taken for 50% germinationWhereas the days taken by those mango stones for 50%germination, inoculated with G. leptotichum, A. laevis,G. intraradices, G. fasciculatum, G. monosporum, andGi. margarita (17.00) were statistically on par, (15-17days). Control seedlings attained 50% germinationwithin 20 days of but those inoculated with G. mosseae(24.00 days), S. dussii (23.00 days), and G. bagyarajitook much longer (22 days).

Days taken for completion of germinationThe data (see table below) indicate that the mangostones inoculated with Gi. margarita and A. laevis tooksignificantly fewer days to complete germination(100%) whereas those inoculated with G. leptotichum,G. intraradices , G. mosseae, G. fasciculatum,G. monosporum, and S. dussii took longer, and thosenot inoculated at all (control) or with G. bagyaraji tookthe longest time.

Effect of different arbuscular mycorrhiza fungi on germination of mango stones and microbial parameters

Number of days taken forgermination

Germination Spore count RootTreatment Initiation 50% Completion (%) Germination index colonization (%)

T1 – Sclerocystis dussii 10.75 23.00 44.75 28.76 52.89 399.00 86.75

T2 – Glomus leptotichum 7.00 15.00 44.00 57.25 169.76 412.00 86.50

T3 – Acaulospora laevis 7.00 17.00 42.00 65.00 192.91 657.00 92.75

T4 – Glomus intraradices 7.00 17.00 44.00 55.93 179.36 411.75 86.00

T5 – Glomus bagyaraji 13.00 22.00 46.00 28.00 52.86 387.50 84.25

T6 – Glomus mosseae 10.75 24.00 44.75 46.35 78.48 423.75 85.75

T7 – Glomus fasciculatum 7.50 17.00 44.75 58.43 179.17 656.00 93.00

T8 – Glomus monosporum 7.50 17.00 44.75 64.06 201.79 576.75 88.75

T9 – Gigaspora margarita 7.00 17.00 42.00 65.00 205.36 669.75 93.00

T10

– Control 8.00 20.00 47.25 49.72 160.90 60.50 20.00S. Em ± 0.77 1.01 0.855 0.73 1.13 50.65 1.93C.D. at 5% 2.29 2.92 2.47 2.09 3.25 146.27 5.59

C.D. – critical difference; S.Em – standard error of mean


Germination percentageInoculation of mango stones with A. laevis andGi. margarita resulted in significantly maximumgermination percentage (65.00%) followed byinoculation with G. monosporum (64.06%) andG. fasciculatum (58.43%). These treatments weresignificantly superior over control and rest of thetreatments. Significantly minimum germinationpercentage was recorded in stones inoculated withG. bagyaraji (28.00%), S. dussii (28.76%), andG. mosseae (46.35%) when compared to control(49.72%) and other treatments.

Germination vigour indexGermination index ranged from 52.86 to 205.36.Gi. margarita had shown significantly highestgermination index (205.36) followed byG. monosporum (201.79) and A. laevis (192.91),which were significantly higher than control(160.90). Significantly lower values of germinationindex were recorded in the treatments with S. dussii(52.89) and G. bagyaraji (52.86) followed byG. mosseae (78.48), even much lower than thegermination index values obtained in control.

ChlamydosporesSignificantly maximum extramatricalchlamydospores were present in the germinatingmedia inoculated with Gi. margarita (669.75/50 g ofsoil), which were statistically similar with the countsin the media inoculated with A. laevis (657.00/50 gof soil), G. fasciculatum (656.00/50 g of soil), andG. monosporum (576.75/50 g of soil). Significantlylower numbers of chlamydospores were recorded incontrol soils (60.50/50 g of soil). Least numbers ofchlamydospores were recorded in the potting mixtureinoculated with G. bagyaraji (387.50/50 g of soil)and S. dussii (399/50% g of soil).

Per cent root colonizationRoot colonization by the AM fungi ranged from84.25% to 93%. Stones inoculated with Gi.margarita and G. fasciculatum (93.00%) recordedsignificantly higher root colonization(mycorrhization), which was statistically at par withA. laevis (92.75%) and G. monosporum (88.75%).The uninoculated stones showed significantly leastroot infection (20.00%).

The stones inoculated with Gi. margarita,A. laevis, G. monosporum, and G. fasciculatumrecorded higher percentage of germination, withhigher germination index of 205.36 to 169.76 whichwas attained in a span of 42 to 44.75 days, recordingsignificantly in much lower time span whencompared to the germination in control stones.

Although there are no reports available to explainthe role of AM fungi in initiation of earlygermination of mango stones, the increased

germination, germination index, and fewer daystaken for germination observed in the presentinvestigation can be attributed to a number ofsequential events that occur, beginning with thepresence of chlamydospores in the rhizosphere(germinating medium) of mango stones. Theinitiation of AM association begins with thegermination of the spore and the contact of fungalhyphae with root epidermis. There is a molecularand chemical dialogue that occurs between the AMfungi and host (Giovannetti, Avio, Sbrana, et al.1993; Harborne and Williams 2000). Plant exudatesfrom the appropriate host are necessary to initiatethe symbiosis. The leachates from mango stonesmight have influenced the germination ofchlamydospores and hyphal growth. Further, sporegermination and some independent hyphal growth ofthe AM fungi can occur in absence of roots and thissaprophytic phase is crucial to the fungal life cycle assurvival depends on the ability to rapidly andeffectively infect suitable hosts. Thus, the AM fungigerminate and some independent hyphal growth isachieved until the emergence of the radical, whichemerges much sooner than the plumule does.Probably the radical gets infected as soon as itemerges. Further, certain of the introduced AMfungi, being efficient root colonizers (Table 1),might establish well in the rhizosphere (Manjunath,Mottan, and Bagyaraj 1983). High level of rootinfection by the AM fungi might affect furthergrowth, namely the growth of roots and shoots(Shivashankar and Iyer 1988).

Mycorrhizal fungi are known to produce Auxins,cytokinins, gibberellins, and vitamins in pure culture(Miller 1971; Crafts and Millar 1974; Slankis1975), and the effects of these compounds on plantgrowth and development are well documented(Torrey 1976). Gibberellins have been found to beeffective in increasing mango germination (Ram1997). The increase in germination the percentagewith higher germination index and fastergermination observed with a few AM fungi in thepresent investigation could be attributed to theproduction of gibberellic acid by the AM fungi.Gange, Brown, and Sinclair (1993) experimentallydemonstrated that the AM fungal presence of the AMfungi affects seed germination of a secondarysuccession site.

Results of the present investigation using nine AMfungi on mango stone germination did not reveal anyuniform response, which indicates that the extent ofthe AM fungal association with the host is also amorphogenetically determined phenomenon. In spiteof the fact that the AM fungi are not host-specific(Smith and Read 1997), they vary widely in theireffectiveness. It was surprising to note thatinoculation with Gi. margarita, A. laevis,G. monosporum, and G. fasciculatum significantlyincreased the germination whereas that withG. bagyaraji, S. dussii, and G. mosseae decreased it,compared to the control. Despite the lack of absolute


specificity accorded to the AM symbiosis, thesymbiotic efficiency is under genetic control and isaffected by host and fungal species. Thus, results ofthe present study clearly indicate that G. bagyaraji,S. dussii, and G. mosseae did not assist mango ingermination. Variation in the nature of mycorrhizaleffects on the hosts has been noted by Janos (1980)and Johnson, Graham, and Smith (1997), a conceptsuggested by Lohman (1927) long ago. Thus, thereare notable cases of growth depression apparentlycaused by the AM fungi in non-host species (Francisand Read 1984) or in host species when phosphateavailability is high (Mosse 1973 and Peng,Eissenstat, Graham, et al. 1993) or in other cases(Modjo and Hendrix 1986).

Thus, when we consider the inoculation of seedor plants, selection of the right fungal partner isessential to successful and efficient symbiosis.Mango stone germination can be increased by 10%to 15% by inoculation with Gi. margarita,A. laevis, G. monosporum, and G. fasciculatum.

ReferencesCrafts C B and Millar C O. 1974Detection and identification of cytokininsproduced by mycorrhizal fungiPlant Physiology 54: 586–588

Francis R and Read D J. 1984The contributions of mycorrhizal fungi to thedetermination of plant community structureIn Management of Mycorrhizas in Agriculture, Horticultureand Forestry, edited by A D Robson, L K Abbott, andN MalajczukKluwer, Dordrech (C/o Koide and Mosse. 2004.A history of research on arbuscular mycorrhizaMycorrhiza 14: 145–163

Gange A C, Brown V K, and Sinclair G S. 1993Vesicular-arbuscular mycorrhizal fungi: adeterminant of plant community structure in earlysuccessionFunctional Ecology 7: 616–622

Gerdemann J W and Nicolson J H. 1963Spores of mycorrhiza Endogone species extractedfrom soil by wet sieving and decantingTransactions of the British Mycological Society 46: 235–244

Giovannetti M, Avio L, Sbrana C, Citernesi A S. 1993Factors affecting apperessorium development invesicular-arbuscular mycorrhizal fungus Glomusmosseae (Nicol. & Gerd.) Gerd. & TrappeNew Phytologist 123: 114–122

Harborne J B and Williams C A. 2000Advances in flavonoid research since 1992Phytochemistry 55: 481–504

Janos D P. 1980Mycorrhiza influence tropical successionBiotropica 12: 56–64

Johnson N C, Graham J H, and Smith F A. 1997Functioning of mycorrhizal associations along themutualism-parasitism continuumNew Phytologist 135: 575–585

Lohman M L. 1927Occurrence of mycorrhiza in Iowa forest plantsUniversity of Iowa Studies in Natural History 11: 33–58

Manjunath A, Mottan R, and Bagyaraj D J. 1983Response of citrus to vesicular arbuscularmycorrhizal inoculation in unsterile soilCanadian Journal of Botany 61: 2729–2732

Miller C O. 1971Cytokinin production by mycorrhizal fungiIn Mycorrhizae, edited by E HacskayloGPO, Washington. pp. 168–174

Modjo H S and Hendrix J W. 1986The mycorrhizal fungus Glomus macrocarpum asa cause of tobacco stunt diseasePhytopathology 76: 688–691

Mosse B. 1973Plant growth responses to vesicular-arbuscularmycorrhiza. IVIn Soil Given Additional PhosphateNew Phytologist 72: 127–136

Panse V G and Sukhatme P V. 1967Statistical Methods for Agricultural Workers,pp. 152–161New Delhi: Indian Council of Agricultural Research

Peng S, Eissenstat D M, Graham J H, Williams K,Hodge N C. 1993Growth depression in mycorrhizal citrus athigh-phosphorus supplyPlant Physiology 101: 1063–1071

Phillips J M and Hayman D S. 1970Improved procedure for clearing roots and stainingparasitic and vesicular-arbuscular mycorrhizalfungi for rapid assessment of infectionTransactions of the British Mycological Society 55: 158–161

Ram S. 1997PropagationIn The Mango: botany, production and uses, edited byR E LitzWallingford, Oxford, UK: CAB International, 367 pp.

Reddy B M S. 1991Selection of efficient VA mycorrhizal fungi formango, acid lime, and papayaMSc (Agriculture) Thesis, University of AgriculturalSciences, Bangalore

Ruehle G D and Ledin R B. 1955Mango growing in FloridaFlorida Agriculture Experimental Station Bulletin 574. p. 90


New approaches

Identification of the AMF (arbuscular mycorrhizalfungi) on roots is almost impossible withmorphological methods. Due to the presence ofcontaminating fungi, it is also difficult withmolecular biological techniques. To allow broadinvestigation of the population structure of the AMFin the field, Renker C, Heinrichs J, Kalderf M, andBuscot F (2003) have established a new method toselectively amplify the ITS (internal transcribedspacer) region of most AMF with a unique primerset (Mycorrhiza 13(4): 191–198). Based on availablesequences of the rDNA, a primer pair specific for

the AMF and a few other fungal groups weredesigned and combined in a nested PCR (polymerasechain reaction) with the already established primarpair ITS5/ITS4. Amplification from contaminatingorganism was reduced by an Alu I restriction afterthe first reaction of the nested PCR. The method wasassessed at five different field sites representingdifferent types of habitats. Members of all majorgroups within the Glomeromycota (exceptArchaeosporaceae) were detected at different sites.Gigasporaceae also proved detectable with themethod, based on cultivated strains.

A new approach for characterization of arbuscular mycorrhiza fungi

Centre for Mycorrhizal Culture Collection

User-friendly digital herbaria of arbuscular mycorrhizal fungi forfacilitating identification

Reena Singh and Alok AdholeyaCentre for Mycorrhizal Research, T E R I, Darbari Seth Block, I H C Complex, Lodhi Road,New Delhi – 110 003, India

Species of the AM (arbuscular mycorrhizal) fungiare currently placed in Zygomycetes in the orderGlomales. In the past two decades, there has been anincrease in the descriptions of species in theGlomales and in the enormous amount of researchon the effects some of these fungi have on the plant

growth. The proliferation of species and variation inthe standards of descriptions inevitably cause muchuncertainty in the minds of the researchers who wantto identify their experimental organisms.Unfortunately, the identification of this group offungi is difficult. The AM fungi are obligate

Shivashankar S and Iyer R. 1988Influence of vesicular-arbuscular mycorrhiza ongrowth and nitrate reductase activity of blackpepperIndian Phytopathology 41: 428–433

Slankis V. 1975Hormonal relationships in mycorrhizaldevelopment, pp. 231–298 inEctomycorrhizae, edited by C G Marx and T T KozlowskiNew York: Academic Press

Smith S E and Read D J. 1997Mycorrhizal symbiosis (2nd edn)San Diego: Academic Press (C/o: Koide and Mosse.2004. A history of research on arbuscularmycorrhiza)Mycorrhiza 14: 145–163

Torrey J A. 1976Root hormones and plant growthAnnual Review of Plant Physiology 27: 435–459


symbionts and cannot yet be cultured on artificialmedia except for a few species. Their classificationis based on the morphology of their resting spores.This implies that for identification purposes, thefungi must have sporulated and that spores must beseparated from the substrate or soil in which theystarted their reproductive phase on a host plant.There is an urgent need for a system to identify or atleast, help identify them. Various attempts have beenmade to devise dichotomous or synoptic keys. Thelimited use and success of such keys is the result ofour limited knowledge of taxonomic character andmaterials used for description. Thus, any effort toimprove the knowledge of taxonomy of these fungiwould be an important step. As this field of scienceis fast losing importance, it becomes the necessity ofthe taxonomists to exchange and disseminateinformation about it.

The information can be imparted by either givingformal training to the researchers or bydisseminating the collated information onbiodiversity. The demand for readily accessiblebiological expertise is growing. In the scientificcommunity and in society, there is an increasingneed for up-to-date information. In the past,scientific data were mainly for scientists. Now theinformation is needed by a far larger group of usersfor various purposes, such as pure or appliedresearch, industrial processes, nature conservation,biodiversity inventories, policy-making, etc. In allthese cases, it may be vital that the information is upto date and valid. At the same time, the size of the

required information sets is growing. We need toknow more, update more, and check our data againsta bigger variety of the existing information. Last butnot least, we are now more in a hurry than everbefore. There is a need to access the new data fast aswe have to make quick decisions dictated by deadlinesof the fast-speeding wheels of time. The ever-growinghunger for information among an increasing number ofusers all over the globe makes it clear that traditionalways of dealing with information dissemination nolonger suffice. The need for publishing scientists’results becomes too slow to meet with our demands;the sheer size of the information is too large to be dealtwith on paper, and the information itself (texts,pictures) is too complicated to be dealt with by aprinting press.

Digital media and electronic data disseminationare fast becoming new instruments for economic andefficient dissemination of our knowledge. The newtools offer exciting possibilities not only fordistributing what we know to eager users at large,but also to the science itself and the way we work.Through the media we are able to store and sharemuch more of what we see and experience. Compactdiscs have become a worldwide-accepted data carrierfor digital information.

At the Centre for Mycorrhizal Research, digitalherbaria of 34 AM fungal species were prepared. Inthese herbaria, fact sheets of each species wereprepared in which the diagnostic features are listedalong with the relevant photographs (see Figure 1 asan example).

Sporocarps 460–750 mm broad, globose tosubglobose, minutely verrucose from exposed tips ofspores formed radially in a singly, tightly packed layeraround a central plexus of hyphae; base indented

Chlamydopores 140–185 mm, clavate to subcylindric,tapering to a hyphal attachment 70–10 mm diameter.Spore walls 1.5–5 mm thick on the sides, at the sporeapex thickened to 17–25 mm, at the base thickenedto 5–8 mm and occluding the attachment at maturity.

Figure 1 Fact sheet of Sclerocystis clavispora Trappe


In view of the WTO (World Trade Organization)obligations, the Indian agriculture needs to braceitself to become competitive in the global marketthrough the development of value-added agriculturalproducts. Among the major rapeseed-mustard-growing Asian countries, China is on the way to achange over from high erucic, high glucosinolate-containing traditional varieties to the internationallyaccepted canola quality cultivars, but such a changeover is yet to begin in India.

T E R I has taken an initiative in this direction: abroad-based awareness workshop on Public–PrivatePartnership: A changeover to value added oilseed inIndia was jointly organized by T E R I (The Energyand Resources Institute), New Delhi, and JNKVV(Jawaharlal Nehru Krishi Vishwa Vidyalaya) on26 February 2005. The workshop, co-sponsored bythe CSIR/TMOP&M (Council of Scientific andIndustrial Research / Technology Mission on Oil,Seeds, Pulses and Maize), Ministry of Agriculture,and Syngenta India Ltd, was well-attended throughparticipation from all stakeholders, comprisingscientists, agriculture experts,nutritionists, processing andpost-harvest engineers,industrialists, food technologists,farmers, and field extensionofficers of the Department ofAgriculture, who shared their viewson various issues pertaining to achange over to value-addedoilseeds in view of the post-WTO(World Trade Organization)scenario emerging in India.

Dr V S Tomar, Director-Research Services, JNKVV, Jabalpur, assisted asChairman and Dr C B Singh, Dean, College ofAgriculture, Jabalpur, acted as co-chairman for allsessions. Dr D P Singh, Vice Chancellor, JNKVV,presided over the plenary session. He appreciated theefforts undertaken for development of nutritionallyimproved quality variety of rapeseed-mustard, which isone of the few exceptions where varietals improvementhas been undertaken for the purpose of domesticconsumption as well as making an export-orientedproduct; this concept should be extended to other cropstoo. This was further substantiated by Prof. S K Rao,Head, Department of Plant Breeding and Genetics; andDirector, Farms, JNKVV, and his fellow scientists.

Dr Abha Agnihotri, Fellow, Plant Biotechnologyand Group Leader, Quality Improvement, T E R I,New Delhi, presented the success story: Developmentof nutritionally improved quality rapeseed-mustardvariety TERI Uttam–Jawahar. Intelligent selection ofplants having desired quality parameters as well asgood yielding attributes play the most critical part indevelopment of rapeseed-mustard of canola-qualityvariety. Thus, T E R I scientists employed

biotechnological approaches to supplement theconventional methods of plant breeding to developnew strains of improved quality, seven of which areregistered at the ICAR (Indian Council of AgriculturalResearch). One of them, (TERI[OO]R9903; INGRNo.04077), formed the first double-low variety ofBrassica napus and is released for cultivation in MadhyaPradesh. Attributes of this high-value crop, based onmulti-location testing under the AICRP R&M (AllIndia Coordinated Research Project on Rapeseed-Mustard) of the ICAR, and the data procured at theJNKVV centres and on farm trials in MP (MadhyaPradesh), were enumerated. Dr Agnihotriemphasized that the main focus is not only ondeveloping nutritionally superior quality varieties butalso on developing systems to involve farmers,extractors, and promoters/exporters for ultimatebenefits to all. Processing engineers were of the viewthat the existing models of small oil expeller at thevillage level may be adapted for efficient extraction ofoil. Mr Goutia, the Padma Bhushan recipient andfarmers’ representative was of the view that the

workshop has been extremelybeneficial to promote the linkagebetween farmers and scientists. Itis therefore very valuable forfuture betterment of agriculture.

The release of a new variety,T E R I Uttam–Jawahar havinginternationally acclaimed canolaquality for cultivation across MPis a landmark achievement forthe agriculture sector in general,and oil seeds sector in particular,as this variety is rich in oil

(approximately 43%) and has high levels of oleicacid (over 60%), which prevents it from gettingrancid soon. Moreover, it tolerates pod shatteringand white rust, and has shorter crop cycle for thebenefit of the farming community. It has proven itssuitability to grow in the mustard-growing belts,moreover it has been found to be economical ascompared to other crops grown under limitedirrigations, and can be an alternative crop in soybean-based cropping systems. Gobhi Sarson, which hasearlier been restricted to only a few northern states, hasnow been developed as a versatile crop for the mustard-growing belts of MP. Even with a conservativeestimate, the oil and meal together will fetch almostdouble the price per hectare, and it has good potentialin the domestic as well as global markets as a fodder-cum-oilseed-cum oil meal crop for added benefits. Therelease of superior quality mustard variety waswelcomed by all farmers who showed an interest togrow the high-quality oilseed crop from the nextseason. Establishing linkages for contract farming foran assured marketing system and premium to farmers isbeing explored for benefits to all stakeholders.

Improved variety rapeseed mustard: TER I cultivation Uttam–Jawahar

A broad-based awareness workshop on a changeoverto value-added oilseeds in India (jointly organized byT E R I and JNKVV)


Dr Andre Fortin is theworld’s leadingmycorrhizologist activelyassociated with mycorrhizaresearch for over the pastforty years. Born in 1937in Quebec, Canada,Dr Fortin did his MScfrom the University ofWisconsin in 1964 andPhD from Laval University(Quebec) in 1966. Though

now retired, Dr Fortin is presently quite active inthe R&D on mycorrhizae. He was scientificchairman for fourth International Conference onMycorrhizae held in 2004. He is presently busy infinalizing the foundation of the InternationalMycorrhiza Society. Dr Fortin was one of the keyscientists who planned and helped in theestablishment of Mycorrhiza Network-Asia at TER Iin 1988. Out of his varied types of researchactivities, his five most significant contributions toresearch on mycorrhizae are highlighted below.

Aseptic cultivation of mycorrhizal fungi onroot organ culturesDr Fortin (1966) developed an aseptic technique forformation of ectomycorrhiza (ECM) on root-organculture (PhD thesis). This work became theforerunner in synthesis of the AM (arbuscularmycorrhiza) on genetically transformed carrot roots.Development of this cultivation technique for theAM fungi allowed investigations into manyfundamental aspects of the complex biology of thissymbiosis, which, according to Dr Fortin, arehighlighted in a book by Declerck Strullu andFortin (in press). These contributions allowed thedevelopment of a unique group of industrial productsby Premier Tech in Canada and T E R I in India,thus making large-scale use of the AM inocula anaccessible technology for sustainable agriculture.

The eco-physiological relationshipbetween ectomycorrhizal fungi andconifer trees of boreal forestsDr Fortin and his team of workers have shown thatseasonality of carbon allocation rather than lowtemperature and high humidity was the primaryfactor explaining why basidiomes of the ECM fungiare produced in late summer and early fall in borealforest. They showed that in these regions, themaximum physiological activity (32 p absorption) ofmycorrhizal roots was reached by mid-August andthat this peak of activity was concomitant with a

massive flow of sugars to the roots. This shift in thepattern of carbon allocation from the aerial to theunderground parts of the tree followed theestablishment of dormancy. Dr Fortin and his teamhave tested this hypothesis under controlledconditions wherein they were able to demonstratethat it was possible to trigger fruit body developmentby reducing day length momentarily to induce buddormancy. When young white pines thus pretreated,were exposed to full sunlight under a longphotoperiod and at normal temperatures (that is,without a cold treatment or an excess of nitrogen),they kept producing basidiomata for up to 15months. The cumulative weight of these fruit bodieswas 3.5 times greater than the weight of the hosttrees. They are currently pursuing R&D on wildedible ECM mushrooms as well as their productionunder controlled conditions.

Significance of arbuscular mycorrhiza inagricultureDr Fortin has conducted research on physiology ofthe AM and his research group developed the firstsoil-free AMF inoculum. This permitted pioneeringwork under field conditions without soilsterilization. The results obtained were largelyresponsible for recent advances in the R&D made byPremier Tech in the development of inocula for usein horticultural and agricultural production.Dr Fortin is the scientific advisor for this company.In collaboration with C. Planchette, Dr Fortincontributed to a comprehensive paper on themanagement of AMF in sustainable agriculture.

Role of mycorrhizal fungi in biocontrol ofsoil-borne plant root diseasesOne of the first studies of Dr Fortin and hiscolleagues on mycorrhizal fungi as biocontrol agentswas on the relationship between ectomycorrhizalfungi and the nematode, Aphelenchus avenae. Thestudies revealed that several of the ECM fungiallowed the growth and reproduction of thenematode. Rhizopogon roseolus, however, proved to behighly toxic, killing the nematode within minutes.These investigations were followed by thedemonstration of antibiotic production that inhibitedgrowth of several pathogenic fungi leading to theidentification of two active substances. Investigationson the AMF demonstrated that these fungi protectplant roots against disease. The mechanism is notonly due to the triggering of plant defencemechanism but also due to the reduction in thenumber of propagules of pathogenic fungi in therhizosphere even before they contact the roots.

Biography of Dr Andre Fortin

Dr Andre Fortin


Synergism between AM fungi and PSB(phosphate solubilizing bacteria)Dr Fortin and his colleagues demonstrated underaseptic conditions using the split-plate method thatneither the AM fungus, Glomus intraradices, nor thebacteria, Pseudomonas aeruginosa, were able toindividually solubilize tricalcic phosphorus. Theywere, however, able to do so in concert. It was also

shown that either ammonium on nitrate could beused for this activity. Also, pH changes could notexplain the results. Dr Fortin observed for the firsttime, the formation of biofilm on extramatrical AMFhyphae. Currently, R&D is being pursued on thepresence, species composition, and functions ofbacterial biofilms on the surface of the AM fungalmycelium under controlled laboratory conditions.

Recent references

The latest additions to the network’s database on mycorrhiza are published here for members’ information. TheMycorrhiza Network will be pleased to supply any of the available documents to bonafide researchers at anominal charge. This list consists of papers from the following journals.

P Applied Soil EcologyP Canadian Journal of Botany-Revue Canadienne

De BotaniqueP Canadian Journal of Plant ScienceP Ecological ApplicationsP Ecology LettersP Environmental StudiesP Fems Microbiology LettersP Folia MicrobiologicaP Forest PathologyP Functional EcologyP GeodermaP Journal of Applied Botany and Food Quality-

Angewandte Botanik

P Journal of EcologyP Journal of Plant PhysiologyP Microbial EcologyP MycotaxonP NatureP New PhytologistP PhytochemistryP Plant and SoilP Plant JournalP Plant Soil and EnvironmentP Soil Science and Plant Nutrition

Copies of papers published by mycorrhizologists during this quarter may please be sent to the Network forinclusion in the next issue.

Ahonen-Jonnarth U*,Roitto M, Markkola A M,Ranta H, Neuvonen S. 2004

Bagger J and Madsen T V*.2004

Blum L K*, Roberts M S,Garland J L, and Mills A L.2004

Effects of nickel and copper on growth and mycorrhiza of Scots pineseedlings inoculated with Gremmeniella abietinaForest Pathology 34(6): 337–348[Department of Mathematics, University of Gavle, Natural & Computer Science,SE-80176 Gavle, Sweden]

Morphological acclimation of aquatic Littorella uniflora to sediment CO2

concentration and wave exposureFunctional Ecology 18(6): 946–951[Department Plant Biology, Aarhus University, Nordlandsvej 68, DK-8240Risskov, Denmark]

Distribution of microbial communities associated with the dominant highmarsh plants and sediments of the United States east coastMicrobial Ecology 48(3): 375–380[Department of Environmental Science, University of Virginia, Microbial EcologyLaboratory, Charlottesville, VA 22904, USA]

Name of the author(s) andyear of publication

Title of the article, name of journal, volume no., issue no., page nos[corresponding address given for author whose name is highlighted in asterisk]


Colgan W* and Trappe J M.2004

Cornejo P, Azcon-AguilarC, Barea J M, Ferrol N*.2004

Das K*, Sharma J R, andMontoya L. 2004

de la Providencia I E,de Souza F A, Fernandez F,Delmas N S, Declerck S*.2005

Entz M H*, Penner K R,Vessey J K, Zelmer C D,Martens J R T. 2004

Garcia C*, Roldan A, andHernandez T. 2005

Gormsen D, Olsson P A*,and Hedlund K. 2004

Hijri M and Sanders I R*.2005

Isayenkov S*, Fester T, andHause B. 2004

NATS truffle and truffle-like fungi 10: Pachyphloeus thysellii sp nov(Pezizaceae, Pezizomycotina)Mycotaxon 90(2): 281–284[School of Biological Sciences, Louisiana Tech University, Ruston, LA 71272,USA]

TTGE (temporal temperature gradient gel electrophoresis) as a tool for thecharacterization of arbuscular mycorrhizal fungiFEMS Microbiology Letters 241(2): 265–270[Dept Microbiol Suelo & Sist Simbiot, CSIC (Consejo Superior de InvestigacionesCientificas), Estacion Experimental Zaidin, eC Professor Albareda 1, Granada18008, Spain]

Lactarius in Kumaon Himalaya 3: A new species of subgenus LactifluusMycotaxon 90(2): 285–290[Botanical Survey of India, 192 Kaulagarh Road, Dehra Dun – 248 195, India]

Arbuscular mycorrhizal fungi reveal distinct patterns of anastomosisformation and hyphal healing mechanisms between different phylogenicgroupsNew Phytologist 165(1): 261–271[University of Catholique Louvain, MUCL (Mycotheque de l’Universite Catholiquede Louvain), Unite Microbiol, 3 Pl Croix Sud, B-1348 Louvain, Belgium]

Mycorrhizal colonization of flax under long-term organic and conventionalmanagementCanadian Journal of Plant Science 84(4): 1097–1099[Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2,Canada]

Ability of different plant species to promote microbiological processes insemiarid soilGeoderma 124(1–2): 193–202[Department of Soil & Water Conservation, CSIC (Consejo Superior deInvestigaciones Cientificas), CEBAS (Centro de Edafología y Biología Aplicada delSegura), POB 4195, Murcia 30080, Spain]

The influence of collembolans and earthworms on AM fungal myceliumApplied Soil Ecology 27(3): 211–220[Department of Ecology, Lund University, Ecology Building, SE-22362 Lund,Sweden]

Low gene copy number shows that arbuscular mycorrhizal fungi inheritgenetically different nucleiNature 433(7022): 160–163[Department Ecology & Evolution, University of Lausanne, CH-1015 Lausanne,Switzerland]

Rapid determination of fungal colonization and arbuscule formation inroots of Medicago truncatula using RT (real-time) PCRJournal of Plant Physiology 161(12): 1379–1383[Department of Metabolism 2, Institute of Plant Biochemistry, Weinberg 3,D-06120 Halle Saale, Saale, Germany]




Koide R T*, Xu B, ShardaJ, Lekberg Y, Ostiguy N.2005

Koukol O*, Gryndler M,Novak F, Vosatka M. 2004

Kula A A R, Hartnett D C,and Wilson G W T. 2005

Lalli G*, Leonardi M, andPacioni G. 2004

Liu Q, Loganathan P*,Hedley M J, Skinner M F.2004

Noldt G*, Bauch J, andKoch G. 2004

Oba H, Shinozaki N,Oyaizu H, Tawaraya K,Wagatsuma T, Barraquio W L,Saito M*. 2004

Olsson P A*, Eriksen B E,and Dahlberg A. 2004

Ponce M A*, Scervino J M,Erra-Balsells R, Ocampo JA, Godeas A M. 2004

Reinhart K O* andCallaway R M. 2004

Evidence of species interactions within an ectomycorrhizal fungalcommunityNew Phytologist 165(1): 305–316[Department of Horticulture, Pennsylvania State University, University Park,Pennsylvania 16802, USA]

Effect of Chalara longipes on decomposition of humic acids from Picea abiesneedle litterFolia Microbiologica 49(5): 574–578[Academic Science, Czech Republic, Institute of Botany, CS-25243 Pruhonice,Czech Republic]

Effects of mycorrhizal symbiosis on tallgrass prairie plant-herbivoreinteractionsEcology Letters 8(1): 61–69[Division of Biology, Kansas State University, Ackert Hall, Manhattan, KS 66506,USA]

A multiple approach to the taxonomy of Lactarius rubrozonatus(Basidiomycota, Russulales)Mycotaxon 90(2): 399–408[Dipartimento Sci Ambientali, University of Aquila, I-67100 Laquila, Italy]

The mobilisation and fate of soil and rock phosphate in the rhizosphere ofectomycorrhizal Pinus radiata seedlings in an Allophanic soilPlant and Soil 264(1–2): 219–229[Natural Resources Institute, Massey University, Private Bag 11 222, PalmerstonNorth, New Zealand]

Structure of the fine roots of Metasequoia glyptostroboides Hu et Cheng andits adaptability to various sitesJournal of Applied Botany and Food Quality-Angewandte Botanik 78(3): 178–184[University of Hamburg, Zentrum Holzwirtschaft, Abt Holzbiol, Division WoodBiology, Department Wood Science, Leuschnerstr 91, D-21031 Hamburg, Germany]

Arbuscular mycorrhizal fungal communities associated with some pioneerplants in the Lahar area of Mt. Pinatubo, PhilippinesSoil Science and Plant Nutrition 50(8): 1195–1203[National Institute of Agroenvironm Science, Tsukuba, Ibaraki 3058604, Japan]

Colonization by arbuscular mycorrhizal and fine endophytic fungi inherbaceous vegetation in the Canadian High ArcticCanadian Journal of Botany-Revue Canadienne De Botanique 82(11): 1547–1556[Department of Microbial Ecology, Lund University, Ecology Building, SE-22362Lund, Sweden]

Flavonoids from shoots, roots and roots exudates of Brassica albaPhytochemistry 65(23): 3131–3134[Fac Ciencias Exactas & Nat, Dept Quim Organ, University of Buenos Aires,CIHIDECAR, CONICET, Pabellon 2,3P Ciudad Univ, RA-1428 Buenos Aires,D F, Argentina]

Soil biota facilitate exotic Acer invasions in Europe and North AmericaEcological Applications 14(6): 1737–1745[Department of Biology, Indiana University, Jordan Hall, Room 127,1001 E third St, Bloomington, IN 47405, USA]




Ribosomal RNA gene sequence diversity in arbuscular mycorrhizal fungi(Glomeromycota)Journal of Ecology 92(6): 986–989[University of Hohenheim, Inst Pflanzenbau & Gunland 340, D-70593 Stuttgart,Germany]

Pine microsatellite markers allow roots and ectomycorrhizas to be linked toindividual treesNew Phytologist 165(1): 295–304[Macaulay Land Use Research Institute, Aberdeen AB15 8QH, Scotland]

Phosphorus and intraspecific density alter plant responses to arbuscularmycorrhizasPlant and Soil 264(1–2): 335–348[Department of Crop Science, North Carolina State University, Campus Box7620, Raleigh, NC 27695, USA]

The tomato carotenoid cleavage dioxygenase 1 genes contribute to theformation of the flavor volatiles beta-ionone, pseudoionone, andgeranylacetonePlant Journal 40(6): 882–892[Horticultural Science Plant Molecular & Cellular Biology Program, University ofFlorida, POB110690, Gainesville, FL 32611, USA]

Performance and gene effects for wheat yield under inoculation ofarbuscular mycorrhiza fungi and Azotobacter chroococcumPlant Soil and Environment 50(9): 409–415[Haryana Agricultural University, Hisar, Haryana, India]

Functioning of ectomycorrhizae and soil microfungi in deciduous forestssituated along a pollution gradient next to a fertilizer factoryPolish Journal of Environmental Studies 13(6): 715–721[Institute of Botany, Zaliuju Ezeru 49, LT-2021 Vilnius, Lithuania]

Microbial inoculation for improving the growth and health ofmicropropagated strawberryApplied Soil Ecology 27(3): 243–258[MTT Agrifood Research Finland, Laukaa Res and Elite Plant Station, FIN-41330Vihtavuori, Finland]

Host plant differences in arbuscular mycorrhizae: Extra radical hyphaedifferences between an invasive forb and a native bunchgrassPlant and Soil 264(1–2): 335–344[Department of Land Resources & Environmental Science, Montana StateUniversity, Bozeman, MT 59717, USA]

Genetic structure of Cenococcum geophilum populations in primarysuccessional volcanic deserts on Mount Fuji as revealed by microsatellitemarkersNew Phytologist 165(1): 285–293[Nihon University, Coll Bioresource Science, Department of Plant Science andResources, Kanagawa, Japan]

Rodriguez A*, Clapp J P,and Dodd J C. 2004

Saari S K, Campbell C D,Russell J, Alexander I J,Anderson I C*. 2005

Schroeder M S* and JanosD P. 2004

Simkin A J, Schwartz S H,Auldridge M, Taylor M G,Klee H J*. 2004

Singh R*, Behl R K, SinghK P, Jain P, Narula N. 2004

Stankeviciene D* andPeciulyte D. 2004

Vestberg A*, Kukkonen S,Saari K, Parikka P, HuttunenJ, Tainio L, Devos D,Weekers F, Kevers C,Thonart P, Lemoine M C,Cordier C, Alabouvette C,Gianinazzi S. 2004

Walling S Z and Zabinski CA*. 2004

Wu B Y*, Nara K, andHogetsu T. 2005




FORM IV (as per Rule 8)

Statement regarding ownership and other particulars about newsletter: Mycorrhiza News

1 Place of Publication: New Delhi

2 Periodicity of its publication: Quarterly

3 Printer’s Name: Dr R K PachauriWhether Citizen of India: YesAddress: T E R I

Darbari Seth Block, I H C ComplexLodhi Road, New Delhi – 110 003

4 Publisher’s Name: Dr R K PachauriWhether Citizen of India: YesAddress: T E R I


5 Editor’s Name: Dr Alok AdholeyaWhether Citizen of India: YesAddress: T E R I


6 Name and address of individuals T E R Iwho own the newsletter and partners Darbari Seth Block, I H C Complexor shareholders holding more than Lodhi Road, New Delhi – 110 003one per cent of the total capital:

I, R K Pachauri, hereby declare that the particulars given above are true to the best of my knowledge and belief.

Signature of Publisher

Date: 1 April 2005 Dr R K Pachauri

CAB Direct is the electronic platform developed by CABI Publishing, the publishing arm of CAB International <http://www.cabi.org>, for access to the organization’s varied abstracting and indexing database. CABI Publishingis one of the leading publishers of bibliographic database, books, CD-ROMs, and Internet resources in applied lifesciences, and as part of its mission it provides information solutions to support sustainable development.CABI’s range of information products covers over 90 years of research into the applied life sciences and relateddisciplines, including animal sciences, entomology, plant sciences, environmental sciences, human health,parasitology, mycology, crop protection, rural development, economics, and tourism. All articles of Mycorrhiza Newsare indexed and abstracted in CAB Direct.

We would like to invite you to avail of this opportunity to reach out globally by contributing articles inMycorrhiza News.

Please send your contributions toThe EditorMycorrhiza News Tel. 2468 2100 or 2468 2111T E R I (The Energy and Resources Institute) Fax 2468 2144 or 2468 2145Darbari Seth Block India +91 • Delhi (0) 11IHC Complex, Lodhi Road E-mail [email protected] Delhi – 110 003 Web www.teriin.org/pub

Inviting contributions Mycorrhiza News indexed and abstracted in CAB Direct

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Cancún, México17–22 July 2005

San Francisco, California, USA23–28 July 2005

ISSN 0970-695X Regd No. 49170/89

Printed and published by Dr R K Pachauri on behalf of the The Energy and Resources Institute, Darbari Seth Block, IHC Complex,Lodhi Road, New Delhi – 110 003, and printed at Multiplexus (India), C-440, DSIDC, Narela Industrial Park, Narela, Delhi – 110 040.

Editor Alok Adholeya • Associate Editor Reeta Sharma • Assistant Editors Yateendra Joshi, Pritika Kalra

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