About TER IA dynamic and flexible organization with a global vision and a local focus,TERI (The Energy and Resources Institute, formerly known as the TataEnergy Research Institute) was established in 1974. TER I’s focus is onresearch in the fields of energy, environment, and sustainable development,and on documentation and information dissemination. The genesis of theseactivities lie in TER I’s firm belief that the efficient utilization of energy,sustainable use of natural resources, large-scale adoption of renewableenergy technologies, and reduction of all forms of waste would move theprocess of development towards the goal of sustainability.

Biotechnology and Management of Bioresources DivisionFocusing on ecological, environmental, and food security issues, theactivities of the Biotechnology and Management of Bioresources Divisioninclude working with a wide variety of living organisms, sophisticatedgenetic engineering techniques, and, at the grass-roots level, with villagecommunities.

Mycorrhiza NetworkThe Biotechnology and Management of Bioresources Division’sMycorrhiza Network works through its three wings—the MIC (MycorrhizaInformation Centre), the CMCC (Centre for Mycorrhizal CultureCollection), and Mycorrhiza News.

The MIC is primarily responsible for establishing databases on Asianmycorrhizologists and on mycorrhizal literature (RIZA), which allowsinformation retrieval and supplies documents on request. The CMCC hasthe main objectives of procuring strains of both ecto and vesiculararbuscular mycorrhizal fungi from India and abroad; multiplying andmaintaining these fungi in pure cultures; screening, isolating, identifying,multiplying, and maintaining native mycorrhizal fungi; developing adatabase on cultures maintained; and providing starter cultures on request.Cultures are available on an exchange basis or on specific requests atnominal costs for spore extraction or handling.

Mycorrhiza NewsMycorrhiza News – a quarterly newsletter publishing articles compiled fromRIZA – invites short papers from budding and senior mycorrhizologists,giving methodologies being used in mycorrhizal research, providinginformation on forthcoming events on mycorrhiza and related subjects,listing important research references published during the quarter, andhighlighting the activities of the CMCC.

Vol. 18 No. 2

July 2006

2 Mycorrhiza News 18(2) • July 2006

Production of phosphatases bymycorrhizal fungi/rootsMany isolates of ectomycorrhizal fungi excreteenzymes, which are capable of digesting organicmatter. Phosphates become available following thedegradation of litter components by fungalphosphatases. Phytates and various other forms oforganic phosphorus are degraded and phosphates areabsorbed in the soil.

Acid phosphatases contribute to the mineralizationof organic phosphorus compounds in soil, thusenhancing the biological availability of releasedinorganic phosphorus. Both the roots andmycorrhizal symbionts produce acid phosphatases.

Production of phosphatases byectomycorrhizae

Studies conducted at the Institute of TerrestrialEcology, Merlewood Research Station, Grange-over-Sands, Cumbria, UK, on acid phosphataseproduction (determined by phenol release fromdi-sodium phosphate) by non-mycorrhizal andectomycorrhizal roots of pine (Pinus contorta) andbirch (Betula pubescens) seedlings in liquid culturesof six mycorrhizal and two saprophytic fungi(Agaricales), showed that phosphatase production bymycorrhizal roots was lower than in non-mycorrhizalroots of birch. But in roots of pine, phosphataseproduction of mycorrhizal and non-mycorrhizal rootswas not significantly different, except for mycorrhizaof Hebeloma crustuliniforme where it was as in birch(Dighton 1983).

Studies conducted at the Universitat Hohenheim,Institute fur Planzenernahrung, Stuttgart, Germany,on inorganic and organic phosphate, measured insoil solutions of bulk soil, rhizosphere soil, andmycorrhizal rhizoplane soil in 80-year-old Norwayspruce (Picea abies) stands showed that about 50%(range of 35%–65%) of total phosphate was presentas organic phosphate. Compared with bulk soil, theconcentration of readily hydrolysable organicphosphorus was lower in rhizosphere and rhizoplanesoils, and this difference was particularly marked inthe humus layer. In contrast, the concentration ofinorganic phosphate was the same or greater. Acidphosphatase activity present was 2–2.5 times greaterin the rhizoplane soil than in the bulk soil. Also,there was a positive correlation between phosphataseactivity and the length of mycelial hyphae. Thestudies stress the role of organic phosphate and acidphosphatase in the rhizosphere in phosphate uptakeby mycorrhizal roots of Norway spruce trees on acidsoils (Haussling and Marschner 1989).

Studies conducted at the Laboratoire de Biologiedes Ligneux, Universite de Nancy, Vandoeuvra-les-Nancy, Cedex, France, showed that the interface ofPisolithus tinctorius mycelium (strain H 445) with thePinus sylvestris root system was characterized by thedevelopment of polysaccharide fibrillae, which jointogether fungal and root surfaces. The developmentof such fibrillae is the result of mutual recognitionand corresponds to the phase of fungal attachment tothe host plant, leading to mycorrhizal formation.Parietal, plasmalemma, vacuolar, and cytoplasmicphosphatases, localized using immunocytochemistry,were evident only in the fungus. Extracellular

Phosphorus mobilization by mycorrhizal fungi—Part 1. Production and

detection of phosphatasesSujan Singh*TER I, Darbari Seth Block, IHC Complex, Lodhi Road, New Delhi – 110 003, India

* This paper has been compiled from TER I records in RIZA.

Contents

Phosphorus mobilization by mycorrhizal fungi—Part 1.

Production and detection of phosphatases 2

Research findings

Impact of arbuscular mycorrhizal fungus (Glomus

fasciculatum) and phosphate solubilizingbacterium (Pseudomonas striata) on growth and

nutrient status of Azadirachta indica L. 10

Arbuscular mycorrhizal fungi in the reclamationand restoration of soil fertility 13

Effect of bio-inoculant organisms on growth and yield of

Coleus forskohlii Briq.—an endangered medicinal plant 15

New approachesSustained in-vitro pine root development 18

Centre for Mycorrhizal Culture Collection

Functioning and relevance of heavy metal uptake andtranslocation by mycorrhizal fungi 18

Recent references 23

Forthcoming events 27

Mycorrhiza News 18(2) • July 2006 3

phosphatases secreted in the medium were detectedaround hyphae; however, they were absent in thespace separating hyphae from the root surface(Gourp and Pargney 1991).

Studies conducted at the Department of Biology,Clarkson University, Potsdam, USA; Department ofScience, Blubbton College, Blafftion, Ohio, USA;and Institute of Terrestrial Ecology, Grange-over-Sands, Cumbria, UK, on American beech (Fagusgrandifolia), occurring in a climax stand and graybirch (Betula populifolia), growing in mid-successional stands, showed that all the root typescollected early or late in the growing season on acommon soil type demonstrated similar oxygenconsumption rates on both a root length basis anddry weight basis. All the root types demonstratedmeasurable phosphatase activities at pH 5.0 withP-nitrophenyl phosphate, bis-p-nitrophenylphosphate, and phytic acid as substrates, and thus,the potential to mineralize monoester and diesterforms of organic phosphorus. The activity variedseasonally and among ectomycorrhizal morphotypes.Comparison of uptake of 32P from inositolphosphate by gray birch roots suggested thatphosphatase activity was linked to phosphorus uptakefrom this source (Antibus and Dighton 1993;Antibus, Bower, and Dighton 1997).

In studies conducted at the Department of Botany,University College Dublin, Belfield, Dublin,Ireland, ectomycorrhizal fungi (Paxillus involutus,Suillus grevillei, and two unidentified basidiomycetesfrom excised Sitka spruce mycorrhizae) wereisolated from stands of Sitka spruce either inmonoculture or in mixture with Japanese larch in anIrish conifer plantation. They were grown for 35days on modified Melin-Norkran liquid mediumcontaining ferric phytate as the phosphorus source.The cultures were then separated into wall andmembrane-bound, cytoplasmic and extracellularfractions and assayed for phosphatase. Wall- andmembrane-bound fractions contained the most activeacid phosphatase. The unidentified basidiomycetesshowed a lower substrate affinity and higher velocityof reaction than P. involutus and S. grevillei. Wall-and membrane-bound and cytoplasmic phosphataseactivities were optimum over a broad pH range(4.0–6.0). Various methods were used to releasewall-bound phosphatase and a high proportion(49%–88%) appeared to be tightly held within thewall. Phosphatase released from the wall bysonication had a similar Km to wall-boundphosphatase, but Vmax was lower. The use of anumber of substrates demonstrated a high affinity forinorganic pyrophosphate and sodiumbeta-glycerophosphate, but a low phytase activity(McElhinney and Mitchell 1993).

In studies conducted at the Department of Botany,University of Helsinki, Unioninkatu, Helsinki,Finland, on morphologically similarectomycorrhizae between Scotspine (Pinus sylvestis)and different geographical isolates of Suillus

variegatus and Suillus bovinus were synthesized undersemi aseptic conditions in sand-peat growth mixturecontaining urea formaldehyde as the organicnitrogen source. Acid phosphatase isozyme activitieswere detected in the ectomycorrhizae but wererestricted to a single isozyme known to be commonto both fungal species (Sen 1990).

Production of phosphatases byVA-mycorrhizal fungiStudies conducted at the ICRISAT (InternationalCrops Research Institute for the Semi-Arid Tropics),Patancheru, Andhra Pradesh, India, showed that insterilized soil, acid and alkaline phosphataseactivities in the rhizospheres of mycorrhizal and non-mycorrhizal plants were higher than in non-rhizosphere soil. No significant differences werenoted between the rhizospheres of mycorrhizal andnon-mycorrhizal plants. In unsterile soil, differencesin alkaline phosphatase activity among the threetreatments were not significant. With acidphosphatase, the activity was highest in therhizosphere of mycorrhizal plants, followed byrhizosphere of non-mycorrhizal plants and non-rhizosphere soil (Krishna and Bagyaraj 1985).

Studies conducted at the Department of Biology,Clarkson University, New York, USA and Institute ofTerrestrial Ecology, Cumbria, UK, on red maple (Acerrubrum), growing in a mid-successional stand,demonstrated oxygen consumption rates similar toAmerican beech and gray birch root material collectedearly and late in the growing season on a common soiltype. All the root types examined demonstratedmeasurable phosphatase activity at pH 5 withp-nitrophenyl phosphate, bis-p-nitrophenyl phosphate,and phytic acid as substrates, with rates of hydrolysisgreatest with p-nitrophenyl phosphate. VAM (vesiculararbuscular mycorrhiza) infected roots of red mapleconsistently demonstrated lowest phosphatase activityon both length and dry weight basis. Comparison of32p uptake from inositol phosphate by red maple andgray birch roots suggested that the phosphatase activitywas linked to phosphorus uptake from this source(Antibus and Dighton 1993).

Comparative effect of differentmycorrhizal fungi on phosphatase activityin soil and/or plant rootsIn studies conducted at the Biological Laboratory,University of Kent, Canterbury, Kent, UK, theplants of rape, wheat, and onion were inoculatedsingly with Glomus geosporum, G. mosseae, andG. monosporum or were left uninoculated and weregrown in sand with little available phosphorus. Rootand rhizosphere levels of acid phosphatase activitywere higher for plants inoculated with G. geosporumand G. mosseae than for control plants. Infection byG. monosporum did not result in a similar increase inphosphatase activity, but plant growth was improved.

4 Mycorrhiza News 18(2) • July 2006

In further experiments on wheat, increases inphosphatase induced by G. geosporum or G. mosseaebecame apparent 25–51 days after sowing (Dodd,Burton, Burns, et al. 1987).

Studies conducted at the Indian Institute ofHorticultural Research, Hessaraghatta, Bangalore,India, on phosphatase activity in papaya (Caricapapaya) roots showed that inoculation with AMF(arbuscular mycorrhizal fungi) enhanced the activityof both acid and alkaline phosphatases (25%–114%)on the root surface as determined in enzyme extractsfrom root and soil surrounding the root region. Acidphosphatase activity was much greater than alkalinephosphatase activity. Plants inoculated with Glomusmosseae showed better activity than those withG. fasciculatum (Mohandas 1990).

In studies conducted at the Faculty ofHorticulture, Chiba University, Matsudom, Chiba,Japan, marigold (Tagetes patula) and leek (Alliumporrum) were inoculated with each of the three AMF– namely, Glomus mosseae, G. etunicatum, andGigaspora rosea – to compare the phosphataselocalization in the intraradical hyphae. Themycorrhizal roots were harvested at 3, 4, 5, and6 weeks after sowing, treated with a digestionsolution containing cellulase and pectinase, andthen stained for phosphatase activities at pH 5.0 andpH 8.5. G. mosseae formed fine-branched (mature)arbuscules only at the early phase of infection(3–4 weeks after sowing). Mature arbuscules ofG. etunicatum and G. rosea were observed from theearly phase (4 weeks after sowing) up to the end ofexperiment. At pH 5.0, the localization of thephosphatase activities of the three fungi was similar,irrespective of the host plant species. The activityappeared in the mature arbuscules and intercellularhyphae, whereas the collapsed arbuscules wereinactive. Ten millimolar NaF, an acid phosphataseinhibitor, inhibited the phosphatase activities ofG. mosseae and G. etunicatum, but did not inhibit theactivities of G. rosea. At pH 8.5, a difference amongthe fungal species was found in the localization ofphosphatase activity while there was no suchdifference between host species. The maturearbuscules were also the active sites in all threespecies. Only G. rosea showed the activity in theintercellular hyphae while the two Glomus spp. didnot. Five millimolar EDTA (ethylene diaminetetraacetic acid), asynthetic chelate, inhibited theactivity of G. rosea at pH 8.5 while the activities ofG. mosseae and G. etunicatum were not affected byeither 5 mM EDTA or 10 mM KCN (both arealkaline phosphatase inhibitors) (Ezawa, Saito, andYoshida 1995).

In studies conducted at the Laboratory ofDevelopmental Biology, Institute of Botany,KatholieKe Universiteit Leuven, K Mercierlaan,Leuven, Belgium, activities of extracellular enzymeswere determined in organic matter of beech (Fagussylvatica) leaf litter, colonized by the litterdecomposer Lepista nuda or by vegetative mycelium

from the mycorrhizal fungi, Thelephora terrestris orSuillus bovinus, and buried for up to six months inplant containers in which mycorrhizal or non-mycorrhizal P. sylvestris seedlings were cultivated ata low rate of nutrient addition. After differentperiods of colonization, the activities ofphosphomonoesterase and protease, the two enzymesinvolved in nitrogen and phosphorus mobilization,were determined in colonized and uncolonized litter.The activities of cellulase, beta-xylosidase, beta-glucosidase, and polyphenol oxidase (catecholoxidase) were investigated as indicators of thedecomposing capacity of fungi in the litter. Lowactivities of all enzymes tested occurred inuncolonized beech leaves. Phosphomono-esteraseactivity was high in litter colonized with L. nuda orS. bovinus, and was intermediate in the T. terrestristreatment. For all other enzymes, the activities in theorganic matter inoculated with the white-rot litterdecomposer were considerably larger than thosedetected in litter colonized by ectomycorrhizalbasidiomycetes. Cellulase activity was low in thecontrol, as well as in the mycorrhizal treatments.Beta-xylosidase and beta-glucosidase were detectedin the litter with mycorrhizal mycelium, whereaspolyphenol oxidase activity was only clearlyincreased in the S. bovinus treatment. These resultsdemonstrated the low lignocellulase activity of bothmycorrhizal fungi. This reduced the capacity of themycorrhizal fungi to exploit fresh beech leaf litter,whose endogenous nitrogen was associated with orshielded by refractory compounds (Colpaert andLaere 1996).

Studies conducted at the CNRS (Centre nationalde la recherche scientifique), Centre de PedologieBiologique, Vandoeurize, Nancy, France, onextraradical hyphae of Glomus intraradices orG. claroideum, extracted from root-free sand of two-compartment pot cultures and used to determinefungal phosphatase activity (p-nitrophenyl phosphatehydrolysis) showed that G. intraradices had thehighest external phosphatase activity in twoexperiments and G. claroideum had the same activityin one experiment. The activity reached 184 µ molp-nitrophenyl phosphates hydrolysed per mg dryweight H-1 (Joner and Johansen 2000).

Detection methods for phosphataseproduction by mycorrhizal fungiEctomycorrhizal fungiIn studies conducted at the Laboratoire deRecherches dur les Symbiotes des Racines, INRA-ENSA (Institut National de la RechercheAgronomique–Centre national de la recherchescientifique), Place Vala, Montpellier, France, acidphosphatase activities were measured in a phosphate-depleted medium of Hebeloma cylindrosporummycelia. These activities showed a great variabilitydepending on the substrate (sodium para nitrophenylphosphate or sodium polyphosphate) and the strain

Mycorrhiza News 18(2) • July 2006 5

of H. cylindrosporum (wild dikaryotic strain orhomokaryotic strains issued from this dikaryon). Thepurification tests were carried out on the phosphatasesecreted by the wild dikaryon. After filtration,concentration, and desaltation of the culturesolution, the phosphatases were separated by a cationexchange chromatography (carboxy methyl trisacryl).Two fractions of phosphatase activities werecollected, one of which was eluted using a gradientof sodium chloride. The electrophoretograms of thefractions showed a high purity and phosphatase weredirectly injected to a rabbit. The appearance ofantibodies in the serum was detected by the ELISA(Enzyme-Linked Immunosorbent Assay) test. It wasconcluded that the antibodies were useful to study theregulation of the fungal phosphatase activities in thepresence of orthophosphate or the phanerogam host(Deransart, Chaumat, Cleyet-Marel, et al. 1990).

In further studies conducted at the above institute,an acid phosphatase of Pisolithus tinctorius waspurified and used to prepare polyclonal antibodies.These antibodies were applied in homologous testson the mycelium and mycorrhizae of several fungi.This immunochemical approach seemed to be a wayto characterize and detect fungi when a particularprotein is used as a marker (Clayet-Marel,Bousquent, and Mousain 1990).

In the studies conducted at the Department ofBiochemistry and Microbiology, G B PantUniversity of Agriculture and Technology, PantNagar, Uttar Pradesh, India, alkaline phosphatasepurified from mycorrhizal and non-mycorrhizalFrench bean (Phaseolus vulgaris) gave a single bandon 5%–6% gradient PAGE (polyacrylamide gelelectrophoresis). A single protein peak was observedby UV spectrophotometry with an absorptionmaximum at 275 nm. The Vmax and Km differedslightly for the two preparations but they had similarpH (8.5) and temperature 40 °C optima. Thealkaline phosphatase from non-mycorrhizal beanshad wider substrate specificities than that frommycorrhizal beans (Kumari, Mishra, and Johri 1990).

The pNPPase (p-nitrophenolphosphomonoesterase assay), commonly used tomeasure cell-wall-associated and extracellularphosphatase activity of soil fungi, is usually done inthe context of fungal nutrition, where inorganicphosphorus supply may be enhanced bymineralization of organic phosphorus in the soil.Series of experiments were conducted at theBournemouth University School, ConservationScience, Talhot Campus, Dorset, UK, with theectomycorrhizal basidiomycete Hebelomacylindrosporum, that highlight components ofaccepted methodology that might impinge on thereliability of the assay. These include the loss ofpNPPase after filtration, inaccuracies in measuringwall-associated enzyme, and the ample pool ofintracellular pNPPase can be mistakenly measured asexternal pNPPase if cells are accidentally damaged(Tibbett, Sanders, Grantham, et al. 2000).

VAM fungi

In studies conducted at the Institute fur Pflanzenbauand Tierhygience in den Tropen and Subtropen,University of Gottingen, Grisebachstrabe,Gottingen, Germany, methods are reported forseparation and identification of organic acids,including phenolic acids, and on the determinationof phosphatases from VAM roots. For gas-chromatographic preparation of the samplescontaining the organic acids, silylation andmethylation were tested, as well as various solventsand silylating reagents. Columns of different polaritywere examined for their separation properties. Gas-chromatographic separation of the acids wasincomplete on the packed column OV 7. With thecapillary column PVMS 54, separation was muchbetter, but improvements are still needed. MSP(mycorrhiza specific phosphatases) appearing asthree bands were found by electrophoresis withporosity gradient gels in mycorrhizal roots ofAllium, Sorghum, and Eupatorium plants. In thesethree species, each MSP was located at the sameposition. Inoculated onion roots enzymaticallydigested with cellulase and pectinase showed no rootspecific phosphatases, but still revealed MSP (Fabig,Vielhauer, Moawad, et al. 1989).

In further studies conducted at the aboveuniversity, Eupatorium odoratum, Sorghum, andonion plants were inoculated with Acaulosporalongula. The plants were harvested after 8 and 16weeks, respectively, and cell-free root extracts wereprepared for electrophoresis. The improvedseparation of mycorrhizal proteins through a3%–30% polyacrylamide gradient gel revealed threeMSP instead of only one diffuse MSP band, which isto be seen in a 10% homogeneous gel. It issuggested that fluctuations in total MSP activityduring the vegetation period are the result offluctuations in single phosphatases sharplydistinguished from one another (Vielhauer 1990).

In the studies conducted at the InstituteColtivazioni Arboree dell’ Universita, Via Giuria 15,Turin, Italy, activities of acid and alkalinephosphatase, esterase, and succinate dehydrogenasewere assessed in extraradical mycelium of Glomusclarum by histochemical methods. Staining wasobserved for all enzymes with the exception of acidphosphatase. The amount of mycelium showingactivity decreased after initial infection. In olderhyphae, stained deposits were irregularly distributedinside the hyphae. It is suggested that in some hyphaltracts, vacuolization takes place and enzymaticactivity is reduced (Schubert and Mazitelli 1990).

In studies conducted at the Central Arid ZoneResearch Institute, Jodhpur, India, filter paperstreated with a mixture of 1-naphthyl phosphate assubstrate and the diazonium salt Fast Red TR as anindicator were used for the nondestructive visualdemonstration of the release of acid phosphatase byVAM fungi. Wheat was grown for six weeks in

6 Mycorrhiza News 18(2) • July 2006

sterilized soils in specially designed pots with fivecompartments with or without hyphal and rootbarriers. The plants were inoculated with G. mosseaeand then treated filter paper was placed at the outersurface of the hyphal compartment. Acid phosphataseactivity was visualized as a red-coloured hyphal printon the filter paper (Tarafdar 1995).

Studies conducted at the National GrasslandResearch Institute, Nishinasuno, Tochigi, Japan,showed that an alkaline phosphatase in theintraradical hyphae of VAM fungi was closely relatedto an improvement of plant growth. To detect thephosphatase activity in a crude extract of mycorrhizalroots, phosphatase isozymes in mycorrhizal and non-mycorrhizal onion roots were compared with those inGigaspora margarita by electrophoresis. Amycorrhiza-specific band was found when thephosphatase was stained under alkaline conditions.To clarify the origin of this phosphatase, thephosphatase extracted from intraradical hyphae wasalso compared with the phosphatase frommycorrhizal roots by electrophoresis. Theintraradical hyphae were isolated from mycorrhizalroots by enzyme digestion followed by Percollgradient centrifugation. The soluble protein wasextracted from the hyphae by ultrasonication aftertreatment with chitinase. A phosphatase in the hyphalsoluble protein showed a similar, but slightly higher,relative mobility on the gel, compared with themycorrhiza-specific phosphatase from roots. Byadding the hyphal extract to the root extract, therelative mobility of the mycorrhiza-specificphosphatase was slightly changed and becameidentical to that of the phosphatase in the hyphae.This indicates that the specific band of phosphatasefound in the crude extract from mycorrhizal rootswas of intraradical hyphal origin (Kojima, Hayatsu,and Saito 1998).

Inter- and intra-strain variations ofphosphatase activity in ectomycorrhizalfungiIn studies conducted at the Centre de Recherche enBiologie Forestiere, Universite Laval, Sainte-Foy,Quebec, Canada, the variation in acid phosphataseactivity among the monokaryotic F1 progeny fromtwo different synthesized dikaryotic cultures of theectomycorrhizal basidiomycete, Laccaria bicolor, wasexamined. The progeny of one of the dikaryonsshowed variation in acid phosphatase activity up to10 times that of the lowest value. The progeny of theother dikaryon were much less variable, showingdifferences of up to five times the lowest value. Bothsets of monokaryotic progeny showed distributionsindicative of polygenic inheritance for acidphosphatase activity in this fungus (Kropp 1990a).

Further studies conducted at the above universityon acid phosphatase activity of monokaryoticprogeny from controlled crosses in L. bicolorindicated that acid phosphatase activity in this fungus

was polygenic. A biometric analysis was done topartition genetic and environmental components ofthe variations among dikaryons created by pairingselected monkaryotic cultures. The results showedthat a relatively small proportion of the phenotypicvariability observed in this study was due toenvironmental factors. Further partitioning showedthat the genetic variation could be attributedprimarily to additive rather than non-additivecomponents. The heretability of acid phosphataseactivity in L. bicolor was thus quite high, indicatingthat strains having an elevated acid phosphataseactivity could be created by breeding (Kropp 1990b).

Studies conducted at the Universite ClaudeBernard Lyon 1, Unite d’Ecologie Microbienne,Associee au Centre National de la RechercheScientifique, Cedex, France, on interstrain variationwith 11 wild strains and intra-strain variability with20 sib-monokaryons and 50 reconstituted dikaryonsprogeny of Hebeloma cylindrosporum strain showedthat the range of variation of acid phosphataseactivity among wild dikaryotic mycelia was the sameas that among sib-monokaryons or dikaryonsbelonging to the progeny of a single strain. The totalphosphatase activity of the wild strains ranged from5.7 to 96.0 TmU (total milliunits). It ranged from11.1 to 120.5 TmU within sib-monokaryons andfrom 34.2 to 178.1 TmU for reconstituteddikaryons. Specific phosphatase activity of wilddikaryons ranged from 48.5 ImU (internationalmilliunits) to 675.6 ImU, whereas the ranges ofvariation among sib-monokaryons and reconstituteddikaryons were, respectively, 85.3 to 71.0 and 270.7to 816.1 ImU. On average, sib-monokaryons andreconstituted dikaryons had lower activity than theirparental dikaryon. However, four reconstituteddikaryons had a higher specific activity than theoriginal dikaryon, H. cylindrosporum. The growth ofthe studied mycelia also varied, but in a narrowerrange (from 97.1 to 151.6 microg protein perculture for wild dikaryons, from 130.1 to 199.1microg for sib-monokaryons, and from 160.6 to275.9 microg for reconstituted dikaryons). Nocorrelation could be detected between specific acidphosphatase activity and growth rate in pure culturewithin the different monokaryotic or dikaryoticpopulations studied. The results demonstrate thepossibility of obtaining, by intra-strain crossing,mycelia having higher phosphatase activity than theparental wild strains (Meysselle, Gay, and Deband1991).

Studies conducted at the Department of ForestScience, University of Alberta, Edmonton, Canada,on mycelial extracts of 43 isolates of Suillustomentosus from forest regions subjected to starch gelelectrophoresis showed that a total of 21 bands wereresolved from eight different enzymes presumablyrepresenting 13 loci. Six loci were polymorphicamong these isolates. Cluster and principalcomponents analysis demonstrated that the intra-specific genetic variation existed among and within

Mycorrhiza News 18(2) • July 2006 7

the forest regions. Polymorphic loci of acidphosphatase and alkaline phosphatase exhibited thegreatest genetic similarity among the isolates withinforest regions. Habitat isolation and host selectioncould be the sources of the isozyme variation amongforest regions in this fungal species (Zhu,Higginbotham, and Dancik 1987).

In studies conducted at the United StatesDepartment of Agriculture, Pacific NorthwestResearch Station, Forestry Sciences Laboratory,Corvallis, Oregon, USA, six isolates of Laccarialaccata (S-167, from a forest nursery and S-238,S-283, S-236, S-444, and S-472 isolates fromnatural forests) were analysed for acid phosphatase,alkaline phosphatase activity, acid phosphataseisozyme patterns, production of other enzymes andhormones. Differences in enzyme activity andphytohormone production were prominent among theisolates. The patterns of acid phosphatase isozymecould be clearly divided into three host-relatedgroups. Two polymorphic gene loci could beidentified as coding for enzymes of acid phosphatase.Two of these gene loci, Acp-b and Acp-c, werecharacterized by mostly constant, host dependentfrequencies. The other, Acp-d, exhibits allelefrequencies related to different habitats. Five isolatesshare the same Acp-a habitat, four isolates share theAcp-b habitat, two the Acp-c habitat, and only oneisolate, S-238, was from a high elevation at theAcp-d habitat. The isolate from a forest nurserydiffered strikingly in several characteristics fromother isolates, all of which were from a naturalforest. This suggested that nursery soil managementpractices may select isolates for particular edaphicecotypes of mycorrhizal fungi (Iwan 1987).

Relation of phosphatases with mycorrhizalinfection in plantsIn studies conducted at the Laboratoire dePhytoparasitologie, INRA-CNRS, Station deGenetique et d, Ameliozation des Plants, INRA,Dijon, Cedex, France, a histochemical procedurewas developed to visualize and estimate theproportion of arbuscular mycorrhizal infectionsshowing alkaline phosphatase activity and wascompared with the total amount of fungal tissue(indicated by trypan blue staining) and of livingmycelium (indicated by succinate dephyclrogenaseactivity). In roots of leeks and Platanus acerifolia,only a small proportion of living intraradicalmycelium showed alkaline phosphatase activityduring early infection by Glomus spp. but thisincreased greatly just before the mycorrhizal growthresponse of the host plant. The infection revealed byall three strains reached a maximum at six weeksafter inoculation, after which the level of trypanblue-stained infection remained constant, while theproportion showing succinate dehydrogenase andalkaline phosphatase activity declined as theinfection aged. Alkaline phosphatase activity was

absent from virtually all abortive entry point hyphaeformed on roots of a resistant myc (-) nod (-) mutantof pea, although succinate dehydrogenase activitywas detected. These observations suggest that thealkaline phosphatase activity is induced bycolonization of host roots and that this fungalenzyme could find a useful marker for analysing thesymbiotic efficiency of arbuscular mycorrhizalinfection (Tisserant, Gianinazzi-Pearson,Gianinazzi, et al. 1993).

In studies conducted at the Western RegionalResearch Centre, USDA-ARS (United StatesDepartment of Agriculture–Agriculture ResearchService), Albany, California, USA, two cultivars ofPhaseolus vulgaris (Mexico 309 responsive to VAM,Rio Tibagi less responsive to VAM) were grownin a greenhouse in Leonard jars containingsand/vermiculite supplied with nutrient solutioncontaining low concentrations of nitrogen andphosphorus, and were inoculated or not with soil thatcontained spores and hyphae of Glomus etunicatum.The plants were harvested three and four weeks postemergence. Mexico 309 beans grew faster supportedproportionately more VAM fungi and assimilatedmore phosphorus than did Rio Tibagi plants. SPUR(specific phosphorus uptake rate) was 35% greater innon-inoculated Rio Tibagi compared with Mexico309 roots. Colonization by G. etunicatum more thandoubled the SPUR of each cultivar compared tocontrols. Acid and alkaline phosphatase activitieswere significantly higher in Mexico 309 mycorrhizaethan in controls but in VAM and control Rio Tibagiroots, there were no significant differences. New acidphosphatase isoenzymes that were not present incontrols appeared in VAM colonized roots in bothcultivars and high molecular weight acidphosphatases decreased concomitantly in the host.Polyphosphate hydrolase activity increased inmycorrhizae of both cultivars compared touninfected roots. Malate dehydrogenase, peroxidase,and esterase activities were also higher in VAMcolonized roots than in controls and usually newisozymes appeared in mycorrhizae that were eitherundetectable in control roots or were expressed toonly a very small extent. Total peroxidase activitywas higher although two host bands disappeared inmycorrhizae and one new band having more than50% of total activity appeared. These resultsindicated that the dependence of a host on VAMfungi increased when nutrients that mycorrhizae canprovide were limited. The greater the increase inabsorption or utilization capacity following VAMcolonization, the greater would be the dependence ofthe host. More importantly, by identifying enzymeactivities that affect these plant microbe associations,specific genes can be targeted that code for theseenzymes for future manipulation (Pacovsky, Silvada,Carvalho, et al. 1990).

In studies conducted at the Faculty ofHorticulture, Chiba University, Matsudo 271, Japan,Tagetes patula cv. Bonanza spray, Tagetes patula ev

8 Mycorrhiza News 18(2) • July 2006

Bonanza spray, T. patula cv. Disco and T. erecta cv.Discovery were inoculated with Glomus etunicatumand in additional experiments, Bonanza spray wasinoculated with G. mosseae or Gigaspora spp. also inorder to investigate the origin and properties of thephosphatase specific to VAM infection. Solublephosphatases were extracted from six-week-oldplants and analysed using electrophoresis. Theinfection specific phosphatases were commonlydetected in all these associations and the Rf valueswere almost the same (0.11–0.12). Weakphosphatase activity was observed at the same Rfvalue as that of infection specific phosphatase innon-mycorrhizal plants. No major phosphatase bandwas observed at Rf 0.11–0.12 in the extracts ofgerminating and resting spores of G. etunicatum. Itwas thus suggested that infection specificphosphatase was of the host origin. The infectionspecific phosphatase was partly purified from theroots of Bonanza spray infected with G. etunicatumabout 170-fold and characterized. The optimum pHof 5.0, the hydrolysis of various phosphate esters,and the inhibition of fluoride, molybdate, phosphate,and vandate were indicative of the typicalcharacteristics of non-specific acid phosphates (E.C.3.1–3.2). In addition, since the enzyme hydrolysedthe pyrophosphate bond effectively, it was suggestedthat the enzyme was an acid phosphatase originatingfrom the plant (Ezawa and Yoshida 1994).

Studies conducted at the University of WestIndies, Centre for Biotechnology, Kingston 7,Jamaica, on the effect of VAM fungus, Glomuspallidum, on the phosphatase activity in cowpea(Vigna unguiculata) roots at successive stages ofplant growth showed that both acid and alkalinephosphatase activities were significantly higher(P=0.05) in mycorrhizal than in non-mycorrhizalroots 30 days after inoculation (Thiagarajan andAhmad 1994).

Studies conducted at the Laboratoire dePhytoparasitologie, INRA-CNRS, Station deGenetique et al. Amelioration des plantes, Dijon,Cedex, France, on soyabean and pineapple (havingdifferent growth rates and response to phosphate) tosee the effects of phosphate fertilization onphysiological activities of VAM fungi showed thattotal mycorrhizal infection estimated by trypan bluestaining was reduced by phosphate fertilization inphosphorus sufficient soils. In phosphorus deficientsoil, fungal infection in pineapple roots was notmodified by phosphate application. The level of totalmycorrhizal infection was not related to plantgrowth. Fungal alkaline phosphatase staining, and toa lesser extent, succinate dehydrogenase activityshowed a relationship with plant growth. There isthus a potential of this procedure for estimatingendomycorrhizal infection as a marker of efficiencyof the symbiosis (Guillemin, Orozco, Gianinazzi-Pearson, et al. 1995).

ReferencesAntibus R K, Bower D, and Dighton J. 1997Root surfact phosphatase activities and uptake of32P-labelled inositol phosphate in field-collectedgray birch and red maple rootsMycorrhiza 7: 39–46

Antibus R K and Dighton J. 1993Acid phosphate activities and oxygen uptake inmycorrhizas of select deciduous forest trees, pp. 65In Proceedings of the Ninth North American Conference onMycorrhizae[Guelph, Ontario, Canada, 8–12 August 1993]Ontario: University of Guelph

Cleyet-Marel J C, Bousquent N, and Mousain D. 1990The immunochemical approach for thecharacterization of ectomycorrhizal fungiAgriculture, Ecosystems and Environment 28(1–4): 79–83

Colpaert J V and Van Laere A. 1996A comparison of the extracellular enzyme activitiesof two ectomycorrhizal and a leaf-saprotrophicbasidiomycete colonizing beech leaf litterNew Phytologist 134(1): 133–141

Deransart C, Chaumat E, Cleyet-Marel J C, Mousain D,Labarere J. 1990.Purification assay of phosphatases secreted byHebeloma cylindrosporum and preparation ofpolyclonal antibodiesSymbosis (Rehovot) 9(1–3): 185–194.

Dighton J. 1983Phosphatase production by micorrhizal fungi: treeroot systems and their micorrhizas In Tree rootsystems and their mycorrhizas. et al. Atkison D. (Eds.)The Hague, Netherlands 1983. pp. 455–462

Dodd J C, Burton CC, Burns R G, Jeffries P. 1987Phosphatase activity associated with the roots andthe roots and the rhizosphere of plants infectedwith vesicular-arbuscular mycorrhizal fungiNew Phytologist 107(1): 163–172

Ezawa T, Saito M, and Yoshida T. 1995Comparison of phosphatase localization in theintraradical hyphae of arbuscular mycorrhizalfungi, Glomus spp. and Gigaspora spp.Plant and Soil 176(1): 57–63

Ezawa T and Yoshida T. 1994Acid phosphatase specific to arbuscularmycorrhizal infection in marigold and possible rolein symbiosisSoil Science and Plant Nutrition 40(4): 655–665

Fabig B, Vielhauer K, Moawad A M, Achtrich W. 1989Gas-chromatographic separation of organic acidsand electrophoretic determination of phosphatasesfrom VA mycorrhizal rootsZ. Pflanzenernatr Bodenk 152: 261–265

Mycorrhiza News 18(2) • July 2006 9

Gourp V and Pargney J C. 1991Acid phosphatase immunocyto localization ofpisolithus tinctorius L. during its confrontationwith Pinus sylvestris (Pers.) Desv. root systemCryptogammie Mycologre 12(4): 293–304

Guillemin J P, Orozco M O, Gianinazzi–Pearson V,Gianinazzi S. 1995Influence of phosphate fertilization on fungal alkalinephosphatase and succinate dehydrogenase activities inarbuscular mycorrhiza of soybean and pineappleAgriculture, Ecosystems and Environment 53(1): 63–70

Haussling M and Marschner H. 1989Organic and inorganic soil phosphates and acidphosphatase activity in the rhizosphere of 80-year-old Norway spruce (Picea abies (L) Karst.) treesBiology and Fertility of Soils 8(2): 128–133

Iwan H O. 1987Enzyme activity and phytohormone production ofa mycorrhizal fungus, Laccaria laccataCanadian Journal of Forest Research 17(8): 855–858

Joner E J and Johansen A. 2000Phosphatase activity of external hyphae of twoarbuscular mycorrhizal fungiMycological Research 104: 81–86

Kumari K, Mishra D P, and Johri B N. 1990Characterization of mycorrhiza-specific alkalinephosphatase from French beanHisar: Agricultural University. pp. 57–58

Kojima T, Hayatsu M, and Saito M. 1998Intraradical hyphae phosphatase of the arbuscularmycorrhizal fungus, Gigaspora margaritaBiology and Fertility of Soils 26(4): 331–335

Krishna K R and Bagyaraj D J. 1985Phosphatases in the rhozospheres of mycorrhizaland non-mycorrhizal groundnutJournal of Soil Biology and Ecology 5(2): 81–85

Kropp B R. 1990aVariation in acid phosphatase activity amongprogency from controlled crosses in theectomycorrhizal fungus Laccaria bicolorCanadian Journal of Botany 68(4): 864–866

Kropp B R. 1990bInheritance of acid phosphatase activity inLaccaria bicolorWyoming: University of Wyoming. pp. 176[Proceedings of the Eighth North American Conference onMycorrhiza Innovation and Hierarchical Integration,5–8 September 1990, Jackson, Wyoming, USA]

McElhinney C and Mitchell D T. 1993Phosphatase activity of four ectomycorrhizal fungifound in a Sitka spruce-Japanese larch plantation inIrelandMycological Research 97(6): 725–732

Meysselle J P, Gay G, and Deband J C. 1991Intraspecific genetic variation of acid phosphataseactivity in monokaryotic and dikaryotic populations ofthe ectomycorrhizal fungus Hebeloma cylindrosporumCanadian Journal of Botany 69(4): 808–813

Mohandas S. 1990Enhanced phosphatase activity in mycorrhizalpapaya (Carica papaya cv. Coorg Honey Dew) rootsIn Current Trends in Mycorrhiza Research, edited by JalaliB L and Chand HIn Proceedings of the National Conference on Mycorrhiza,pp. 55–56[Hisar, India, 14–16 February 1990]Hisar: Haryana Agricultural University

Pacovsky R S, Silvada P, Carvalho M T V, Tsai S M.1990Increased nutrient assimilation and enzymeactivities in field beans inoculated with GlomusetunicatumWyoming: University of Wyoming. pp. 230[Proceedings of the Eighth North American Conference onMycorrhiza Innovation and Hierarchical Integration,5–8 September 1990, Jackson, Wyoming, USA]

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

Sen R. 1990Isozymic identification of individual ectomycorrhizassynthesized between scots pine (pinus sylvestris l.) andisolates of two species of suillusNew Phytologist 114(4): 617–626

Tarafdar J C. 1995Visual demonstration of in vivo acid phosphataseactivity of VA mycorrhizal fungiCurrent Science 541–543

Thiagarajan T R and Ahmad M H. 1994Phosphatase Activity and Cytokinin Content inCowpeas (Vigna Unguiculata) Inoculated with aVesicular Arbuscular Mycorrhizal FungusBiology and Fertility of Soils 17(1): 51–56

Tibbett M, Sanders, F E, Grantham K, Cairney J W G.2000Some potential inaccuracies of the p-nitrophenylphosphomonoesterase assay in the study of thephosphorus nutrition of soil borne fungiBiology and Fertility of Soils 31(1): 92–96

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

Vielhauer K. 1990Enzymatic studies and techniques on mycorrhizalroots of different plant speciesAgriculture, Ecosystems and Environment 29(1–4): 435–438

Zhu H, Higginbotham K O, and Dancik B P. 1987Intraspecific genetic variability of isozymes in theectomycorrhizal fungus Suillus tomentosus7th North American Conference on Mycorrhiza,3–8 May 1987, Gainesville, Florida

10 Mycorrhiza News 18(2) • July 2006

AMF (arbuscular mycorrhizal fungi) are obligatesymbionts that colonize the roots of more than 90%of plants in all terrestrial environments. Thebeneficial effect of AMF on plant healthimprovement is now well established (Prasad 2000,2002). Recent investigations have brought to lightinstances where biological activities are markedlyenhanced in two or three member associations oforganisms. The phosphate solubilizing micro-organisms interact well with AMF in phosphorus-deficient soils. So far, no systematic investigationshave been made on the growth response ofAzadirachta indica to AMF individually and/or incombination with PSB (phosphate solubilizingbacteria). The present research work deals with theeffects of single and dual inoculation of Glomusfasciculatum and phosphate solubilizing bacterium(Pseudomonas striata) on Azadirachta indica plant.

Materials and methodsPreparation of AM fungal inoculum: Azadirachtaindica (neem) trees were surveyed and AMF sporeswere isolated from the rhizospheric soil by the wetsieving and decanting method (Gerdmann andNicolson 1963). Quantification of AM fungal sporedensity from the air-dried soil was carried out usingthe method given by Gaur and Adholeya (1994).Single AM fungal spores were purified andmaintained as pure cultures by the standardtechnique using maize (Zea mays) as the host plant.Diagnostic slides of spores were prepared usingPVLG (polyvinyl alcohol lactoglycerol) as amountant. AMF spores were identified using relevantliterature (Mortan and Benny 1990; Schenck andPerez 1990; Prasad and Rajak 1999). Soil basedG. fasciculatum AMF inocula was prepared bygrowing sterilized seeds of Paspalum notatum in potscontaining sterilized sandy loam soil (sand:soil, 1:1,w/v). The substrate containing spores, sporocarps,and root pieces served as a stock culture of AMFinoculum.Procurement of PSB: The culture of PSB wasobtained from the IARI (Indian AgriculturalResearch Institute), New Delhi, India.Earthen pot preparation and inoculation: A potculture experiment was conducted to study the effectof Glomus fasciculatum and PSB (P. striata) on

A. indica seedlings under nursery conditions. Thesoil was ground, passed through a 4 mm sieve, andwas autoclaved for 2 consecutive days. The soil wasdeficient in phosphorus. Eight kilograms of suchprocessed soil was used to fill in the earthern pots(30 cm diameter).

The topsoil was mixed with 200 ml soilcontaining finely chopped, heavily infected rootpieces of G. fasciculatum inoculum comprised of150–200 spores/plant. Healthy seeds of A. indicawere surface sterilized and treated with soil andcharcoal mixture (1:3) based carrier inoculant ofP. striata, wherever required. Autoclaved soil withoutany microbial inoculum served as control. Thetreatment was replicated ten times and arranged in arandomized block design. The plants in pots weremaintained to grow for 90 days in a mist house withtemperature of 27–35 oC, 70%–80% humidity, andlight intensity of 15000Lux–19000Lux. Plantsamples were taken at intervals of 30 days. Growthparameters were observed by well-known methods.Estimation of AM fungal infection: Freshlycollected roots were washed in water, cleaned with10% KOH (potassium hydroxide), acidified with 1NHCL (hydrochloric acid) and stained in 0.05%trypan blue (Phillips and Hayman 1970).Quantification of AMF root infection was carried outusing the slide method (Giovannetti and Mosse 1980).Estimation of shoot NPK (nitrogen, phosphorus,and potassium): Nitrogen was estimated by theKjeldhal method, phosphorus was determined by theAmmonium molybdate vandate method, andpotassium was determined using the Flamephotometer as described by Jackson (1973).

Results and discussionThe inoculation with AMF in general resulted inhigher root infection than the uninoculated plants(Table 1). Dual inoculation of G. fasciculatum andP. striata has significantly increased root infection,spore population, stem height, number of leaves,total dry weight compared to single inoculations, andcontrol. The stem height, number of leaves, and dryweight were directly proportional to percentagecolonization in roots, and AMF spore population insoil during the plant growth period and data werestatistically significant at P=0.05. Effect of AMF and

Research findings

Impact of arbuscular mycorrhizal fungus (Glomus fasciculatum) and

phosphate solubilizing bacterium (Pseudomonas striata) on growth and

nutrient status of Azadirachta indica L.

Kamal PrasadFlat 401, Surya Tower, New Ashok Nagar, Eluru, West Godawari District, Andhra Pradesh, India

Mycorrhiza News 18(2) • July 2006 11

P. striata inoculation statistically increased the stemheight of A. indica plants when compare withcontrol. Maximum (136.54%), (92.98%), and(79.62%) increase in stem height was registered inthe plants raised after dual inoculation withG. fasciculatum + P. striata and single inoculation ofG. fasciculatum and P. striata in 90 days of plantgrowth, respectively. A higher number of leaves(16.4) were recorded in the plants raised after dualinoculation of G. fasciculatum + P. striata after 90days of plant growth followed by G. fasciculatum(13.65) and P. striata (11.8) in individualinoculation. Similarly, variable enhancementcompared to the control was also noticed in total dryweight ranging from 27.69% to 41.86%, 80.96% to98.26%, and 64.14% to 105.75% in 30 days,60 days, and 90 days of plant growth, respectively,after inoculation of AMF and P. striata singly and/ordually. As evident from Table 1, the highest dryweight (415.4 mg/plant) was recorded inG. fasciculatum and P. striata inoculated plants.A statistically significant difference in dry weightcompared to control was found in the A. indica plantsraised after singly and/or dually treated plants.

Seed inoculation with P. striata together with soilinoculation with AMF increased the NPK uptake inA. indica. Inoculation of soil with G. fasciculatumresulted in an increase in the percentage of NPKover control. However, the results were notsignificant further. The seed inoculation withP. striata in conjunction with soil inoculation withG. fasciculatum produced significantly higher NPKuptake than the soil inoculation with AMF alone.The interaction of both the organisms brought abouta significant increase in nutrient uptake by the plant(Table 2).

Overall assessment of the data indicates thatinoculation of AMF and/or in dual combination withP. striata caused an increase in stem height, numberof leaves, biomass, shoot NPK, AMF infection inroots, and population in soil of A. indica. Dualinoculation of G. fasciculatum and P. striata broughtmaximum improvement in the studied parametercompared to the other single inoculations.

On the basis of the aforementioned results, it canbe concluded that single inoculation of AMF orbacterium did not significantly enhance plantgrowth, but dual inoculation of AMF and bacteria

Table 1 Influence of Glomus fasciculatum and Pseudomonas striata on root colonization, spore density, stem height, number ofleaves, and dry weight of Azadirachta Indica under nursery conditions.

No. of % root No. of AMF spores/ Stem height Total dry weightTreatment days colonization 50g of soil (cm) No. of leaves (mg/plant)

Control 30 — — 5.18c ± 0.106 4.3 c ± 0.260 74.3 c± 0.26360 — — 6.46 c ± 0.040 6.4 c ± 0.371 155.5 b ± 0.34290 — — 7.80b ± 0.157 8.8 c ± 0.221 201.9 c ± 0.25130 25 ± 0.472 56 ± 1.248 8.41 b ± 0.072 8.3 a± 0.260 98.6 b ± 0.234

(62.36) (93.02) (32.71)

Glomus 60 35 ± 0.944 68 ± 2.162 11.81 a± 0.136 10.7 b ± 0.260 285.5 c ± 0.265fasciculatum (82.95) (67.19) (83.60)

90 45 ± 0.944 89 ± 1.416 15.05 b ± 0.151 13.65 b ± 0.334 342.4 a ± 0.214(92.98) (55.11) (69.59)

P. striata 30 — — 7.72 b ± 0.181 6.8 b ± 0.243 94.88 c ± 0.223 (49.03) (58.14) (27.69)

60 — — 10.84 b ± 0.144 8.7 c ± 0.266 281.4 a ± 0.211(67.80) (35.94) (80.96)

90 — — 14.01 b ± 0.154 11.8 b ± 0.243 331.4 b ± 0.172(79.62) (34.09) (64.14)

Glomus fasciculatum 30 45 ± 0.144 65 ± 1.248 10.42 a ± 0.145 10.4 a ± 0.344 105.4 b ± 0.145+ P. striata (101.16) (141.86) (41.86)

60 55 ± 0.148 85 ± 2.162 13.37 a ± 0.247 13.2 a ± 0.139 308.3 a ± 0.185(106.97) (106.25) (98.26)

90 85 ± 0.188 105 ± 1.416 18.45 a ± 0.133 16.4 a ± 0.134 415.4 a ± 0.194(136.54) (86.36) (105.75)

AMF - Arbuscular mycorrhizal fungiEach figure represents the mean of ten replicates; ±SE values are mean of ten observations;Figures in the parantheses denote per cent increase over the control; values without common letters differ significantly at LSD=0.05

12 Mycorrhiza News 18(2) • July 2006

significantly enhanced plant growth characteristics.The present study also confirmed that significantcombinations (AMF and PSB) can be used asbiofertilizer for improved production of A. indica.Hence, this dual inoculation technology will be usefulin successful afforestation and waste land managementprogrammes where neem is extensively included.

ReferencesGaur A and Adholeya A. 1994Estimation of VAM spores in the soil—a modifiedmethodsMycorrhiza News 6(1): 10–11

Gerdmann J W and Nicolson T H. 1963Spore of mycorrhizal Endogone species extractedfrom soil by wet sieving and decantingTransactions of the British Mycological Society 46: 235–244

Giovannetti M and Mosse B. 1980An evaluation of technique for measuring vesicularcarbuncular infection in rootsNew Phytologist 84: 489–500

Jackson M L. 1973Soil chemical analysisNew Delhi: Prentice Hall of India Ltd

Mortan J B and Benny J L. 1990Revised classification of arbuscular mycorrhizaefungi (Zygomycetes), a new order Glomales, twonew suborders, Glomineae Gigasporineae and twonew families, Acaulosporaceae and Gigasporaceaewith and emendation of GlomaceaeMycotaxon 37: 471–491

Phillips J M and Hayman D S. 1970Improved procedures for clearing root and stainingparasitic and vesicular arbuscular mycorrhizalfungi for rapid assessment of infectionTransactions of the British Mycological Society 55: 58–161

Prasad K. 2000Growth responses of Acacia nilotica (L.) Del.inoculated with Rhizobium and Glomus fasciculatumVAM fungiJournal of Tropical Forestry 16(1): 22–27

Prasad K. 2002Effect of arbuscular mycorrhizae on biomass,yield, uptake and translocation of nitrogen,phosphorus and potassium in Azaditacta indica L.In Frontiers in Microbial Biotechnology and Plant Pathologypp. 187–191. Edited by C Manoharachary,D K Purohit, S R Reddy, M A Singaracharya, andS Girisham.Jodhpur: Scientific Publishers (India)

Prasad K and Rajak R C. 1999Recent advances in mycorrhizal taxonomy:Morphological and molecular criteriaIn Microbial Biotechnology for Sustainable Development andProductivity pp. 62-72. Edited by R C RajakJodhpur, India: Scientific publisher (India) Jodhpur

Schenck N C and Perez Y. 1990Manual for the identification of VA mycorrhizalfungi 3rd edition, Edited by N C Schenck and Y Perez,Gainesville, Florida, USA: INVAM, University ofFlorida

Table 2 Influence of Glomus fasciculatum and Pseudomonas striata on NPK status of Azadirachta indica under nursery conditions

Parameter/ No. of Nitrogen Phosphorus Potassiumtreatment days (mg/plant) (mg/plant) (mg/plant)

Control 30 0.85 c ± 0.043 0.181 c ± 0.094 0.910 c ± 0.12060 3.95 c ± 0.243 0.385 c ± 0.043 3.620 c ± 0.09590 5.04 c ± 0.970 0.641 c ± 0.067 4.650 b ± 0.093

Glomus fasciculatum 30 2.19 b ± 0.029 (157.65) 0.458 a ± 0.085 (153.04) 2.12 a ± 0.058 (132.97)60 6.15 b ± 0.141 (55.70) 0.656 b ± 0.093 (70.39) 6.041 b ± 0.690 (66.88)90 7.66 b ± 0.041 (51.98) 0.914 c ± 0.131 (42.59) 7.455 b ± 0.071 (60.32)

P. striata 30 7.98 b ± 0.145 (132.94) 0.425 a ± 0.094 (134.81) 2.10 a ± 0.456 (130.77)60 5.91 b ± 0.149 (49.62) 0.645 b ± 0.0.67 (67.53) 6.019 ba ± 0.098 (66.27)

90 6.34 b ± 0.089 (25.80) 0.814 c ± 0.139 (26.99) 7.310 b ± 0.075 (57.20)Glomus fasciculatum 30 2.43 b ± 0.133 (185.88) 0.554 a ± 0.143 (206.08) 2.25 a ± 0.195 (147.25)+ P. striata 60 7.45 a ± 0.199 (88.61) 0.759 a ± 0.083 (97.14) 7.14 a ± 0.119 (97.24)

90 8.96 a ± 0.143 (77.78) 1.41 a ± 0.591 (119.97) 8.744 a ± 0.079 (88.04)

NPK – nitrogen, phosphorus, and potassiumEach figure represents the mean of three replicates; ±SE values are mean of three observations;Figures in the parantheses denote per cent increase over the control; Values without common letters differ significantly at LSD=0.05

Mycorrhiza News 18(2) • July 2006 13

IntroductionWastelands remain unexploited due to scarcemarginal soil, severe drought conditions, and variousanthropogenic activities. Such sites usually supportxerophytic vegetation, which can adapt to harsh soilconditions. Present studies were conducted toevaluate the role of VA (vesicular-arbuscular)mycorrhizal fungi in adaptation of xerophyticvegetation in wastelands, which may ultimately helpin the reclamation of wastelands by restoration ofsoil fertility.

Materials and methods

The research work was carried out at a site inChunkankadai Hills supporting thin, xerophyticvegetation. It is located in Kanyakumari district, thesouthern most part of the state of Tamil Nadu. Thewasteland under study has Cymbopogon flexuous s Neesex Steud, Jatropha glandulifera Roxb and Calotropisprocera, R. Br., as dominant vegetation. AM(arbuscular mycorrhizal) fungal association and sporecontent of the rhizosphere soil of the pioneer plantsand two tuber yielding plants, Dioscorea alata andManihot esculenta, were examined during this study.

Soil samples from the root zone of each plantspecies grown in different locations of the site wererandomly collected; physical and chemical propertiesof the soil such as texture, pH, electricalconductivity, available nitrogen, phosphorus, andpotassium were determined by standard analyticalmethods (Jackson 1973).

Rhizosphere soil samples with tender roots ofplants were collected randomly. The first samplingwas done in August 2003, and the second inFebruary 2004. Roots were separated, washed, andcut into small segments, cleared in KOH (potassiumhydroxide), and stained with trypan blue (Phillipsand Hayman 1970). VA mycorrhization of each plantspecies was determined by estimating the percentroot colonization (Giovannetti and Mosse 1980).

For AM fungal spore enumeration, 100 g of thesubstratum was dispersed in 1 litre water. After 15minutes, the suspension was decanted through 710 and48 µm sieves, and the remains on the sieves werewashed into beakers. The supernatant was filteredthrough grided filter papers. Each filter paper wasspread onto a micro-slide and observed under adissecting microscope at x40 magnification. The intactAMF spores were counted. The AM fungal spores werepicked up with a wet needle and mounted in poly-vinylalcohol lactophenol on a micro-slide for identification.

The intact and the crushed spores were examined undera compound microscope and identified according toSchenck and Perez (1987).

Results and discussionJatropha glandulifera Roxb is a small evergreen plantof 4–8 feet, with a stout trunk and 3–5 lobedglandular leaves. Cymbopogon flexuosus Nees exSteud is a perennial grass, while Calotropis procera,R. Br., is an erect shrub with a normal root system.VA mycorrhizal association was observed in all theplants examined. Vesicles, arbuscules, andextramatrical spores characteristic of AMF wereobserved in the roots. The highest amount of AMcolonization was found in roots of Cassava (80%)and the lowest in Calotropis (47%) (Table 2). Theroots of introduced plants showed appressoria, welldeveloped arbuscules, and vesicles of various shapes.Both thick walled and thin walled hyphae wereobserved. In Dioscorea species, arbuscules showeddeeply stained bead like aggregations. Degeneratearbuscules were seen with yellow pigmentation.Cassava showed small vesicles of varied shapes.

The pioneer plant Cympopogon flexuosus producedfinely branched roots. Root squash showed extensiveAMF hyphae, uniformly thin, running parallel to thelong axis. Extramatrical hyphae were moreabundant. Penetration points more then 10/mm wereobserved. These plants thrived well under extremeconditions, and survival capacity was directlyproportional to the extent of AMF mycorrhization.Examination of rhizosphere soils of all plants

Arbuscular mycorrhizal fungi in the reclamation and restoration of

soil fertility

P Charles, A D S Raj, and S KirubaScott Christian College, Nagercoil, Tamil Nadu – 629 003, India

Table 1 Soil characteristics of the study site

Parameter Unit Mean value

Mechanical composition Percentage

Coarse sand ” 52Fine sand ” 20Silt ” 18Clay ” 10Texture Yellowish red

sandy claypH 5.9Organic carbon Percentage 0.32Available nitrogen Kg/ha– 152.8Available phosphorus ” 38.5Available potassium ” 192.5

14 Mycorrhiza News 18(2) • July 2006

revealed the presence of a large population ofextramatrical spores of AM fungi (Table 2).

The largest population of AM spores was observedin the rhizosphere soil of native Cymbopogon (450spores/100 g soil). The average spore count in therhizosphere soils of all the five plants was 280spores/100 g soil. Spores of seven AMF – namely,Acaulospora scrobiculata Trappe, Glomus aggregatumSchenck and Smith emend Koske, G. mosseae (Nicol.and Gerd) Gerd and Trappe, G. geosporum (Nicol. andGerd) Walker, Gigaspora margarita Becker and Hall,Sclerocystis sp., and Scutellospora pellucida (Nicol. andSchenck) Walker and Sanders – were observed.

The study site was a unique habitat where thevegetation was adapted to stress arising from marginalsoil and anthropogenic activities. The results of thepresent investigation indicate that the dominantvegetation of the area had significant AMF association.The most important beneficial effect of mycorrhizalassociation is its role in the increased uptake of certainnutrients, especially phosphorus by plants. Soil pHplays an important role in phosphorus availability insoil and uptake by plants (Wang, Stribley, Tinker,et al. 1985). Soil pH, thus, has significant importancein VA mycorrhizal symbiosis and distribution of AMF(Janardhanan, Abdul-Khaliq, Naushin et al. 1994). Thestudy site became unexploited due to scarce marginalsoil and drought. Among the AMF associated with theplants of the area, Glomus constitute the dominantgenus. G. geosporum and G. aggregatum could thrivewell under stressful conditions.

Plants belonging to the Poaceae family were found tobe extensively associated with AMF under stressful andecologically unfavourable conditions (Stahl, Williams,and Christian 1988). Present observations support thisfindings. According to Dodd (2000), AMF areprimarily responsible for nutrient transfer from soil toplant, soil aggregation, and protection of plants againstdrought stress. This study also confirmed the possiblerole of VAM fungi in the restoration of soil fertility ofunexploited marginal soil. Mycorrhizal networks mightlink many of the plants within this habitat because ofthe general lack of host specificity of these fungi.

In Cymbopogon, extensive and finely branched rootswith abundant AMF greatly influenced the survivalcapacity of the grass during stressful conditions. AMpropagules and its inoculation in drought tolerant rootcrops help in the efficient utilization of marginal soilsand, subsequently, its restoration.

ReferencesDodd J C. 2000The role of arbuscular mycorrhizal fungi in agro andnatural eco systems Outlook on Agriculture 29(1): 55–62

Giovannetti M and Mosse B. 1980An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in rootsNew Phytolologist 84: 489–500

Jackson M L. 1973Soil chemical analysisNew Delhi: Prentice Hall of India Pvt. Ltd

Janardhanan K K, Abdul-Khaliq, Naushin F,Ramaswamy K. 1994.Vesicular-arbuscular mycorrhiza in an alkalineusar land eco system.Current Science 67(6): 465–469

Phillips J M and Hayman D S. 1970Improved procedures for clearing roots andstaining parasitic and vesicular-arbuscularmycorrhizal fungi for rapid assessment of infectionTransactions of the British Mycological Society 55: 158–160

Schenck N C and Perez Y. 1987Manual for the identification of VA mycorrhizal fungiGrainsville, Florida: University of Florida

Stahl P D, Williams S E, and Christian M. 1988Efficacy of native vesicular arbuscularmycorrhizal fungi after severe soil disturbanceNew Phytologist 114: 1–38

Wang G M, Stribley D P, Tinker P D, Walker C C.1985Ecological interaction in soil (edited by Fitter A H)Oxford: Blackwell, pp. 219–224.

Table 2 Native AMF found associated with plants under study

AMF ColonizationSpore No 100/g

Name of the plant Family HC AC VC soil AMF species

Jatropha glandulifera Euphorbiaceae 51.32 13.92 11.38 210.00 Acaulospora scrobiculate± 2.3 ± 1.31 ± 3.1 Glomus aggregatum

Cymbopogon flexuosus Poaceae 62.36 15.18 20.86 450.50 G. mosseae± 5.2 ± 1.36 ± 3.05 G. geosporum

Calotropis procera Asclepiadaceae 47 ± 5.1 27.38 5.17 250.4 Gi. margarita± 5.18 ± 3.95 Sclerocystis Sp

Manihot esculenta Euphorbiaceae 80.0 36.27 22.15 365.75 Scutellosproa pellucida± 7.5 ± 5.63 ± 2.13

Dioscorea alata Dioscoreaceae 71.98 18.96 32.55 143.75± 5.01 ± 3.21 ± 2.50

AMF – arbuscular mycorrhizal fungi; HC – hyphal colonization; AC – arbuscules; VC – vesicles

Mycorrhiza News 18(2) • July 2006 15

Material and methodsThe investigation was carried out at the Departmentof Medicinal and Aromatic Plants, Kittur RaniChannamma College of Horticulture, Arabavi duringthe year 2003/04. Seven AM (arbuscular mycorrhiza)fungal species, viz., Glomus intraradices,G. fasciculatum, G. monosporum, G. mosseae,Sclerocystis dussii, Gigaspora margarita, andconsortia-I formed the eight treatments along withcontrol. Consortia-I consisted of Azotobactorchroococcum, Azospirillum brassilence, Pseudomonasstriata, and Trichoderma harzianum.

Completely randomized block design wasfollowed with three replications. A spacing of60 × 20 cm was followed. The crop was harvested

after 150 days and various morphological and yieldcharacters were recorded (Tables 1 and 2).Chlorophyll content in leaves was estimated as perthe procedure given by Shoaf and Lium (1976).

Forskolin content (%) was estimated in dry tubersof different treatments using HPLC (highperformance liquid chromatography).

Results and discussionThe plants inoculated with different bioinoculantsshowed more vigorous growth than the uninoculatedcontrol plants (Table 1). Among differenttreatments, establishment of cutting was better inconsortia-I treated plants (91.66%) and minimumwas in control (80%). As Trichoderma harzianum has

Effect of bio-inoculant organisms on growth and yield of Coleus forskohlii

Briq.—an endangered medicinal plant

Santosh Dharana, Laxminarayan Hegde, A K Rokhade, C P Patil, and M S KulkarniKittur Rani Chennamma College of Horticulture, University of Agricultural Sciences, Arabavi – 591 310,Belgaum District, Karnataka, India

Table 1 Effect of AM fungi on growth characters in Coleus forskohlii Briq.

Per cent Plant spread (cm)establish- Plant No. of Leaf Leaf Inter-nodal Stem Petiole Totalment of height East- North- branches length breadth length diameter length chlorophyll

Treatment cuttings (cm) West South per plant (cm) (cm) (cm) (cm) (cm) (mg/g)

Glomus 85.00 52.66 52.80 55.20 58.40 5.54 2.58 1.23 2.57 1.16 0.39intraradices

Glomus 86.66 53.40 50.63 56.96 57.66 5.82 2.45 1.24 2.26 1.17 0.37fasciculatum

Glomus 83.33 50.73 51.86 51.20 52.50 5.37 2.27 1.26 2.40 1.22 0.43monosporum

Glomus 91.66 56.76 56.00 52.50 52.50 6.12 2.76 1.58 2.35 1.32 0.61mosseae

Gigaspora 91.66 61.23 55.16 52.00 55.83 6.24 2.70 1.57 2.34 1.33 0.24margarita

Sclerocystis 90.00 57.76 53.50 53.46 53.50 5.76 2.60 1.41 2.33 1.32 0.30dussii

Consortia-I 93.33 58.36 56.16 52.36 53.46 5.59 2.56 1.54 2.35 1.29 0.21

Control 80.00 49.23 54.30 54.16 48.13 5.39 2.41 1.26 2.24 1.17 0.41

Mean 55.02 54.17 53.48 54.00 5.73 2.54 1.38 2.35 1.25

S.Em± 2.530 1.406 1.190 1.122 0.769 0.113 0.080 0.047 0.020 0.031 0.003

C.D. at 5% 7.673 4.265 3.609 3.402 2.334 0.343 0.242 0.144 0.062 0.094 1.24

CV (%) 5.00 4.43 3.80 3.63 2.47 5.91 5.43 5.91 1.50 4.30 —

16 Mycorrhiza News 18(2) • July 2006

pathogen controlling activities (Sukhada 1999) andplant growth promoting substances (Crafts andMiller 1974), the consortia-I inoculated plantsshowed better rooting.

Plants inoculated with Gigaspora margarita hadmaximum plant height (61.23 cm) and leaf length(6.24 cm); the influence on leaf breadth andinternodal length was also good, followed byconsortia-I, Glomus mosseae, and Sclerocystis dussii.The plants inoculated with Glomus mosseae recordedthe highest plant chlorophyll (0.61 mg/g), leafbreadth (2.76 cm), internodal length (1.58 cm), andalso had a good influence on plant spread, leaflength, and petiole length, which was on par withGigaspora margarita and consortia-I overuninoculated plants. Increased plant height withapplication of Glomus mosseae has been reported byBoby and Bagyaraj (2003) in Coleus forskohlii.

Among different treatments, plants inoculatedwith consortia-I, Gigaspora margarita, andSclerocystis dussii exhibited significant influence onyield attributes (Table 2). The plants inoculated withconsortia-I recorded maximum length of tuber(13.52 cm), number of tubers (16.8), and also had agood influence on fresh tuber weight, dry tuberweight, and tuber diameter over uninoculated ones.

In the present experiment, the fresh weight oftubers per plant (g) and dry weight of tubers perplant (g) were found to be higher in the plantsinoculated with Gigaspora margarita and consortia-I,which also had good influence of tuber diameter and

tuber length, compared to the uninoculated plants.An increase in dry tuber yield of more than 25% wasrecorded due to the application of these organisms.This is mainly due to higher expression ofmorphological characters in inoculated plants, whichmight have, in turn, helped in higher photosynthesisand better accumulation of dry matter. Increasedeconomic yields were reported in different crops dueto the application of bio-inoculant organisms byother workers in chilli (Bagyaraj and Sreeramulu1992) and vetiver (Neelima, Gautam, and Verma2002).

Bio-inoculant organisms are known to producegrowth-promoting hormones (Allen, Moove, andChristensen 1980; Azcon, Azcon-Aguilar, and Barea1978) and to increase the availability ofmicronutrients like copper, manganese, iron,magnesium, and zinc in addition to higherphosphorus-uptake (Sreenivasa 1992; Gurumurthyand Sreenivasa 1996; Adivappar, Patil, Patil, et al.2004). A similar beneficial effect of consortia-Icould be attributed in the present study also for theincreased dry weight of tuber per plant in the treatedplants. Similar results were reported by Earanna,Malikarjuniah, Bagyaraj, et al. (2001) in Coleusaromaticus, Priyarani, Aggarwal, and Mehrotra(1999) in Acacia nilotica, and Abdul–Khaliq, Gupta,and Anskumar (2001) in peppermint.

Among the different treatments where forskolinestimation was done (Table 2), the forskolin contentand yield was the maximum in consortia-I (0.403%

Table 2 Effect of AM fungi on tuber yield and forskolin content in Coleus forskohlii

Number of Length of Diameter of Fresh tuber yield Dry tuber yield Forskolintubers per tubers tubers content Forskolin yield

Treatment plant (cm) (cm) (g/plant) (q/ha) (g/plant) (q/ha) (%) (mg/plant)

Glomus intraradices 9.26 11.33 1.25 107.78 89.82 14.08 11.85 — —

Glomus fasciculatum 11.60 11.03 1.27 120.74 100.62 15.78 13.15 0.329 19.30

Glomus monosporum 10.73 11.24 1.25 126.26 105.21 16.43 13.69 — —

Glomus mosseae 9.53 11.54 1.32 125.40 104.48 16.39 13.65 — —

Gigaspora margarita 10.53 13.28 1.36 160.09 133.40 20.92 17.36 0.307 17.28

Sclerocystis dussii 13.53 13.42 1.37 133.06 110.89 17.39 14.62 0.274 15.56

Consortia-I 16.80 13.52 1.34 159.46 132.89 20.85 17.35 0.403 26.07

Control 9.80 10.38 1.30 127.60 106.33 16.69 13.91 0.330 20.75

S.Em± 1.119 0.576 0.020 7.01 5.84 0.94 0.80 — —

C.D. at 5% 3.393 1.746 0.061 21.25 17.70 2.86 2.44 — —

CV (%) 4.709 2.424 0.085 9.16 9.15 9.43 9.65 — —

Mycorrhiza News 18(2) • July 2006 17

and 26.07 mg/plant), followed by Gigasporamargarita (0.307% and 17.28 mg/plant), andSclerocystis dussii (0.274% and 15.56 mg/plant), asagainst control (0.33% and 20.75 mg/plant).The application of consortia-I recorded an increaseof more than 22% of forskolin over control.

The present investigation thus confirmed the roleof microbial consortia-I and AM fungi in theimprovement of both tuber yield and forskolincontent in Coleus forskohlii.

ReferencesAbdul-Khaliq, Gupta M L, and Anskumar S. 2001The effect of vesicular arbuscular mycorrhizalfungi on growth of peppermintIndian Phytopathology, 54(1): 82–84

Adivappar N, Patil P B, Patil C P, Swamy G S K,Athani S I. 2004Effect of AM fungi on growth and nutrient contentof container grown papaya plantsIn Organic Farming in Horticulture for SustainableProduction. Ed. Pathak Ramakrishnan R K, Khan R M,and Ram R A. 29–30 August, Central Institute forSubtropical Horticulture, Ramenkhera, Lucknow,pp. 166–169.

Allen M F, Moove T S Jr, and Christensen M. 1980Phytohormone changes in Bentelona gracilisinfected by VAM mycorrhiza I. Cytokinin increasesin the host plantCanadian Journal of Botany 58: 371–375

Azcon R, Azcon-Aguilar G, and Barea J M. 1978Effect of plant hormones present in bacterialcultures on the formation and response to VAmycorrhizaNew Phytologist 80: 3559–3569

Bagyaraj D J and Sreeramulu K R. 1982Pre-inoculation with VA mycorrhiza improvesgrowth and yield of chilli transplanted in the fieldand save fertilizerPlant and Soil 69: 365–381

Boby V U and Bagyaraj D J. 2003Biological control of root rot of Coleus forskohliiBriq. using microbial inoculantsWorld Journal of Microbiology and Biotechnology19: 175–180

Crafts S B and Miller C O. 1974Detection and identification of cytokininsproduced by mycorrhizal fungiPlant Physiology 54: 586–588

Earanna N, Malikarjuniah R B, Bagyaraj D J,Suresh C K. 2001Response of Coleus aromaticus to Glomusfasciculatum and other beneficial soil microfloraJournal of Spices and Aromatic Crops 10(2): 141–143

Gurumurthy S C and Sreenivasa M N. 1996Response of chilli to different inoculum levels ofGlomus macrocarpus in two soil types of northernKarnatakaKarnataka Journal of Agricultural Sciences9(2): 254–259

Neelima R, Gautam S P, and Verma H M. 2002Impact of four Glomus species on growth, oilcontent, P content and phosphatase activity ofVetiveria zizanioidesIndian Phytopathology 55(4): 434–437

Priya R, Aggarwal A, Mehrotra R S. 1999Growth responses in Acacia nilotica inoculatedwith VAM fungi (Glomus mosseae), Rhizobium sp.and Trichoderma harzianumIndian Phytopathology 52(2): 151–153

Shoaf T W and Lium B. 1976Improved extraction of chlorophyll a and b fromalgae using dimethyl sulphoxideLimnol. Oceanogr 21: 916–926

Sreenivasa M N. 1992Selection of an efficient VAM fungus for chilli(Capsicum annuum L.)Scientia Horticulturae 50: 53–58

Sukhada M. 1999Biofertilizers for horticultural cropsIndian Horticulture 43(4): 32–36

18 Mycorrhiza News 18(2) • July 2006

New approaches

Sustained in-vitro pine root development

Oliveira P, Barriga J, Cavaleiro C, Peixe A, and PotezA Z (2003) succeeded in sustained in-vitro rootdevelopment of stone pine (Pinus pinea) by the use ofectomycorrhizal fungi (Forestry Proceedings 76 (5):579–587, 2003). Stone pine is an economicallyimportant forest tree that grows in Mediterraneanclimate and has been the target for selection effortsthrough micro propagation. Previous attempts onmicro shoots, derived from mature seed cotyledons,reached incipient rooting after induction with acombination of auxin and hypertonic shock, but theirdevelopment in-vitro was not sustained. At this stage,the authors succeeded in overcoming this barrier byco-culturing plantlets with some fungi isolated from

the ectomycorrhizae, enabling satisfactory developmentin vermiculite and later in soil. About half the fungalisolates tested helped the plants resume root growth.Although control plants (in the absence of the fungi)developed roots at a later stage–that is, during the post-transplanting acclimation in vermiculite–their growthwas weaker. The root systems of some inoculatedplants had ectomycorrhizae from the introduced fungibeing carried over when the plants were transferredfrom the co-cultures to vermiculite.

In conclusion, co-cultured rooted micro shoots withectomycorrhizal fungi can be an effective means toovercome the difficulties encountered in the use ofmicro propagation methods on this species.

Centre for Mycorrhizal Culture Collection

IntroductionHeavy metal, by definition, refers to the metallicelements with a specific mass higher than 5 g/cm3,and the ability to form sulphides (Adriano 1986).Trace element would, therefore, be a more correctform since the latter is based only on concentration(less than 0.1% in the soil or 100 mg/kg in drymatter of biological samples). However, the term‘heavy metal’ is generally used and accepted inenvironmental studies. The toxicity of metals in thesoil is dependant on their bioavailability, defined astheir ability to be transferred from the soilcompartment to a living organism (Juste 1988).According to Berthelin, Munier-Lamy, and Leyval(1995), metal bioavailability is a function not only oftheir total concentration, but also their physio-chemical (pH, Eh, organic matter clay content) andbiological (for example, biosorption,bioaccumulation, and solublization) factors.Soil microorganisms, including mycorrhizal fungi,are affected by the presence of high metalconcentration in the soil (Giller, Witter, andMacgrath 1998). But the organisms, in turn,influence the availability of the metals in soil either

directly, through alterations of pH, Eh, biosorptionor uptake, or indirectly in the rhizosphere throughtheir effect on plant growth, root exudation, andresulting rhizosphere chemistry. Because plantsfunction as the principle entry point of heavy metalsinto the food chain leading to animal and man(Rauser 1990), it is very important to analyse thedistribution of various heavy metals in plants. Aunique trait of mycorrhizal fungi is that they form adirect link between the soil and the root, providing apath for movement of elements in the rhizosphere.These aspects of interaction between mycorrhizaeand heavy metals in soil are the topic of this article.

Heavy metal uptake by arbuscularmycorrhizal fungiArbuscular mycorrhizal fungi are the root symbiontsfound in most plant species and habitats (Harley andSmith 1983). While the enhanced absorption of tracemetals (Zn, Cu, Co) from deficient or non-enrichedsoils by AM (arbuscular mycorrhizal) plants hasbeen documented (Faber, Zasoski, Burau, et al.1990), relatively little attention has been paid to therole of AM fungi in metal contaminated soils.

Functioning and relevance of heavy metal uptake and translocation by mycorrhizal fungiPrasun Ray and Alok AdholeyaCentre for Mycorrhizal Research, TERI , Darbari Seth Block, IHC Complex, Lodhi RoadNew Delhi – 110 003, India

Mycorrhiza News 18(2) • July 2006 19

Arbuscular mycorrhiza has been shown to improveplant tolerance to heavy metal stress in polluted soils(Leyval, Haselwandter, and Tarnau 1997).Glomelean hyphae may contribute directly to theuptake and translocation of metals to the host roots,including micronutrients such as Cu and Zn, as wellas the toxic element Cd (Burkert and Robson 1994;Li, Marchner, and George 1991b; Leyval,Haselwandter, and Tarnau 1997). AM fungi maysequester toxic elements, thereby reducing theiravailability to the plants (Kaldorf, Khun, Schroder,et al. 1999; Turnau, Kottke, and Oberwinkler 1993).Such metal sequestration may alter metal translocationpatterns in plants, leading to metal accumulation in themycorrhizal roots and reduced metal transfer to theabove ground biomass (Dehn and Schuepp 1989;Shetty, Hetrick, Figge, et al. 1994).

Uptake into hyphae may be influenced byadsorption on hyphal walls as chitin has a metal-binding capacity. More indirect effects of AM fungion rhizospheric soil include changes in pH (Li,Marchner, and George 1991b), microbialcommunities (Olsson, Francis, Read, et al. 1998),and root exudation patterns (Laheurte, Leyval, andBerthelin 1990)—factors that are known to influencemetal mobility or availability. Evidence for heavymetal-binding has also been shown in AM fungi(Turnau, Kottke, and Oberwinkler 1993).

Since AM fungi cannot be cultivated without ahost plant, metal tolerance cannot be evaluatedthrough fungal growth on axenic media. Hence, themechanism of heavy metal tolerance in AM fungi hasnot yet been elucidated. Co-tolerance to Cd and Znwas suggested from one isolate from a soil pollutedwith long-term application of Zn through sewagesludge. Increased metal tolerance has also beenobserved for AM fungi isolated from a soil naturallyhigh in heavy metals (Weissenhorn, Glashoff,Leyval, et al. 1994).

Heavy metal uptake by ectomycorrhizal fungiSince many ectomycorrhizal fungi can be grown inaxenic culture, metal tolerance has been tested asmycelial growth on axenic media containingincreasing concentrations of heavy metal (Ray,Tiwari, Reddy, et al. 2005). In ectomycorrhizae,there are numerous examples of reduced metaluptake relative to non-mycorrhizal controls(Colpaert and Vanassche 1993; Denny and Wilkins1987; Marchner, Goldbold, and Jentschke 1996),although there are cases where no ameliorationoccurs (Jentschke, Fritz, and Goldbold 1997; Jonesand Hutchinson 1988a,b). Again, fungal sorptioncould be a major mechanism, but complexion byelements (P and S) enriched in or on mycorrhizalstructures (Marchner, Jentschke, and Goldbold1998) or specific proteins (Howe, Evans, andKetteridge 1997) and a dilution effect due toenhanced nutrition and plant growth in mycorrhizalplants may also explain some of the differences.

Heavy metal uptake by ectomycorrhizal fungi grownon fly ash amended media clearly indicated that highuptake ability for one metal does not imply highuptake ability for other metals by ectomycorrhizalfungi. Therefore, it can be said that a stronginterspecfic and intraspecfic variation exists in metaltolerance by ectomycorrhizal fungi (Ray, Reddy,Lapeyrie, et al. 2005).

Heavy metal translocation by mycorrhizalfungiThere has been considerable interest in the potentialuse of arbuscular mycorrhiza in agricultural systems.Experiments have shown that mycorrhizal effects canbe explained by mechanisms such as enlargement ofthe absorbing area and volume of accessible soil,decreased soil pH near the fungal hyphae due toexudation of organic acids and other ions, a highaffinity to P on the membranes of arbuscularmycorrhizal fungi and efficient hyphal transport inthe form of polyphosphate, and utilization bymycorrhiza of P sources that are unavailable to plantroots (Smith and Read 1997). In the similar line,transport of mineral elements into plants through thehyphae of AM fungi has been demonstrated for P(Li, Marchner, and George 1991a,b), N (Ames,Reid, Porter, et al. 1983), Zn (Kothari, Marchner,and Romheld 1991; Burkert and Robson 1994), Cu(Li, Marchner, and George 1991b), Cd (Joner andLeyval 1997), and Fe (Caris, Hordt, Hawkins, et al.1998). For other elements, although the uptake ofSr, Co, and Cs were found to be increased by AMcolonization (Jackson, Miller, and Franklin 1973;Rogers and Williams 1986), the contribution of thehyphae of the AM fungus to the uptake of theseelements has not been elucidated to a great extent.Suzuki, Kumagari, Oohashi, et al. (2001) suggestedthat hyphae of the AM fungal could absorb Zn, Na,Rb, Se, Sr, and Y from the soil and transport theseelements to the plants. They further suggested that theabsorption and transport ability of Be, Sc, Cr, Mn, Fe,Co, Zr, and Tc by AM fungus hyphae may be low.

Since AM fungi cannot be cultivated without ahost plant, it is more difficult to demonstrate theintrinsic metal uptake by their hyphae. Using culturesystems with separate extra radical hyphae fromroots, it has been shown that extra radical hyphaecan accumulate and translocate 65Zn that may differbetween species (Cooper and Tinker 1978). Adding109Cd to a hyphal compartment, Joner and Leyval(1997) showed that extraradical hypahe maytransport Cd from soil to roots.

Functioning and relevanceHeavy metals cannot be chemically degraded.Therefore, remediation of metal polluted soils ismainly limited to immobilization or extraction/concentration techniques. Among remediationoptions for metal contaminated sites,

20 Mycorrhiza News 18(2) • July 2006

phytoremediation methods have recently attractedmuch attention (Anderson and Coats 1994). Theprinciple of phytoremediation methods is to promoteplant growth, reduce or eliminate bioavailability ofmetals, minimize wind and water erosion, improvesoil quality, and reduce leaching of metals. Treatmentsinclude appropriate fertilization, either reduction inmetal availability using different amendment and/orusing metal tolerant plant species.

Ectomycorrhiza has been proposed as abioindicator of air pollution (Fellner 1989). Fungalfruit bodies are potentially useful as bioindicators ofradioactive contaminants of the environment(Berreck, Ohenoja, and Haselwandter 1992). Thesuitability of wild growing mushrooms, includingectomycorrhizal species as bioindicators of heavymetal pollution, has been investigated (Gast, Jansen,Bierling, et al. 1988). A study carried out in theChernobyl area of Ukraine revealed a closecorrelation between the radiocesium content in thearea where the fruit bodies are collected(Grodzinskaya, Berreck, Wasser, et al. 1995). Thisunderlines the potential use of fungal fruit bodies asbioindicators for the heavy metal pollution of thebiosphere as long as the number of samples perspecies and the species collected are representative.

Like ectomycorrhizal fungi, AM species havepotential which can be employed in biomonitoringprogrammes. The decline of AM fungal occurrenceand infectivity in metal polluted soils can be used asbioindicators of soil contamination (Leyval, Sing,and Joner 1995). Mycorrhizal colonization of plantroots after soil remediation can be a sign that themetal concentration or bioavailability has decreased.Since metal tolerance evolves in some fungi frommetal contaminated soils, a sensitive AM fungi canbe used and tested for its ability to colonize roots inthe metal polluted soils, providing usefulinformation about their metal toxicity. In variousinvestigations on the long-term effect of metals onAM fungi in arable fields where metal contaminationsludge has been applied for ten years, preliminaryresults have shown that the diversity of AM fungichanges even at low metal concentrations.

The use of mycorrhizal fungi as bioremediationagents has been reviewed by Donnelly and Fletcher(1994). Mycorrhizal fungi appear to partially protectplants against the toxicity of heavy metals. On theother hand, the host plant may give the fungus aselective survival advantage at a contaminated site.This mutual benefit would make this mycorrhizalassociation superior to the application of singleorganisms, either non-mycorrhizal plants or freeliving microorganisms, for bioremediation purposes.

ConclusionTo improve the understanding of the interactionsbetween the heavy metals, mycorrhizal fungi, androots in contaminated soils, parameters like the

tolerance of the mycorrhizal fungi and host plant, thenutritional status of both organisms, soil propertiesof the metals and their specific behaviour should beconsidered. The extent to which mycorrhizal fungican alleviate metal toxicity to plants under fieldconditions remains to be established.

The protective effect of ectomycorrhizal andericoid mycorrhizal fungi against metal toxicity inplants is quite clear and various mechanisms havebeen demonstrated, the main one being fungalstructures acting like barriers to metal uptake inplants. More work remains to be done on AM fungito understand the mechanism involved in metalimmobilization in the hyphae inside and/or outsidethe roots. The compartment system mentionedalready is a promising approach for such studies.Relatively large amounts of extraradical hyphae canbe produced and separated from the roots, whichshould allow the study of metal adsorption anduptake by AM hyphae, transfer to the roots, andtranslocation to the shoots. The functioning ofdifferent fungi could be compared in such systems.Other experimental devices including normal ortransformed roots might also be useful to study metaltolerance of AM fungi. Results with ectomycorrhizalfungi show metal specific tolerance mechanisms andcompetition between metals for uptake. This shouldalso be investigated for all kind of mycorrhizae, andespecially for the possible use of mycorrhizal fungiin phytoremediation.

There is no doubt that genetic engineering aimedat producing plants which are more tolerant andresistant to heavy metals may play an important rolein the future. But such attempts must not overlookthe possible changes in the susceptibility of theplants to mycorrhizal infection. A metal-resistanceplant breed should still be susceptible to mycorrhizalsymbiosis, and when colonized by a metal-resistantmycorrhizal fungus, genetically modified or derivedfrom the natural resources, should be of great valuefor the rehabilitation of metal contaminated soils.The management of soil microorganisms includingmycorrhizal fungi is a prerequisite for the success offuture restoration programmes.

ReferencesAdriano D C. 1986Trace elements in the terrestrial environmentBerlin Heidelberg, New York: Springer

Ames R N, Reid C P P, Porter L K,Cambardella C.1983Hyphal uptake and transport of nitrogen from 15 Nlabeled sources by Glomus mosseae, a vesicular-arbuscular mycorrhizal fungusNew Phytologist 95: 381–396

Anderson T A and Coats J R. 1994Bioremediation through rhizosphere technologyA C S Symposium series, Washington D C, USA.

Mycorrhiza News 18(2) • July 2006 21

Berreck M B, Ohenoja E, and Haselwandter K. 1992Mycorrhizal fungi as bioindicators of radioactivityIn Responses of forest ecosystems to environmental changes,edited by Teller A, Mathy P, and Jeffers J N R (editors)Elsevier: London pp. 800–802

Berthelin J, Munier-Lamy C, and Leyval C. 1995Effects of microorganisms on mobility of heavymetals in soilIn Environmental impacts of soil componentinteractions: Vol 2 Metals, other inorganics andmicrobial activities, edited by Huang P M, et al.pp. 3–17Florida: LEWIS Publishers

Burkert B and Robson A. 199465Zn uptake in subterrenean clover (Trifoliumsubterraneum L.) by three vesicular-arbuscularmycorrhizal fungi in a root free sandy soil.Soil Biology and Biochemistry 26: 1117–1124

Caris C, Hordt W, Hawkins H J, Romheld V,George E. 1998Studies of iron transport by arbuscularmycorrhizal hyphae from soil to peanut andsorghum plantsMycorrhiza 8: 35–39

Colpaert J V and Vanassche J A. 1993The effects of cadmium on ectomycorrhizal Pinussylvestris L.New Phytologist 123: 325–333

Cooper K M and Tinker P B. 1978Translocation and transfer of nutrients invesicular arbuscular mycorrhiza II. Uptake andtranslocation of Phosphorous, Zinc and SulphurNew Phytologist 81: 43–52

Dehn B and Schuepp H. 1989Influence of VA-mycorrhizae on the uptake anddistribution of heavy metals in plantsAgriculture, Ecosystems, and Environment 29: 79–83

Denny H J and Wilkins D A. 1987Zinc tolerance in Betula ssp. IV. The mechanism ofectomycorrhizal amelioration of Zinc toxicityNew Phytologist 106: 545–553

Donnelly P K and Fletcher J S. 1994Potential use of mycorrhizal fungi asbioremediation agentsAmerican Chemical Society Symposium Series 563: 93–99

Faber B A, Zasoski R J, Burau R G, Uriu K. 1990Zinc uptake by corn as affected by vesicular-arbuscular mycorrhizaePlant and Soil 129: 121–130

Fellner R. 1989Mycorrhiza-forming fungi as bioindicators of airpollutionAgricculture, Ecosystems and Environment 28: 115–120

Gast C H, Jansen E, Bierling J, Haanstra. 1988Heavy metals in mushrooms and their relationshipwith soil characteristicsChemosphere 17: 789–799

Giller K E, Witter E, and Macgrath S P. 1998Toxicity of heavy metals to microorganisms andmicrobial processes in agricultural soils: A reviewSoil Biology and Biochemistry 30: 1389–1414

Grodzinskaya A A, Berreck M, Wasser S P,Hasselwandter K. 1995Radiocesium in fungi:accumulation pattern in theKiev district of Ukraine including the ChernobylzoneSydowia. Beih. 10: 88–96

Harley J L and Smith S E. 1983Mycorrhizal symbiosisNew York: Academic Press

Howe R, Evans R L, and Ketteridge S W. 1997Copper-binding proteins in ectomycorrhizal fungiNew Pythologist l135: 123–131

Jackson N E, Miller R H, and Franklin R E. 1973The influence of vesicular arbuscular mycorrhizaeon uptake of 90Sr from soil by soybeansSoil Biology and Biochemistry 5: 205–212

Jentschke G, Fritz E, and Goldbold D L. 1991Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlingsPhysiologia Planturum 81: 417–422

Joner E J and Leyval C. 1997Uptake of 109Cd by roots and hyphae of a Glomusmosseae/Trifolium subterraneum mycorrhiza fromsoil amended with high and low concentrations ofcadmiumNew Phytologist 135: 353–360

Jones M D and Hutchinson T C. 1988Nickel toxicity in mycorrhizal birch seedlingsinfected with Lacterius rufus or Sclerodermaflavidum. II. Uptake of Nickel, calcium,magnesium, phosphorous and ironNew Phytologist 108: 461–470

Jones M D and Hutchinson T C. 1988aThe effect of Nickel and Copper on the axenicgrowth of ectomycorrhizal fungiCanadian Journal of Botany 66: 119–124

Juste C. 1988Appreciation de la mobilite et de la biodisponibilitedes elements en traces du soilScience du sol 26: 103–112

Kaldorf M, Khun A J, Schroder W H, Hildebrandt U,Bothe H. 1999Selective element deposits in maize colonized by aheavy metal tolerance conferring arbuscularmycorrhizal fungus.Journal of Plant Physiology 154: 718–728

22 Mycorrhiza News 18(2) • July 2006

Kothari S K, Marschner H, and Romheld V. 1991Contribution of the VA mycorrhizal hyphae inacquisition of Phosphorus and Zinc by maize grownin a calcareous soilPlant and Soil 136: 177–185

Laheurte F, Leyval C, and Berthelin J. 1990Root exudates of maize pine and beech seedlingsinfluenced by mycorrhizal and bacterialinoculationSymbiosis 9: 111–116

Leyval C, Haselwandter K, and Tarnau K. 1997Effect of heavy metal pollution on mycorrhizalcolonization and function: Physiological ecologicaland applied aspectsMycorrhiza 7: 139–153

Leyval C, Sing B R, and Joner E J. 1995Occurrence and infectivity of arbuscularmycorrhizal fungi in some Norwegian soilsinfluenced by heavy metals and soils propertiesWater, Air and Soil Pollution 84: 203–216

Li X-L, Marchner H, and George E.1991aExtension of phosphorous depletion zone inVA-mycorrhizal white clover in a calcarious soilPlant and Soil 136: 41–48

Li X-L, Marchner H, and George E.1991bAcquistion of phosphorous and copper byVA-mycorrhizal hyphae and root-to-shoottransport in white cloverPlant and Soil 136: 49–57

Marchner H. 1995Mineral nutrition of higher plantsLondon: Academic Press

Marchner P, Jentschke G, and Goldbold D L. 1998Cation exchange capacity and Lead sorption inectomycorrhizal fungiPlant and Soil 205: 93–98

Marchner P, Goldbold D L, and Jentschke G. 1996Dynamics of lead accumulation in mycorrhizaland non-mycorrhizal Norway spruce (Picea Abies(L.) karstPlant and Soil 178: 239–245

Olsson P A, Francis R, Read D J, Soderstrom B. 1998Growth of arbuscular mycorrhizal mycelium incalcareous dune sand and measurement of specificfatty acidsPlant and Soil 201: 9–16

Rauser W E. 1990PhytchelatinsAnnual Review of Biochemistry 59: 61–86

Ray P, Reddy U G, Lapeyrie F, Adholeya A. 2005Effect of coal ash on growth and metal uptake bysome selected ectomycorrhizal fungi invitro.World Journal of Microbiology and Biotechnology21: 309–315.

Ray P, Tiwari R, Reddy U G, Adholeya A. 2005Detecting the heavy metal tolerance level inectomycorrhizal fungi in vitro.International Journal of Phytoremediation 7: 199–216

Rogers R D and Williams S E. 1986Vesicular-arbuscular mycorrhiza: Influence onplant uptake of Cesium and cobaltSoil Biology and Biochemistry 18: 283–289

Shetty K G, Hetrick B A D, Figge D A H,Schwab A P. 1994Effects of mycorrhizae and other soil microbes onrevegetation of heavy metal contaminated minespoilEnvironmental Pollution 86: 181–188

Smith S E and Read D J. 1997Mycorrhizal symbiosisCalifornia: Academic Press

Suzuki H, Kumagari H, Oohashi K, Sakamoto K,Inubushi K, Enomoto S. 2001Transport of trace elements through the hyphae ofan arbuscular mycorrhizal fungus into Marigolddetermined by the multitracer techniquesSoil Science Plant Nutrition 47: 131–137

Turnau K, Kottke I, and Oberwinkler F. 1993Element localization in mycorrhizal roots ofPteridium aquilinum (L.) Kuhn collected fromexperimental plotstreated with cadmium dustNew Phytologist 123: 313–334

Weissenhorn I, Glashoff A, Leyval C, Berthelin J. 1994Differential tolerance to Cd and Zn of arbuscularmycorrhizal fungal spores isolated from heavymetal polluted soilsPlant and Soil 167: 189–196

Mycorrhiza News 18(2) • July 2006 23

Recent referencesLatest additions to the network’s database on mycorrhiza are published here for the 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 Agriculture Ecosystems & EnvironmentP American NaturalistP Annals of Forest ScienceP Applied Soil EcologyP Aquatic BotanyP Basic and Applied EcologyP ChemosphereP Ecological ResearchP Environmental MicrobiologyP Fems Microbiology Reviews

Title of the article, name of the journal, volume no., issue no., page nos(address of the first author or of the corresponding author, marked with an asterisk)

Diversity of arbuscular mycorrhizal fungi in Tetraclinis articulata(Vahl) Masters woodlands in MoroccoAnnals of Forest Science 63(3): 285–291[*Abbas Y, Centre of Forest Research, Root Symbiosis Laboratory,Sylviculture Department, B P 763, Rabat, Morocco]

Using the incomplete genome of the ectomycorrhizal fungus Amanitabisporigera to identify molecular polymorphisms in the relatedAmanita phalloidesMolecular Ecology Notes 6(1): 218–220[*Adams R I, Department Plant and Microbial Biology, University ofCalifornia Berkeley, Berkeley, CA 94720, USA]

Ectomycorrhizal ecology under primary succession on coastal sanddunes: interactions involving Pinus contorta, suilloid fungi and deerNew Phytologist 169(2): 345–354

Importance of mycorrhization helper bacteria cell density andmetabolite localization for the Pinus sylvestris Lactarius rufussymbiosisFEMS Microbiology Reviews 56(1): 25–33[*Aspray T J, Environm Reclamat Services Ltd, Westerhill Road, GlasgowG64 2QH, Lanark, Scotland]

Interactions between arbuscular mycorrhizal fungi and intra-specificcompetition affect size, and size inequality, of Plantago lanceolata LJournal of Ecology 94(2): 285–294[*Ayres R L, Centre for Academic Practice, University of Warwick, CoventryCV4 8UW, W Midlands, England]

Arbuscular mycorrhizal fungi associated with Populus-Salix stands in asemiarid riparian ecosystemNew Phytologist 170(2): 369–380[*Beauchamp V B, School of Life Sciences, Arizona State University, Tempe,AZ 85287, USA]

Name of the author(s) andyear of publication

Abbas Y*, Ducousso M,Abourouh M, Azcon R,Duponnois R. 2006

Adams R I*, Hallen H E,and Pringle A. 2006

Ashkannejhad S andHorton T R. 2006

Aspray T J*, Jones E E,Whipps J M, Bending G D.2006

Ayres R L*, Gange A C,and Aplin D M. 2006

Beauchamp V B*,Stromberg J C, andStutz J C. 2006

P Journal of EcologyP Journal of Plant PhysiologyP Microbiological ResearchP Molecular Ecology NotesP Molecular Phylogenetics and EvolutionP Mycological ResearchP MycorrhizaP New PhytologistP Plant Molecular Biology

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

24 Mycorrhiza News 18(2) • July 2006

Title of the article, name of the journal, volume no., issue no., page nos(address of the first author or of the corresponding author, marked with an asterisk)

Name of the author(s) andyear of publication

Benjdia M, Rikirsch E,Muller T, Morel M,Corratge C, Zimmermann S,Chalot M, Frommer W B,Wipf D*. 2006

Bennett A E*, Alers-Garcia J,and Bever J D. 2006

Bertini L, Rossi I,Zambonelli A, Amicucci A,Sacchi A, Cecchini M,Gregori G, Stocchi V*. 2006

Bois G*, Bertrand A,Piche Y, Fung M,Khasa D P. 2006

Cakan H* and Karatas C.2006

Chen B D, Zhu Y G*, andSmith F A. 2006

Cho K H, Toler H, Lee J,Ownley B, Stutz J C,Moore J L, Auge R M*. 2006

de la Pena E*, Echeverria SR, Putten W H, Freitas H,Moens M. 2006

Peptide uptake in the ectomycorrhizal fungus Hebelomacylindrosporum: characterization of two di- and tri-peptidetransporters (HcPTR2A and B)New Phytologist 170(2): 401–410[*Wipf D, ZMBP, Plant Physiol, Morgenstelle 1, D-72076 Tubingen,Germany]

Three-way interactions among mutualistic mycorrhizal fungi, plants,and plant enemies: Hypotheses and synthesisAmerican Naturalist 167(2): 141–152[*Bennett A E, Department of Ecology and Evolution, University of CaliforniaDavis, 4348 Storer Hall, Davis, CA 95616, USA]

Molecular identification of Tuber magnatum ectomycorrhizae in the field.Microbiological Research 161(1):59–64[*Stocchi V, Institute of Chimica Biologica Giorgio Fornaini, University ofUrbino, Via Saffi 2, I-61029 Urbino, PU, Italy]

Growth, compatible solute and salt accumulation of five mycorrhizalfungal species grown over a range of NaCl concentrationsMycorrhiza 16(2): 99–109[*Bois G, Laboratory of Mycology, University of Laval, Local 2110, PavillonCE Marchand, Quebec City, PQ G1K 7P4, Canada]

Interactions between mycorrhizal colonization and plant life formsalong the successional gradient of coastal sand dunes in the easternMediterranean, Turkey.Ecological Research 21(2): 301–310[*Cakan H, Faculty of Science and Letters, Department Biology, CukurovaUniversity, TR-01330 Adana, Turkey]

Effects of arbuscular mycorrhizal inoculation on uranium and arsenicaccumulation by Chinese brake fern (Pteris vittata L.) from a uraniummining-impacted soil.Chemosphere 62(9): 1464–1473[*Zhu Y G, Chinese Academy of Science, Ecoenvironmental Science ResearchCentre, State Key Laboratory of Environmental Chemistry and Ecotoxicology,Department of Soil Environmental Sciences, Beijing 100085, Peoples Republicof China]

Mycorrhizal symbiosis and response of sorghum plants to combineddrought and salinity stressesJournal of Plant Physiology 163(5): 517–528[*Auge R M, Department of Plant Sciences, University of Tennessee, 2431Joe Johnson Dr, Knoxville, TN 37996, USA]

Mechanism of control of root-feeding nematodes by mycorrhizal fungiin the dune grass Ammophila arenariaNew Phytologist 169(4): 829–840[*de la Pena E, CLO, Agricultural Research Centre, Burg Van Gansberghelaan96, B-9820 Merelbeke, Belgium]

Mycorrhiza News 18(2) • July 2006 25

Title of the article, name of the journal, volume no., issue no., page nos(address of the first author or of the corresponding author, marked with an asterisk)

Name of the author(s) andyear of publication

Fujiyoshi M*, Kagawa A,Nakatsubo T, Masuzawa T.2006.

Gehring C A* andConnell J H. 2006

Gosling P*, Hodge A,Goodlass G, Bending G D.2006

Gryta H*, Carriconde F,Charcosset J Y, Jargeat P,Gardes M. 2006

He X H, Bledsoe C S,Zasoski R J, Southworth D,Horwath W R*. 2006

Hol W H G* andCook R. 2005

Iotti M and Zambonelli A*.2006

Izumi H, Anderson I C*,Alexander I J, Killham K,Moore E R B. 2006

Lins C E L,Cavalcante U M T,Sampaio E V S B,Messias A S, Maia L C*.2006

Effects of arbuscular mycorrhizal fungi and soil developmental stageson herbaceous plants growing in the early stage of primary successionon Mount FujiEcological Research 21(2): 278–284[*Fujiyoshi M, School of Humanities and Culture, Department of HumanDevelopment, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa2591292, Japan]

Arbuscular mycorrhizal fungi in the tree seedlings of two Australianrain forests: occurrence, colonization, and relationships with plantperformanceMycorrhiza 16(2): 89–98[*Gehring C A, Department of Biological Sciences, Northern ArizonaUniversity, Box 5640, Flagstaff, AZ 86011, USA]

Arbuscular mycorrhizal fungi and organic farmingAgriculture Ecosystems & Environment 113(1–4): 17–35[*Gosling P, Warwick HRI, University of Warwick, Warwick CV35 9EF, England]

Population dynamics of the ectomycorrhizal fungal species Tricholomapopulinum and Tricholoma scalpturatum associated with black poplarunder differing environmental conditionsEnvironmental Microbiology 8(5): 773–786[*Gryta H, University of Toulouse 3, UMR 5174, Laboratory for Evolutionand Divers Biol, CNRS, ENFA, Bat 4R3,118 Route Narbonne, F-31062Toulouse 9, France]

Rapid nitrogen transfer from ectomycorrhizal pines to adjacentectomycorrhizal and arbuscular mycorrhizal plants in a California oakwoodlandNew Phytologist 170(1): 143–151[*Horwath W R, Department of Land, Air and Water Resources, University ofCalifornia Davis, Davis, CA 95616, USA]

An overview of arbuscular mycorrhizal fungi-nematode interactionsBasic and Applied Ecology 6(6): 489–503[*Hol W H G, Agricultural Research Centre, Burg van Gansberghelaan 96,B-9820 Merelbeke, Belgium]

A quick and precise technique for identifying ectomycorrhizas by PCRMycological Research 110(1): 60–65[*Zambonelli A, Dipartimento Protez and Valorizzaz Agroalimentaire,University of Bologna, Via Fanin 46, I-40127 Bologna, Italy]

Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris)FEMS Microbiology Reviews 56(1): 34–43[Anderson I C*, Macaulay Land Use Research Institute, Aberdeen AB15 8QH,Scotland]

Growth of mycorrhized seedlings of Leucaena leucocephala (Lam.) deWit. in a copper contaminated soilApplied Soil Ecology 31(3): 181–185[*Maia L C, Departmento de Biologia, Univrsidade Federal de Pernambuco,BR-50670420 Recife, PE, Brazil]

26 Mycorrhiza News 18(2) • July 2006

Title of the article, name of the journal, volume no., issue no., page nos(address of the first author or of the corresponding author, marked with an asterisk)

Name of the author(s) andyear of publication

Phylogeny of the ectomycorrhizal mushroom genus Alnicola(Basidiomycota, Cortinariaceae) based on rDNA sequences with specialemphasis on host specificity and morphological charactersMolecular Phylogenetics and Evolution 38(3): 794–807[*Moreau PA, Faculty of Pharmaceutical Science and Biology, LaboratoryBot, 3 Rue Prof Laguesse, BP 83, F-59006 Lille, France]

PIP aquaporin gene expression in arbuscular mycorrhizal Glycine maxand Lactuca sativa plants in relation to drought stress tolerancePlant Molecular Biology 60(3): 389–404[*Ruiz-Lozano J M, CSIC, Estac Expt Zaidin, Department of MicrobiologySuelo and Sistemas Simbiot, Prof Albareda 1, E-18008 Granada, Spain]

Effects of water-level fluctuations on the arbuscular mycorrhizalcolonization of Typha latifolia LAquatic Botany 84(3): 210–216[*Ray A M, Centre for Ecological Research and Education, Idaho StateUniversity, Pocatello, ID 83209, USA]

Drought effects on fine-root and ectomycorrhizal-root biomass inmanaged Pinus oaxacana Mirov stands in Oaxaca, MexicoMycorrhiza 16(2): 117–124[*Asbjornsen H, Department of Natural Resource Ecology and Management,Iowa State University, Ames, IA 50011, USA]

Moreau P A*, Peintner U,and Gardes M. 2006

Porcel R, Aroca R,Azcon R, Ruiz-Lozano J M*.2006

Ray A M* and Inouye R S.2006

Valdes M, Asbjornsen H*,Gomez-Cardenas M,Juarez M, Vogt K A. 2006

Publication of research findings

Mycorrhiza News invites short papers on the subject forpublication by mycorrhizologists. Papers may be sent to:

The EditorMycorrhiza NewsT E R IDarbari Seth BlockIHC Complex, Lodhi RoadNew Delhi – 110 003, India

Papers sent in print form should be followed by soft copies,carrying the complete mailing address, telephone number, and faxnumber of the author/s. E-mails may be sent to<aloka@teri.res.in> or <tpsankar@teri.res.in>.

Mycorrhiza News 18(2) • July 2006 27

Granada, Spain23–27 July 2006

Melbourne, Australia6–9 August 2006

Kuala Lumpur, Malaysia9–11 August 2006

Brisbane, Australia16–19 August 2006

Mexico City, Mexico20–25 August 2006

Madurai, India18 September–21 October 2006

Bangalore, India9–11 November 2006

Boston, USA16–17 November 2006

New Delhi, India2–4 February 2007

5th International Conference on Mycorrhiza Professor Albareda 1, E-18008 Granada, SpainTel. +34 958 181600 • Fax +34 958 129600 E-mail francisca.gonzalez@eez.csic.es

Agricultural Biotechnology International Conference‘Unlocking the potential of agricultural biotechnology’ABIC 2006 Conference ManagersLevel 1, 322 Gelnferrie Road, Malvern Victoria 3144Tel. +61 2 9265 0700 • Fax +61 2 9267 5443E-mail hcheeseman@ausbiotech.org • Website http://www.abic2006.org

Biotechnology Asia 2006 Conference and ExhibitionBiotechnology Asia 2006 Conference Secretariat60B, Jalan SS21/58 Damansara Utama, 47400 Petaling Jaya, Selangor Darul Ehsan,MalaysiaTel. (603) 7727 0619 • Fax (603) 7727 0614Website http://www.biotechexpo.com.my

Tropical Crop Biotechnology ConferenceHoteliers InternationalPO Box 12563, George St Post Shop, Brisbane QLD 4003, AustraliaTel. +61 7 3210 1646 • Fax +61 7 3210 1606E-mail hoteliers@hoteliersint.com • Website http://www.tcbc2006.com.au/

International Plant Breeding SymposiumHotel Sheraton Centro HistóricoMexico CityE-mail intlplantbreeding@cgiar.org • Website http:// www.intlplantbreeding.com

International Conference on Biotechnology in Water ManagementProf. P S Navaraj, Yadava College, MaduraiE-mail navaraj678@sify.com

5th Conference of the Asian Federation for Information Technology inAgriculture AFITA 2006Dr V C Patil, National Science Seminar Complex, Indian Institute of Science,Bangalore, IndiaWebsites http://www.afita2006.org; http://www.insait.org

NanoBiotech World Congress and ExhibitionTel. 203 926 1400 • Fax 203 926 0003E-mail naenquiries@selectbiosciences.comWebsite http://www.nanobiotechcongress.com

30th All-India Cell Biology Conference and Symposium on Molecules toCompartments: Cross-talks and networksDepartment of Zoology, University of Delhi (North Campus), DelhiTel. +91-11 27666051 • E-mail ashrivastava@zoology.du.ac.in

Forthcoming eventsConferences, congresses, seminars, symposia, andworkshops

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 T P Sankar • Assistant Editor Jaya Kapur

ISSN 0970-695X Regd No. 49170/89 Rs 38/-

VAM - For the first time in the worldProduced and processed through Sterile Technology

Mycorrhiza (Fungus root) is the symbiotic associationbetween plant roots and soil fungus. The Endomycorr-hiza (VAM) is an obligate symbiont. VAM (VesicularArbuscular Mycorrhiza) for Agriculture, Forestry andHorticulture crops is being produced through the mostadvanced technology, is now commercially available forfarming applications.

The Mass production technology of VAM has beendeveloped by TERI-DBT for the first time in the world.We are the first in the world to produce VAM incommercial scale using the sterile technology of TER I-DBT.

Named as ECORRHIZA-VAM (Powder form) & NURS-ERRHIZA-VAM (Tablet form), the biotic root inoculantgives the following benefits to the crop plants.

• Increased phosphorus uptake • Increased micronutri-ent uptake • Enhanced water uptake • Increasedresistance to pathogens and pests • Enhancedtolerance to soil stress viz. high salt levels, heavy metaltoxicity, drought, high temperatures etc. • Enhancedtransplant survival • Enhanced beneficial microbialpopulation in the root zone.

We are also producing the Bio-fertilizers forNitrogen fixation (Azospirillum, Azotobacter),Phosphate solubilization (Bacillus megateriumvar.phosphaticum).

1. AZOSPIRILLUM: This is an associative symbiotic Nitrogen fixing bacteria. It fixes the atmospheric Nitrogen throughthe action of Nitrogenase enzyme. It secretes auxin, cytokinin & gibberellin like substances which promote the growthof plants. It is useful for non-leguminous crops. DOSAGE: 4 kgs. per acre 2. AZOTOBACTER: This is a free living,Non-symbiotic Nitrogen fixing bacteria. It fixes atmospheric Nitrogen through the action of Nitrogenase enzyme. It isspecially recommended for Paddy crop. DOSAGE: 4 kgs. per acre. 3. BACILLUS MEGATERIUM VAR.PHOSPHATICUM (PHOSPHOBACTER): This bacteria solubilizes the unavailable phosphorus in the soil through thesecretion of weak organic acids such as formic, acetic, lactic acids etc. These acids reduce the pH and bring about thedissolution of bound the forms of phosphate. Some of the hydroxy acids may chelate with Calcuim and Iron resulting ineffective solubilization and utilization of phosphorus.DOSAGE: 4 kgs. per acre

All the above Bio-fertilizers are compatible with each other. Chemical fertilizer use can be reduced by 25%. UseAzospirillum / Azotobacter, Phosphobacter and Ecorrhiza-VAM together for better results.

ECORRHIZA-VAM (Mycorrhizalinoculum): In Powder formDosage: 3-5 kgs. per acreApplication Details: Mix 3-5 kgs.of Ecorrhiza-VAM in 200-250kgs. of organic manure & applyuniformly to all plants near the roots. Irrigate the field to maintain enough

moisture for the Mycorrhizal spore germination andintegration with the root system.

N U R S E R R H I Z A - V A M(Mycorrhizal inoculum): In Tabletform Dosage: 1 Tablet / Polybag orpot in NurseriesApplication Details: Place 1 tabletin 2-4 inch deep hole near the plantroots in polybags or pots. Cover thehole with soil and water the plant. The

tablet will dissociate and Mycorrhiza will integrate withthe root system of the plant.

The above products: • Contain pure, Pathogen freeand viable inoculum • Have long shelf life • Areproduced through soil less production system • Canbe applied and stored easily

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