About TERI The Bioresources and Biotechnology Division The...

About TERIA dynamic and flexible organization with a global vision and a local focus,TERI was established in 1974. While in the initial period the focus was mainlyon documentation and information dissemination activities, research activitiesin the fields of energy, environment, and sustainable development wereinitiated towards the end of 1982. The genesis of these activities lay in TERI’sfirm belief that efficient utilization of energy, sustainable use of naturalresources, large-scale adoption of renewable energy technologies, and reductionof all forms of waste would move the process of development towards the goalof sustainability.

The Bioresources and Biotechnology DivisionFocusing on ecological, environmental, and food security issues, the Division’sactivities include working with a wide variety of living organisms, sophisticatedgenetic engineering techniques, and, at the grassroots level, with villagecommunities. The Division functions through five areas – Centre for MycorrhizalResearch, Microbial Biotechnology, Plant Molecular Biology, Plant TissueCulture, and Forestry/Biodiversity. The Division is actively engaged inmycorrhizal research. The Mycorrhiza Network has specifically been created tohelp scientists across the globe in carrying out research on mycorrhiza.

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

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

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

Vol. 12 No. 1April 2000

2 Mycorrhiza News 12(1) • April 2000

Effect of edaphic and climatic factors on development ofmycorrhiza in tree nurseries (part III): effect of soil toxicity

Sujan Singh ... 2

Research findingsEffect of VAM on root induction in in vitro sugarcane

(Saccharum officinarum L.) seedlings � a new technique ... 13

Distribution of an ectomycorrhizal fungus (Pisolithus tinctorius)in association with different forest tree species in AndhraPradesh, southern India ... 16

Long-term preservation and delivery of ectomycorrhizalfungal inoculum ... 18

New approachesMolecular tools for generic differentiation in Glomales ... 19An improved procedure for mycorrhizal root surface

disinfection ... 19

Recent references ... 20

Forthcoming events ... 23

List of cultures available with the CMCC ... 24

Contents

Many unfavourable edaphic factors may influencedevelopment of mycorrhiza in tree nurseries. Theseinclude toxicity due to metals, release of allelo-pathic compounds in soil, high salt concentrationsin soil, etc.

Toxicity due to metalsMetal toxicity in soil may be induced by dischargeof sewage or industrial pollutants into the soil ordue to the presence of excessive quantities ofmetals in certain categories of soils. Information onthe effect of heavy metal pollution on mycorrhizawas given in an earlier issue of Mycorrhiza News(Sujan Singh 1996). This issue gives the work doneon experimental evidence of the effect of metals onmycorrhiza.

Various physico-chemical reactions in the soildetermine the availability of metals. Of specificimportance are the adsorption of metals onto soilparticles, competition of the metals present in thesoil, and interaction of biological factors in therhizosphere. Increased tolerance of mycorrhizalplants to toxic metal concentrations in the soilmakes mycorrhiza significant (Schepp, Dehn, andSticher 1987).

Aluminium toxicity

Effect of mycorrhiza on aluminium toxicityin plantsStudies were conducted at the Departmento deGenetica Escola, Superior Agricultura Luiz deQueiroz University, Sao Paulo, Brazil, onaluminium-tolerant and -intolerant cultivars ofLeucaena leucocephala. Height, shoot dry weight,and nutrient accumulation increased in both cultivars,65 days after the seedlings were transplanted in the

pots with washed, sterilized sand, irrigated withnutrient solution containing 0 and 9 ppm Al(aluminium) and inoculated with Glomusleptotichum; but the increases were greater inAl-tolerant cultivars. There were no significantdifferences between seedlings given different Altreatments (Melo, Silveira, and Maluf 1987).

In studies conducted at the Department ofNatural Resources and the Boyce–ThompsonInstitute for Plant Research, Cornell University,USA, pitch pine (Pinus rigida) seedlings weregrown with or without Pisolithus tinctorius andexposed to low levels of Al on sand culture. At50 µM, Al reduced non-mycorrhizal seedlinggrowth and increased foliar concentrations but didnot alter photosynthetic gas exchange or other as-pects of seedling nutrition. Non-mycorrhizal seed-lings exposed to 200 µM Al exhibited decreasedgrowth, increased transpiration rates, decreasedwater use efficiency, increased foliar aluminiumand sodium, and reduced foliar phosphorus.Mycorrhizal seedlings when exposed to Al exhib-ited unaltered growth, physiological functions, andionic relations. The studies suggested that thefungal symbiont modulated ionic relations in therhizosphere which reduced Al–phosphorusprecipitation reactions, Al uptake, and subsequentAl exposure of root and shoot tissue (Cummingand Weinstein 1990a).

Studies were conducted with the seedlings ofbirch, pine, and spruce at the School of BiologicalSciences, University of Birmingham, Birmingham,Great Britain. Mycorrhizal associations withspecies of Amanita, Paxillus, Pisolithus, Rhizopogon,Scleroderma, and Suillus decreased the toxicity ofzinc, copper, nickel, and aluminium. Except withlow levels of treatments, the presence ofmycorrhiza decreases metal accumulation in

Effect of edaphic and climatic factors on development of mycorrhiza intree nurseries (part III): effect of soil toxicity

Sujan Singh*TERI, Darbari Seth Block, Habitat Place, Lodhi Road, New Delhi – 110 003, India

* Compiled from TERI database – Riza

Mycorrhiza News 12(1) • April 2000 3

shoots. Concentration in roots is little affected butlarge amount of metal may be found inextramatrical hyphae (Wilkins 1991).

In studies conducted at the Department ofMicrobial Ecology, University of Lund,Helgonavagen, Sweden, intact ectomycorrhizalsystems containing Fagus sylvatica, Betula pendula,or Pinus contorta plants infected with either Paxillusinvolutus or Laccaria bicolor and grown in eitherpeat microcosms or semihydroponic cultureshowed that elevated concentrations of Aldecreased the yield of all plant species and thepotassium concentration in roots at high levels.The interactions involving mycorrhizal infectionwere strongest in P. contorta. Yield of mycorrhizalpine plants was largely unaffected by raised Alalthough mycorrhizal root morphology was alteredin peat microcosm and phosphorus and magnesiumconcentrations significantly increased in themycorrhizal roots (Finlay, Kottke, and Soderstrom1993).

Studies were conducted on Pinus sylvestrisseedlings at the Polish Academy Science Institute,Dendrol, Parkowa, Kornik, Poland. Seedlings weregrown on semisterilized peat parlite medium withaluminium ion (Al3+) concentration of 4.0 mM andinoculated with Al-tolerant strain of Suillus luteus(No. 14). Mycorrhizal abundance was similar inAl-treated and untreated plants. Of the threemycorrhizal morphotypes (single, dichotomousbranched, and coralloid), the coralloid mycorrhizaewere rare in Al-treated plants. Also, mycorrhizae inAl-treated plants had thinner mantle and lackedHarting net as compared to untreated controls.The Al+3 concentration did not reduce growth.Also, mycorrhizal formation with the selectedS. luteus strain caused lower Al3+ translocation tothe upper part of the tested seedlings comparedwith non-mycorrhizal controls (Kieliszewska-Rokicka et al. 1998).

The fixation and removal of phosphorus in thelaterite soil due to iron and Al inducedphosphorus deficiency in sweet potato which couldbe rectified to a certain extent by inoculation withVAM (vesicular–arbuscular mycorrhizal) fungusGlomus microcarpum in studies conducted at theDepartment of Post Graduate Studies andResearch in Botany, Sanatana Dharma College,Alappuzha, India (Haricumar and Potty 1999).

Effect of mycorrhiza on aluminium toxicityunder different nitrogen sourcesPitch pine (Pinus rigida) seedlings were grown insand culture with NO

3−, NH

4NO

3, or NH

4+ and

exposed to 0 or 200 µM Al for six weeks in studiesconducted at the Department of Botany, Universityof Vermont, Burlington, USA. Foliar nitrogen con-centrations, root nitrate reductase activity, and Alinhibition of nitrate reductase activity weredependent on the proportion of nitrate in the nutri-ent solution. The association of Pisolithus tinctoriuswith roots of seedlings reduced both root and foliar

nitrogen reductase activity compared withuninoculated controls suggesting that symbiontsreduced nitrate uptake and translocation to foliage.This was confirmed by using 36 ClO

3− to measure

undirectional plasma membrane nitrate fluxes.Mycorrhizal root tips absorbed 50% less nitratethan non-mycorrhizal root tips. Preferential use ofNH

4+ by ectomycorrhizal roots may thus result in

reduced movement of Al into root tissues andamelioration of Al toxicity (Cumming 1990).

In another study conducted at the same univer-sity, non-mycorrhizal and mycorrhizal (withPisolithus tinctorius) seedlings of Pinus rigida weregrown in a-quarter strength Johnson’s solutionmodified to contain NO

3−, NH

4NO

3, or NH

4+

and

exposed to 0 or 200 µM Al for six weeks. Non-mycorrhizal seedlings grown with NO

3− showed

reductions in height, growth, and final shootweight in response to Al. Aluminium, for allsources of nitrogen, consistently reduced the rootweights. Increasing the proportion of NO

3−

in the

nutrient solution raised cation accumulation inroots and shoots and depressed tissue anionconcentrations. The coprecipitation of Al andphosphate in roots of Al-treated seedlings furtherlimited phosphorus availability in this treatment.Mycorrhizal infection maintained growth and foliarphosphorus levels under Al exposure suggestingthat Al-induced phosphorus limitation was acritical factor in non-mycorrhizal seedlings grownon primarily NO

3−-based nutrient solutions

(Cumming and Weinstein 1990b).In studies conducted at the US Forest Service,

United States Department of Agriculture, NorthEast Research Station, Delaware, USA, Pinus rigidaseedlings, with or without Pisolithus tinctoriusinoculation, were grown in sand irrigated withnutrient solution (pH 3.8) containing 0, 1, or20 mg Al per litre (0, 370, or 740 µM Al) to see theeffect of elevated nitrate nitrogen or ammoniumnitrogen where nitrogen was increased three timesabove ambient levels. Elevated nitrate nitrogen hadno significant effect on Al toxicity in non-mycorrhizal seedlings but Al toxicity at ambientammonium nitrogen was ameliorated by elevatedammonium nitrogen. Symptoms of Al toxicity inroots of mycorrhizal seedlings at ambient nitrogenlevel were reduced by elevated ammonium nitrogenand absent in elevated nitrate nitrogen (Schier andMcQuattie 1999).

Effect of mycorrhiza collected fromdifferent site conditions on aluminiumtoxicityIn studies conducted at the School ofEnvironmental Biology, Curtin University ofTechnology, Perth, Western Australia, Al toleranceof Pisolithus tinctorius isolates collected from threedifferent localities – a 30-year-old abandoned coalmining site (pH 4.3; Al 164 ppm), a 10-year-oldrehabilitated site (pH 5.1; Al 6 ppm), and forestsite (pH 5.3; Al 2 ppm), was determined both in


Al-impregnated MMN (Modified Melin-Norkran’s) medium and in host tissues. Isolatesfrom abandoned coal mining site demonstratedmycelial growth up to 2000 ppm Al with a thresh-old of 90 ppm before significant Al accumulationoccurred. In contrast, growth of isolates from reha-bilitated site and forest site was limited by 22 ppmand 12 ppm Al, respectively; with significant metalaccumulation at 1–4 ppm substrate Al. Electronmicroprobe analysis of eucalyptus seedlings inocu-lated with Al-tolerant (mine site) or Al-sensitive(forest site) isolates and control plants (withoutmycorrhizal inoculation) grown in acidic,Al-contaminated soil, demonstrated that Al accu-mulation within the hyphal wall and presence ofmetal within the cytoplasm affected the degree ofAl tolerance in the host plant. The presence of Alin the cytoplasm coincided with significant accu-mulations of sulphur, indicating a role ofphytochelatins as biochemical (and hence genetic)basis for tolerance to Al in mine site isolates. Re-tention of Al within the hyphal mantle led to de-creased Al levels across the root proper. Incontrast, non-mycorrhizal roots demonstratedconsistently high Al levels in all root tissues withnegligible quantities of calcium and magnesiumindicating a degree of cellular necrosis attributableto Al toxicity. Dry-weight analysis confirmed Alaccumulation within roots of seedlings inoculatedwith Al-tolerant mycorrhizae and limited metaltranslocation to the shoot. In contrast, forest siteand control plants contained as much Al in shootas in root tissues. Also, plants inoculated withAl-tolerant isolates were taller with greater rootand shoot dry mass and increased mycorrhizalinfection and possessed superior nutrient statuswith total nitrogen, phosphorus, and potassiumdouble that of seedlings inoculated with isolatesfrom forest site. Control seedlings possessed lownutritional status (Louise, Brendon, and John 1992).

Effect of mycorrhiza on aluminium toxicityunder elevated CO

2In studies conducted at the US Forest Service,North East Forest Research Station, Delaware,USA, Pinus rigida seedlings inoculated withPisolithus tinctorius were grown for 13 weeks onsand irrigated with two different levels of nutrients(0.1 or 0.2 strength Clark solution) containing0, 6.25, 12.5, or 25 mg/litre Al (0, 232, 463, or927 µM Al) in growth chambers fumigated with350 (ambient) or 700 (elevated) µlitre/litre CO

2. At

ambient CO2 in the absence of Al, mean total dryweight of seedlings at high nutrient level was 164%higher than that at the low level. Total dry weightat the elevated CO2, in the absence of Al, was sig-nificantly greater than that in the ambient CO

2 at

low (+34%) and high (+16%) nutrient levels. Rootand shoot dry weight at both nutrient levelsdecreased with increasing Al concentrations withAl reducing root growth more than shoot growth.Although visible symptoms of Al toxicity in roots

and needles were reduced by CO2 enrichment,

there was no significant CO2–Al interaction for

shoot and root dry weight. The percentage ofseedlings’ roots that became mycorrhizal werenegatively related to nutrient level and was greaterat elevated than at ambient CO

2 levels (Schier and

McQuattie 1998).

Effect of aluminium and calcium onectomycorrhizal fungiIn studies conducted at the Department of Botany,University of Toronto, Ontario, Canada, isolates ofHebeloma crustuliniforme, Rhizopogon rubescens, andSuillus tomentosus obtained from basidiospores ofdifferent-aged jack pine (Pinus banksiana) standswere grown for four weeks in nutrient solutionscontaining 37, 185, 370, or 740 µM Al combinedin a factorial design with 25, 125, 250, or 500 µMcalcium and maintained at pH 3.8. Growthof all fungi was reduced at 370 µM Al. InH. crustuliniforme, stepwise increments in externalAl concentrations resulted in reduced mycelialconcentrations of calcium, magnesium, and potas-sium, and increased mycelial concentrations of Al,phosphorus, and iron. However, stepwise incre-ments in external calcium concentrations from25 to 500 µM stimulated the growth ofH. crustuliniforme. In R. rubescens, high external Alconcentration resulted in reduced mycelial concen-tration of all elements, except Al and phosphorus,while increments of calcium had no effect on thegrowth of this fungus. In S. tomentosus, high exter-nal Al concentration led to increased mycelial con-centration of all elements except calcium andsulphur while high external level of calcium actedsynergistically with high external Al concentrationto reduce the growth of this fungus. The responseof S. tomentosus to Al and calcium could not bepredicted from the soil and basidiocarp analysis.Alterations in calcium–Al ratios of soils may influ-ence the succession of ectomycorrhizal fungi(Browning and Hutchinson 1991).

Effect of aluminium and pH onectomycorrhizaSoil pH affects Al toxicity to mycorrhizal fungi andplants. In acidic forest soils, Al and other heavymetals become water soluble, reducing the absorb-ing capacity of the tree root system, primarilythrough destruction of mycorrhizal fungi andresulting in death of stress trees (oaks) (Jakucs etal. 1986).

In studies conducted at the Institute fürAllgemeine Botanie, Johannes Gutenberg –Universitat, Mainz, Germany, three-year-oldseedlings of Norway spruce (Picea abies) were heldfor two years in spray hydroculture and thentreated for six months with nutrient solution simi-lar in composition to lysimeter solution obtainedfrom below spruce forests. The pH values wereadjusted to 5.0, 3.5, and 2.5, and some of thenutrient solutions with pH 3.5 or 2.5 were


enriched with 1 or 2 mM Al per litre. The acidstress caused damage to fine roots, namely lysis ofthe peripheral root cell walls (acid holes), destruc-tion of the calyptra, lysis of the root meristem, and,in some cases, collapse of the phloem in Norwayspruce (Picea abies) seedlings. Aluminium intensi-fied the damage and also led to swelling of the roottips and made the root cell walls brittle (Vogeleiand Rothe 1988).

In studies conducted at the Department ofForest Science, College of Forestry, Oregon StateUniversity, Corvallis, USA, balsam fir (Abiesbalsamea) seedlings inoculated with Cenococcumgeophilum (isolate 145A), Hebeloma crustuliniforme(isolate S166), and Laccaria laccata (isolate 238B),were grown in potting mixture which was suppliedwith 0, 50, or 100 mg Al per g of substrate andadjusted to pH 3, 4, or 5. Seedlings were wateredwith one-fifth strength Arnon’s solution having apH of 3, 4, or 5 and containing 0, 50, or 100 mg Alper litre. With increase in solution acidity and Alconcentration, ectomycorrhizal formation, rootweight, shoot weight, total weight, root–shootratio, and boron concentration decreased; iron,manganese, phosphorus, and zinc concentrationsincreased; and nitrogen, potassium, calcium, andmagnesium concentrations did not vary signifi-cantly (Entry et al. 1987).

Studies were conducted at the School ofBiological Sciences, University of Birmingham,Birmingham, Great Britain, on Norway spruceseedlings obtained from seedlots collected frompopulations growing in acidic and calcareous soilsand then grown on sterile perlite culture containing0–6 mM Al. Aluminium inhibited hypocotylextension in seedlings in calcareous soils but not inseedlings in acidic soils. Both types of seedlingswere inoculated or not with Paxillus involutus andgrown for further 10 weeks in perlite. Aluminiumsulphate solutions were added to raise Al concen-trations to 0–6 mM. The seedlings were harvestedafter further 10 weeks. The fungus was associatedin roots of inoculated plants but did not formmycorrhiza. The growth of uninoculated acidicplants was somewhat stimulated by Al treatmentbut that of uninoculated calcareous plants was re-duced and the plants were chlorotic. The presenceof mycorrhizal fungus in rhizosphere enhancedgrowth of calcareous plants but had little effect onacidic plants. Shoot analysis suggested that greaterAl sensitivity of calcareous plants involves an in-ability to exclude Al or to maintain normal calciumand magnesium uptake in its presence. The pres-ence of rhizospheric fungi reduced the effect of Al(Wilkins and Hodson 1989).

Nickel toxicityStudies were conducted on birch (Betula papyrifera)seedlings at the Department of Botany, Universityof Toronto, Toronto, Ontario, Canada. The seed-lings were either uninoculated or inoculated withScleroderma flavidum (known to increase nickel

tolerance) or Lactarius rufus (does not increasenickel tolerance), and were exposed to 85 µM Ni(nickel). Taking decreased water uptake byseedlings as an indicator of the progression of Nitoxicity for over 16 weeks, L. rufus was found toprovide some initial protection against Ni toxicity.However, water uptake in S. flavidum infectedseedlings was least affected by Ni and was signifi-cantly higher than L. rufus infected or uninoculatedseedlings after week 10. Total dry weight of theS. flavidum infected seedlings was higher than thatof other seedlings at weeks 12 and 21. The weightof S. flavidum tissue per root, as determined bychitin analysis, was significantly greater than thatof L. rufus in Ni-treated and control seedlings, itincreased with time. L. rufus did not grow once theroots were exposed to Ni but S. flavidum continuedto grow following Ni exposure and this may be animportant characteristic in distinguishing fungi forenhancing metal tolerance. Nickel reduced photo-synthetic rates in seedlings infected withS. flavidum relative to S. flavidum inoculated seed-lings not exposed to Ni but this effect was lackingin uninoculated seedlings. Respiration rates werenot affected by metal treatment or mycorrhizalformation (Jones and Hutchinson 1988).

Betula papyrifera was grown with or without Nifor 22 weeks and then both were exposed to 63Nifor two minutes to 48 hours in further studiesconducted at the same university. After Ni uptake,the roots were desorbed with 50 mM calcium chlo-ride at 5 ºC for 30 minutes to determine exchange-able Ni. Total exchangeable 63Ni uptake in rootswas unaffected by inoculation treatments withLactarius rufus or Scleroderma flavidum either in thepresence or absence of DNP (dinitrophenol), ageneral metabolic inhibitor. The amount of ex-changeable Ni was significantly affected by infec-tion and uptake was least in seedlings inoculatedwith S. flavidum. Application of DNP increased Niuptake in shoots of uninfected seedlings and thoseinoculated with L. rufus but not in seedlings inocu-lated with S. flavidum or those in the absence ofDNP (Jones, Dainty, and Hutchinson 1988).

Cadmium toxicityMycorrhizal associations reduced the uptake ofheavy metals in shoots of plants growing in soilswith high concentrations of readily available metalsin experiments conducted at the Eidg.Forschungsanstalt fhr Obst-Wein-und-Gartenhau,Wadensinl, Germany. In soils with low metalcontent, VAM increased the uptake of zinc butdecreased the uptake of highly toxic Cd (cadmium)in shoots (Schepp, Dehn, and Sticher 1987).

Studies were conducted at the Laboratory ofPlant Ecology, Katholiecke Universiteit, Lenven,Belgium, on Pinus sylvestris plants cultivated in asemihydroponic system that allowed the visualobservation of the root system for comparison ofthe ability of nine ectomycorrhizal fungi (collectedfrom either unpolluted soils or soils polluted with


zinc and Cd) to increase Cd tolerance in plants.Although Cd addition resulted in decreases intranspiration in the non-mycorrhizal plants,disturbances in water relations could be more orless pronounced in the mycorrhizal pines. Severalmycorrhizal fungi hardly survived in the pollutedsubstrate. Fungi producing a large mycelialbiomass showed the greatest effect in overcomingtoxicity. Some strains were able to increase theirgrowth when treated with Cd. In a denseextramatrical mycelium, the potential for Cdretention increases where the individual hyphae areexposed to a lower Cd concentration than in asparse mycelium. A protective effect against Cdtoxicity in the host was observed with allmycobionts since Cd uptake was highest in non-mycorrhizal seedlings. The treatment had no effecton the growth of the seedlings or on the concentra-tions of other cations probably due to the relativelylow toxicity and short observation period (Colpaertand Van Assche1993).

In studies conducted at the University ofWales, School of Agriculture and Forest Science,Bangor, Wales, UK, both mycorrhizal (withLaccaria bicolor or Paxillus involutus) and non-mycorrhizal seedlings of Norway spruce (Piceaabies) were exposed to 0 (control), 0.5, or 5 µMCdSO

4 for nine weeks in a sand culture system

with frequent addition of nutrient solutions. Inpure culture, P. involutus and L. bicolor showedsimilar Cd tolerance. However, in symbiosis, Cdtreatments decreased colonization by L. bicolor butnot by P. involutus which ameliorated the negativeeffect of 0.5 µM Cd on shoot and root growth andchlorophyll content of old needles while L. bicolordid not. Mycorrhizal colonization did not affect Cdconcentration of old needles and roots of seedlings.Both mycorrhizal species reduced Cd concentra-tions of young needles to a similar degree com-pared with non-mycorrhizal seedlings. However, inthe 0.5 µM Cd treatment, Cd content of needles ofP. involutus colonized seedlings was increasedwhereas that of L. bicolor colonized and non-mycorrhizal seedlings were similar. Also, theamount of Cd translocated to the needlesexpressed on the root length basis was similar in alltreatments. All mycorrhizal seedlings were similarlyaffected by 0.5 µM Cd and concentrations of phos-phorus, potassium, calcium, magnesium, and man-ganese were decreased to a similar extent in bothmycorrhizal and non-mycorrhizal seedlings. Incontrast to L. bicolor, P. involutus increased phos-phorus uptake and altered patterns of root branch-ing (Jentschke, Winter, and Godbold 1999).

Copper toxicityIn studies conducted at the Citrus Research andEducation Centre, University of Florida, LakeAlfred, USA, carrizo citrange (Ponicirus trifoliata ×Citrus sinensis) seedlings were planted in a sandysoil (pH 6.8) which was amended with eight Cu(copper) concentrations (0–300 µg per g of soil) as

basic copper sulphate (CuSO4.3Ca(OH)

2.H

2O)

adjusting to double acid extractable Cu concentra-tions in soil from 3 to 248 µg per g of soil. Seedlinggrowth and colonization by the mycorrhizal fungus,Glomus intraradices, reduced logarithmically withCu concentration. Minimum toxic amount of Curanged from 19 to 34 µg per g of soil. Leafphosphorus content decreased linearly with Cu formycorrhizal seedlings without added phosphorusbut not for non-mycorrhizal seedlings with supple-mental phosphorus. The Cu-induced reduction inphosphorus uptake of mycorrhizal plants was moreclosely related to the inhibition of hyphal develop-ment outside the root than to the development ofvesicles and arbuscules inside the root. ThusCu-induced phosphorus deficiency was attributedto inhibition of phosphorus uptake by themycorrhizal hyphae in the soil. In citrus orchardsoil with a double acid extractable Cu 80 µg per gof soil and pH 5, replanted trees were stunted andhad less mycorrhizal colonization than unaffectedtrees (Graham, Timmen, and Fardelmann 1986).

Zinc toxicityIn studies conducted at the PhysiologischePflanzen Anatomie, Universitat Bremen, Germany,Pinus sylvestris seedlings were either uninfected orinfected with Suillus bovinus strain cultured onzinc-enriched medium, S. bovinus strain culturedon normal medium, and an unidentified isolate(BP), and exposed to various external Zn (zinc)concentrations. Non-mycorrhizal seedlings had thecapability to control uptake and threshold level,where no limitation of uptake is possible. ExcessZn accumulated in the root system to protect theshoot against toxic concentrations. Effect of anectomycorrhizal fungus on Zn uptake and distribu-tion depended on the fungal species, external Znconcentrations, and Zn content of fungal culturemedium. Under conditions of low external Znsupply, mycorrhizal infection with S. bovinus led toincreased Zn uptake in roots and needles ofP. sylvestris. In case of high external Zn concentra-tions, mycobionts varied considerably in theircapability to reduce Zn transport to the shoot. Theeffect could be enhanced only by an infection withS. bovinus, pretreated with high Zn concentrations(Bucking and Heyser 1994).

Scandium toxicityStudies were conducted at the Faculty ofEnvironmental and Forest Biology, College ofEnvironmental Sciences and Forest Biology,Syracuse, USA, on loblolly pine (Pinus taeda) andhoney locust (Gleditsia triacanthos) grown insolution culture at pH 3.95 with 10–100 µM and10–50 µM Sc (scandium), respectively. After35 days, root and shoot elongation in both specieswere significantly reduced at 50 µM Sc or higher.With honey locust, reductions in dry weights ofroot, leaf, and stem were measurable at 10 µM Sc.In both species, Sc induced chlorosis and


substantially reduced chlorophyll contents ofyoung leaves at all concentrations. At 100 µM Sc,short roots of loblolly pine developed a distinctlymycorrhizal appearance and Sc significantly re-duced potassium concentration in tissues but hadlittle effect on the concentrations of calcium andmagnesium. Scandium is a transition elementknown to accumulate in combustion products atconcentrations of up to 1000 ppm or in treatedsewage sludge. Its average concentrations in soilare 0.5–45 ppm (Yang, Schaedle, and Tepper 1989).

Lead toxicityIn studies conducted at the University ofGottingen, Institute of Forest Botany, ForestEcosystem Research Centre, Gottingen, Germany,Norway spruce seedlings, non-mycorrhizal ormycorrhizal with Laccaria bicolor or Paxillusinvolutus, were grown in root boxes in a quartzsand / nutrient solution culture system suppliedwith NH

4NO

3 nitrogen or NO

3− nitrogen for 6

weeks and with 5µM Pb (lead) during the last threeweeks. pH was higher with NO

3− nutrition than

with NH4NO

3 nutrition. No Pb was detected in

shoots. Total Pb in roots was reduced bymycorrhizal infection but was not affected by thesource of nitrogen. About 47% and 10% Pb couldbe desorbed from roots by washing in 5 mM CaCl

2or 0.4 mM HCl, respectively. This mainly com-prised apoplastic Pb and was neither affected bythe source of nitrogen nor by the presence ofmycorrhiza. Mycorrhizal infection had no effect onthe pH at the rhizoplane. X-ray micro-analysis ofsemi-thin sections of mycorrhizal and non-mycorrhizal short roots showed that despite thehigher binding capacity of cortex cell walls, Pbcontent was lower with NO3

− nutrition than withNH

4NO

3 nutrition. Lead content in the stele was

also reduced with NO3− nutrition. Mycorrhizal in-

fection had no effect on the Pb content of cell wallsin the cortex and stele. In an accompanying solu-tion culture experiment conducted under similarconditions, increased solution pH (5 vs 3.2) had asimilar effect on Pb concentration in root cortexcell walls as had nitrate nutrition in the sandculture experiment (Jentschke et al. 1998).

Toxicity due to allelopathic compoundsPhenolic compounds identified previously aspotentially responsible for allelopathic interferencesin spruce forest at high altitude were analysed incanopy leachates and snow and soil solutioncollected from the three layers of the podsolic soil(OA, E, and B) at the Department of LifeSciences, Laboratory Altitude EcosystemDynam, Le Bourget, France. Leachates werecharacterized by high tanning capacity and byp-hydroxyacetophenone which was also detected asthe major monomeric compound in snow. At least10 phenolic monomers including vanillic,p-hydroxy benzoic, and protocatechuic acids wereidentified in capillary waters extracted from the OA

layer. In soil solutions, significant decreases inphenolic concentrations with depth were observedbetween the E and B layers; with qualitative modi-fications of the phenolic pattern. Spruce leachatesand soil solutions exhibited high temporal variabil-ity resulting in transitory allelopathic potentialtowards both aerial and subterranean parts ofspruce seedlings (Gallet and Pellissier 1997).

Allelopathic effect of associated floraIn studies conducted at the INRA (InstitutNational de la Recherche Agronomique), BordeauxLaboratoire d’ Ecophysiologie et Nutrition, Cestas,France, the hypothesis that grass, Molinia caerulea,has an allelopathic effect on the growth of youngred oak seedlings, was tested. Oak seedlings weregrown for two years in a greenhouse, with orwithout fertilizer application and alone or withM. caerulea. Stem and root biomass of unfertilizedoak seedlings grown with M. caerulea were signifi-cantly lower (10.3 and 60.0 g dry weights, respec-tively) compared with those of the seedlings grownalone (53.4 and 294.9 g, respectively). Roots ofseedlings grown with M. caerulea had less Laccariaspecies (an efficient ectomycorrhizal fungus) andgreater proportion of Cenococcum geophilum (whichis less efficient) as well as more non-mycorrhizalroots and fewer fine roots (Timbal, Gelpe, andGarbeye 1990).

Studies were conducted on Douglas fir(Pseudotsuga menziesii) and pine (Pinus ponderosa)seedlings at the Department of Forest Science,Oregon State University, Corvallis, USA. Theseedlings were grown in replacement series inpasteurized soil with no ectomycorrhizal fungi; twoectomycorrhizal fungi added (Douglas fir specificRhizopogon vinicolor and pine specificR. ochraceorubens); and four ectomycorrhizal fungiadded (two Rhizopogon species and two generalistsLaccaria laccata and Hebeloma crustuliniforme). Areplacement series in unpasteurized forest soil wasalso included. Tree species mutually inhibited eachother in treatments without ectomycorrhizal fungibut infected with Thelephora terrestris, a greenhousecontaminant which usually colonized controlplants. The mutual inhibition disappeared whenfour ectomycorrhizal fungi species were added.Douglas fir seedlings were significantly larger inmixtures than in monoculture. However, there wasno corresponding decrease in the size of pineseedlings. This was solely due to seedlings withL. laccata which apparently enhanced nitrogen andphosphorus uptake by Douglas fir at the expense ofluxury consumption by pine. Results indicate thatectomycorrhizal fungi can reduce competitionbetween plant species and perhaps increase overallcommunity phosphorus uptake. However, patternswere specific to both ectomycorrhizal fungi andtree species and were different in unpasteurizedsoils (Perry et al. 1989).

Below-ground competition and phytotoxicitybetween Vaccinium myrtillus (which reduces


survival of tree seedling and growth in borealforests) and transplanted Picea abies was reducedby exclusion tubes in studies conducted at theSwedish University of Agricultural Sciences,Faculty of Forestry, Umea, Sweden. Reducedbelow-ground competition by V. myrtillusincreased current-year shoot length, shoot and rootbiomass, and nutrient concentration andmycorrhizal colonization in seedlings of P. abies(Jaderlund et al. 1997).

In studies conducted at the McGill University,Department of Natural Resources Science,Canada, black spruce (Picea mariana) seedlingsclose to (less than one metre) and far from (greaterthan one metre) Kalmia angustifolia were sampledfrom a clear cut to assess mycorrhizal infection andvarious growth parameters. K. angustifolia, anericaceous shrub, frequently invades black spruceclear cuts and on many sites where K. angustifoliagrows, black spruce seedlings become chlorotic andstunted due to allelopathic chemicals ofK. angustifolia. Seedlings close to K. angustifolia, ascompared to those growing far from K. angustifolia,had significantly lower foliage concentrations ofnitrogen and phosphorus, lower rate of mycorrhizalinfection, and were more frequently associated withPhialocephala dimorphospora, a potential root patho-gen of black spruce (Yamasaki et al. 1998).

Allelopathic effect of mulch or extractsof organic matterStudies were conducted in the Laboratory etPedologie Biology, University of Savoie,Chambery, Cedex, France, to verify that the initialgrowth of spruce seedlings is controlled byallelopathic compounds from vegetation and soilorganic horizon and enhanced by mycorrhizaldevelopment. The action of different aqueousextracts under laboratory conditions was studied atdifferent stages of spruce development. Germina-tion was completely inhibited by bilberry leavesand partly by fern foliage and moss. The firststages of seedling growth were limited by bilberryleaves and rhizosphere bilberry roots. A positiveeffect was noticed for rotten wood. Mycorrhiza ofyoung seedlings was inhibited by bilberry leaves(Andre et al. 1987).

Studies conducted at the Agricultural Univer-sity, Biological Station, Kampsweg, Wijster, TheNetherlands, showed that aqueous extracts of Scotspine needles negatively affected growth rates ofLaccaria proxima and Rhizopogon luteolus whereasonly high concentrations of needle extractssufficiently inhibited the growth rates of Paxillusinvolutus and Xerocomus badius. The needle extractssignificantly enhanced the growth rate of L. bicolor.Shoot extracts of the grass, Deschampsia flexuosa,inhibited the growth rates of L. proxima,P. involutus, and R. luteolus, and significantlyenhanced the growth rate of L. bicolor. The

shoot extracts contained about 3–5 times morehigh molecular weight components, aliphaticacids, and phenolics, than the root extracts (Baaret al. 1994).

Potential allelopathic (phytotoxic or beneficial)effect of barley, oat, and wheat straw mulches ongrowth, mineral nutrition, and mycorrhizal statusof black spruce seedlings was evaluated undergreenhouse conditions in studies conducted at theMinistere de l’ Energie et des Resources, Servie del’ Amelioration des Arbes Sainte Foy, Québec,Canada. The various straws did not affect theheight of spruce seedlings over a two-monthgrowth period. The newly formed fine roots oftreated and control seedlings were mycorrhizal.Oat and wheat straw significantly enhanced thephosphorus content of foliage as compared to thatof controls. All treatments significantly depressedfoliar manganese content indicating that the strawcould exert a detrimental effect on manganeseuptake. Monitoring the status of manganese inplanted spruce seedlings when using allelopathiccover straw mulches is suggested as a method forpreventing the establishment of weed species(Jobidon 1989).

Allelopathic effect of other fungi onmycorrhizaStudies were conducted at the University ofWisconsin, Madison, Wisconsin, USA, to observethe possible suppression of mycorrhiza formingfungi by antibiotics excreted by Coprinus ephemeruscommonly used in the production of compostedfertilizers from saw dust and bark and paper millsludge. Additional inoculation with C. ephemerusdid not reduce the growth or enzymatic activity offeeder roots of one-year-old Pinus resinosa andP. ponderosa grown in a greenhouse in sandy soilinoculated with Cenococcum graniforme orThelephora terrestris. Several seedlings showedsinuous morphology or serpentinosis of tap rootwhich probably resulted from excretion ofantibiotics by T. terrestris (Iyer 1981).

Toxicity due to soil salinityStudies conducted at the Laboratoire dePhytonique, FRD Biotechnologies, Angers, Cedex,France, on 60-day-old culture of two birch (Betulapendula) clones, 4 and 5 (isolated in vitro), inocu-lated with Paxillus involutus indicated that theClone 4 showed the most growth stimulation(120%) as compared to Clone 5 (55%). Clone 4was also more tolerant to NaCl (sodium chloride)than Clone 5, their respective decreases in biomassbeing 10% and 40% in a medium containing 50 mMNaCl. Responses to salinity and mycorrhiza werelinked to the osmotic and nutritional status of invitro plants. Translocation of ions towards theshoots thus appeared to be key factor in the observedresponse (Catinus, Guerrier, and Strullu 1990).


Studies were conducted at the University ofWestern Australia, Nedlands, Australia, on theeffect of NaCl on infection of Trifolium resupinatumby hyphae of Gigaspora decipiens. Roots, 1 cm fromthe spore, were infected after 15 days in soilsmoistened with 0 or 75 mM NaCl indicating thatthe initial hyphal growth was not inhibited in soilmoistened with 75 mM NaCl. With time, NaClincreasingly inhibited hyphal growth as roots,2.5 cm from the spores, were not infected after 26days in the soil moistened with 75 mM NaCl butwere infected in the soil without added NaCl. In soilmoistened with 150 mM NaCl, roots, 1 cm from thespores, were infected after 26 days but roots, 2 cmfrom the spores, were not infected after 36 days. In-creasing concentrations of NaCl inhibits either thehyphal growth or the infectivity of G. decipiens hyphaeafter they have grown through soil. The latter may beunlikely because NaCl did not affect the infectivity ofG. decipiens hyphae placed directly on to the roots.Increasing concentrations of NaCl also inhibited thespread of infection after the occurrence of initial in-fection. This is probably due to NaCl in the soil in-hibiting hyphal growth as well as possibly affectingsupply of carbohydrates from the plant to the fungus(McMillen, Juniper, and Abbott 1998).

In studies conducted at the Department ofOrnamental Horticulture, University of Florida,Gainsville, Florida, USA, NaCl tolerance andphosphoorus content in split-root carrizo citrangeseedlings (Ponicirus trifoliata × Citrus sinensis)colonized with Glomus intraradices on zero, one,and two root halves and treated with NaCl at 0,25, 50, and 100 mM doses were examined. Thedegree of stress was measured as reduction of drymatter accumulation and rise in the level ofproline-betaine (stachydrine). Shoot and root dryweight production during this period decreasedwith increasing levels of salt. Absolute reductionswere similar for plants inoculated on one vs twohalf root systems but percentage decreases wereless in the latter due to greater overall growth in alltreatments. Betaine levels in leaf tissues were posi-tively related to salt level of the soil for eachmycorrhizal treatment. Significant differences inbetaine levels were also detected in plants with orwithout mycorrhizal fungi and mean levels tendedto be higher for those colonized on one vs twohalves of their root system. In contrast, a half rootsystem and its fungal symbiont supplied enoughphosphorus to allow concentration of leaf phospho-rus to equal that of fully infected root systems, yetthe two groups did not show equal growth undercontrol conditions or percentage reductions withNaCl (Duke, Johnson, and Koch 1986).

The responses of Pistacia vera (a non-halophytic salt-tolerant deciduous nut tree) inocu-lated with Glomus mosseae and treated withdeionized water and water with electrical conduc-tivities of 3.8, 6.5, and 9.7 ds/m EC at age oneweek was determined in studies conducted at the

Department of Padologie Vegetal and Departmentde Technologia Agraria, Generalitat de Catalunga,Cabrils, Barcelona, Spain. After 12-week growth,mycorrhizal P. vera maintained the leaf turgor andshoot dry weight while uninoculated plants did notmaintain leaf turgor when irrigated with water of9.7 ds/m EC. A significant check in growth withrespect to controls was observed in all thenon-VAM treatments. The results thus show theimproved salt tolerance of VAM plants (Estaunand Save 1990).

Studies were conducted at the InstitutoSperimentale per l’ Olive Culture, Casenza, Italy,on 14-month-old olive trees of cultivars Caroleaand Nacellaria del Belice in pots irrigated withwater with NaCl content of 0%, 0.3%, 0.6%, and0.9%. After one year or 35 irrigations, there wasreduction in root content of phosphorus,potassium, and calcium and rise in sodium and nochange in magnesium as the salinity increased inboth the cultivars. Difference in mycorrhizalresponses were observed between the cultivarsregardless of the irrigation water salinity. However,mycorrhizal parameters were affected by increasingsalinity more in Carolea (with a reduction in bothfrequency and function) than in Nacellaria delBelice which showed significant increase infrequency but no significant change in function(Briccoli–Bati et al. 1994).

The response of young mycorrhizal (inoculatedwith Glomus mosseae) and uninoculated olive (Oleaeuropaea) plants under saline conditions (25, 50,75, and 100 mM NaCl) with or without supple-mental calcium was studied at the Departmento diOrtoflorofrutticoltura, Universita de Firenze,Firenze, Italy. Depolarization caused by NaCl atthe cellular level (cell transmembraneelectropotential) was less in mycorrhizal roots thanin non-mycorrhizal ones. The opposite was foundat the level of the whole root system (transrootelectropotential). Supplemental calcium in thesaline treatments had protective effect on mem-brane integrity cancelling or reducing the differ-ences in depolarization (cell transmembraneelectropotential and transroot electropotential,respectively) between mycorrhizal and non-mycorrhizal plants. After three months, bothmycorrhizal and non-mycorrhizal plants subjectedto saline conditions grew less than control plantsnot exposed to saline conditions. However,mycorrhizal plants when compared with non-mycorrhizal ones demonstrated greater growth ofshoots and leaves. Infected roots accumulatedgreater quantities of sodium, phosphorus, and cal-cium, and exhibited a lower potassium–sodiumratio; but in leaves, mycorrhizal plants had greaterpotassium–sodium ratio than non-mycorrhizalplants (Rinaldelli and Mancuso 1996).

Further studies were conducted at the sameuniversity to measure the electrical impedanceparameters in shoots and leaves of olive plants to


follow variations in extracellular resistance, intra-cellular resistance, and the state of membrane inmycorrhizal (inoculated with Glomus mosseae) andnon-mycorrhizal plants subjected to saline stress. Adouble DCE (double-distributed circuit element)(ZARC) and a single DCE (ZARC) model wereused as equivalent circuit for shoots and leaves,respectively. There was a reduction in extra- andintracellular resistance values for non-mycorrhizalplants with increased NaCl concentration.Mycorrhizal infection brought about a noticeableimprovement in salt tolerance in olive plants whichwas clearly demonstrated by trends in impedanceparameters (Mancuso and Rinaldelli 1996).

Studies conducted at the PomologyDepartment, Faculty of Agriculture, AlexandriaUniversity, Alexandria, Egypt, on one-year-oldsour orange seedlings in black polyethylene bagswith clay loam soil showed that irrigation with sa-line water (up to 4500 ppm salt; NaCl + potassiumchloride) reduced total leaf chlorophyll and chloro-phyll-a concentrations, peroxidase activity inleaves, and starch and total carbohydrate concen-trations in leaves and roots; did not affect chloro-phyll-b concentration and polyphenol oxidase(catechol oxidase); and increased sugar concentra-tions in some cases when compared with controls(irrigated with tap water). Inoculation with Glomusintraradices increased total chlorophyll and chloro-phyll-b content in the first season, chlorophyll-a inthe second season, increased polyphenol oxidaseactivity, leaf and root sugars, and carbohydrateconcentrations but did not affect peroxidase activ-ity (Ezz and Nawar 1994).

Toxicity due to calcareous soilThe term symbiocalcicole is used to define dwarfshrubs or tree species that can only toleratecalcareous soils in association with mycorrhizalfungi (Lapeyrie 1990). Studies conducted at theDepartment of Agricultural Botany, the HabrewUniversity, Rohovot, Israel, on Citrus incanusshowed that the plants transplanted in the labora-tory degenerated in native dark basaltic soils and insterilized, terrarosa calcareous soils where infectionby mycorrhizal fungi did not occur. They grew incalcareous terrarosa when mycorrhiza developed.Successful development of C. incanus inunfertilized soils depended on ectomycorrhizalinfection. Plants developed without mycorrhiza inboth the soils when irrigated with a complete nutri-ent solution. It thus suggests that whenmycorrhizae are absent, C. incanus plants degener-ate in unfertilized soils due to mineral nutrientdeficiency (Berliner, Jacoby, and Zamski 1986).

In studies conducted at the Zagazig University,Faculty of Agricultural Science, Moshtohor-Tukh,Egypt, sour orange (Citrus aurantium) seedlingswere transplanted into pots (containing 18 kgcalcareous soil), fertilized with three differentlevels of rock phosphate, and foliar sprayed withmicronutrient solution (iron, manganese,

molybdenum, zinc, and copper). The VAM inocu-lation and or phosphorus fertilization generallystimulated plant growth characters, shoot and rootlength, leaf numbers, and dry matter accumulation.Nitrogen and phosphorus contents of mycorrhizalplants were greater than non-mycorrhizal plants atall soil phosphorus levels. Micronutrient uptakewas considerably improved in VAM plants overeither phosphorus fertilized, uninoculated, or con-trol plants (El-Maksoud, Boutros, andLofty 1988b).

Studies conducted at the National ResearchCentre, Dokki, Egypt, on sour orange (Citrusaurantium) grown in sandy and calcareous soilsunder greenhouse conditions and inoculated withVAM spores showed that plant height, root length,leaf number, leaf area, and dry mass of inoculatedplants were always significantly greater thanuninoculated controls in both soils. Percentage ofmycorrhizal infection significantly increased andsignificant differences were observed in nitrogen,phosphorus, iron, manganese, and zinc contents ofroots and aerial parts as a result of inoculation.Combined treatment with VAM and phosphorusgave more growth responses than phosphorus aloneor control although phosphorus fertilized plantswere always better than unfertilized plants. Thegrowth characters were significantly larger in cal-careous than in sandy soil (El-Maksoud, Boutros,and Lofty 1988a).

Toxicity by overheated soilStudies conducted at the Instituto deInvestigaciones Agrobiologicas de Galicia, Apdo,Santiago de Composieta, Spain, showed thatgermination rates, root colonization, and intensityof arbuscule formation of Glomus macrocarpumwere reduced following treatment with aqueousextracts of burnt soil or overheated unburnt soiland enhanced by extracts from unburnt soilscompared with the control (distilled water). It isthus concluded that overheated soils contain watersoluble substances which affect VAM colonizationand growth (Vilarino and Arines 1992).

ReferencesAndre J, Gensac P, Pellissier F, Trosset L. 1987Regeneration of spruce communities at altitude: pre-liminary studies on the role of allelopathic compoundsand mycorrhizas in the first stages of developmentRevue d’Ecologie et de biologie du soil 24(3): 301–310

Baar J, Ozinga W A, Sweers I L, Kuyper T W. 1994Stimulatory and inhibitory effects of needle litterand grass extracts on the growth of someectomycorrhizal fungiSoil Biology Biochemistry 26(8): 1073–1079

Berliner R, Jacoby B, and Zamski E. 1986Absence of Citrus incanus from basaltic soils inIsrael: effect of mycorrhizaeEcology 67(5): 1283–1288


Briccoli–Bati C, Rinaldi R, Tocci C, Sirianni T,Iannotta N. 1994Influence of salty water irrigation on mycorrhizaeof young olive trees in containers, edited by S LavicActa Horticulturae 356: 218–220[Proceedings of the Second International Symposium onOlive Growing, 6–10 September 1993, Jerusalem, Israel]

Browning M H R and Hutchinson T C. 1991The effect of aluminium and calcium on the growthand nutrition of selected ectomycorrhizal fungi ofjack pineCanadian Journal of Botany 69(8): 1691–1699

Bucking H and Heyser W. 1994The effects of ectomycorrhizal fungi on Zn uptakeand distribution in seedlings of Pinus sylvestris L.Plant and Soil 167(2): 203–212

Catinus B, Guerrier G, and Strullu D G. 1990Responses of birch clones to mycorrhizas and salinityCanadian Journal of Forest Research 20(7): 1120–1124

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

Cumming J R. 1990Nitrogen source effects on Al toxicity in non-mycorrhizal and mycorrhizal pitch pine (Pinus rigida)seedlings. II. Nitrate reduction and NO3−−−−− uptakeCanadian Journal of Botany 68(12): 2653–2659

Cumming J R and Weinstein L H. 1990aAluminium–mycorrhizal interactions in thephysiology of pitch pine seedlingsPlant and Soil 125(1): 7–18

Cumming J R and Weinstein L H. 1990bNitrogen source effects on Al toxicity innonmycorrhizal and mycorrhizal pitch pine (Pinusrigida) seedlings. I. Growth and nutritionCanadian Journal of Botany 68(12): 2644–2652

Duke E R, Johnson C R, and Koch K E. 1986Accumulation of phosphorus, dry matter andbetaine during NaCl stress of split-root citrusseedlings colonized with vesicular–arbuscularmycorrhizal fungi on zero, one or two halvesThe New Phytologist 104: 583–590

El-Maksoud Abd H K, Boutros B N, and Lotfy A A. 1988aGrowth response of sour orange, Citrus aurantiumL. to mycorrhizal inoculation superphosphatefertilization in sandy and calcareous soilsEgyptian Journal of Soil Science 28(3): 385–395

El-Maksoud Abd H K, Boutros B N, and Lofty A A. 1988bInfluence of vesicular arbuscular mycorrhizalassociation and rock phosphate fertilization ongrowth of Citrus aurantium L. in calcareous soilAnnals of Agricultural Science Moshtohor 26(3): 2105–2117

Entry J A; Cromack K, Jr; Stafford S G; Castellano M A.1987The effect of pH and aluminium concentration onectomycorrhizal formation in Abies balsameaCanadian Journal of Forest Research 17(8): 865–871

Estaun V and Save R. 1990Salt stress and vesicular–arbuscular mycorrhizaeinteractions on Pistacia vera water relations, p. 96In 8th NACOM Innovation and Hierarchical IntegrationWyoming: University of Wyoming. 324 pp.[Proceedings of the 8th North American Conference onMycorrhiza Innovation and Hierarchical Integration,5–8 September 1990, Jackson, Wyoming]

Ezz T and Nawar A. 1994Salinity and mycorrhizal association in relation tocarbohydrate status, leaf chlorophyll and activity ofperoxidase and polyphenol oxidase enzymes in sourorange seedlingsAlexandria Journal of Agricultural Research 39(1): 263–280

Finlay R D, Kottke I, and Soderstrom B. 1993Interactions between raised aluminium concentra-tions and ectomycorrhizal infection of Pinuscontorta, Betula pendula and Fagus sylvaticaIn 9th North American Conference on MycorrhizaeOntario: University of Guelph. 155 pp.[Proceedings of the 9th North American Conference onMycorrhizae, 8–12 August 1993, Ontario, Canada]

Gallet C and Pellissier F. 1997Phenolic compounds in natural solutions of aconiferous forestJournal of Chemical Ecology 23(10): 2401–2412

Graham J H, Timmen L W, and Fardelmann D. 1986Toxicity of fungicidal copper in soil to citrus seed-lings and vesicular–arbuscular mycorrhizal fungiPhytopathology 76: 65–70

Haricumar V S and Potty V P. 1999Effect of inoculation of arbuscular mycorrhizalfungus Glomus microcarpum on phosphoruskinetics in sweet potato soilIn 4th National Conference on Mycorrhiza: Abstracts, p. 18Bhopal: Barkatullah Vishwavidyalaya[Proceedings of the 4th National Conference onMycorrhiza, 5–7 March 1999, Bhopal, India]

Iyer J G. 1981On antibiotic effect of Coprinus ephemerusIn Forestry Research Notes (No. 243)Wisconsin: Department of Forestry, University ofWisconsin. 5 pp.

Jaderlund A, Zackrisson O, Dahlberg A, Nilsson M C.1997Interference of Vaccinium myrtillus onestablishment, growth, and nutrition of Piceaabies seedlings in a northern boreal siteCanadian Journal of Forest Research – Revue Canadiennede Recherche Forestiere 27(12): 2017–2025


Jakucs P, Meszaros I, Papp B L, Toth J A. 1986Acidification of soil and decay of sessile oak in the‘Sikfokut project’ area (N-Hungary)Acta Botanica Hungarica 32(1–4): 303–322

Jentschke G, Marschner P, Vodnik D, Marth C,Bredemeier M, Rapp C, Fritz E, Gogala N,Godbold D L. 1998Lead uptake by Picea abies seedlings: effects ofnitrogen sources and mycorrhizasJournal of Plant Physiology 153(1–2): 97–104

Jentschke G, Winter S, and Godbold DL. 1999Ectomycorrhizas and cadmium toxicity in Norwayspruce seedlingsTree Physiology 19(1): 23–30

Jobidon R. 1989Effect of straw residues on black spruce seedlinggrowth and mineral nutrition, under greenhouseconditionsCanadian Journal of Forest Research 9(10):1291–1293

Jones M D and Hutchinson T C. 1988Nickel toxicity in mycorrhizal birch seedlingsinfected with Lactarius rufus or Sclerodermaflavidum. I. Effects on growth, photosynthesis,respiration and transpirationThe New Phytologist 108(4): 451–459

Jones M D, Dainty J, and Hutchinson T C. 1988The effect of infection by Lactarius rufus orScleroderma flavidum on the uptake of 63Ni bypaper birchCanadian Journal of Botany 66(5): 934–940

Kieliszewska-Rokicka B, Rudawska M, Leski T,Kurczynska E U. 1998Effect of low pH and aluminium on growth of Pinussylvestris L. seedlings mycorrhizal with Suillusluteus (L. ex Fr.) S.F. GrayChemosphere 36(4–5): 751–756

Lapeyrie F. 1990The role of ectomycorrhizal fungi in calcareous soiltolerance by ‘symbiocalcicole’ woody plantsAnnales des Sciences Forestieres 47(6): 579–589

Louise E W, Brendon J G, and John K. 1992Differential aluminium tolerance in theectomycorrhizal fungus Pisolithus tinctorius fromacidic soilsIn International Symposium on Management of Mycorrhizas:Programme and Abstracts, edited by D JasperNedlands: Australian Institute of Agricultural Sciences.164 pp.[International Symposium on Management ofMycorrhizas, 28 September– 2 October 1992, Nedlands,Australia]

Mancuso S and Rinaldelli E. 1996Response of young mycorrhizal and non–mycorrhizal plants of olive tree (Olea europaea L.)to saline conditions. II. Dynamics of electricalimpedance parameters of shoots and leavesAdvances in Horticultural Science 10(3): 135–145

McMillen B G, Juniper S, and Abbott L K. 1998Inhibition of hyphal growth of vesicular–arbuscularmycorrhizal infection from fungus spores in soilcontaining sodium chlorideSoil Biology and Biochemisty 30(3): 1639–1646

Melo I S, Silveira A P D da, and Maluf A M. 1987Effect of aluminium and Glomus leptotichum onthe development of cultivars of Leucaenaleucocephala tolerant and intolerant of aluminiumAnais da escola superior de agricultura luiz de queiroz44(2): 1365–1380

Perry D A, Margolis H, Choquette C, Molina R,Trappe J M. 1989Ectomycorrhizal mediation of competition betweenconiferous tree speciesThe New Phytologist 112(4): 501–511

Rinaldelli E and Mancuso S. 1996Response of young mycorrhizal and non-mycorrhizal plants of olive tree (Olea europaea L.)to saline conditions. I. Short-term electrophysi-ological and long-term vegetative salt effectsAdvances in Horticultural Science 10(3): 126–134

Schepp H, Dehn B, and Sticher H. 1987Interaktionen zwischen VA mycorrhizen undSchwermetallbelastungenAngew Botanik 61: 85–96

Schier G A and McQuattie C J. 1998Effects of carbon dioxide enrichment on responseof mycorrhizal pitch pine (Pinus rigida) toaluminium: growth and mineral nutritionTrees – Structure and Function 12(6): 340–346

Schier G A and McQuattie C J. 1999Effect of nitrogen source on aluminium toxicity innon-mycorrhizal and ectomycorrhizal pitch pineseedlingsJournal of Plant Nutrition 22(6): 951–965

Sujan Singh. 1996Effect of soil pollution on mycorrhizal developmentMycorrhiza News 8(2): 1–8

Timbal J, Gelpe J, and Garbeye J. 1990Preliminary study on the effect of Molinia caeruleaon growth and mycorrhizal status of northern redoak (Quercus rubra) seedlingsAnnales des Sciences Forestieres 47(6): 643–649


Vilarino A and Arines J. 1992The influence of aqueous extracts of burnt orheated soil on the activity of vesicular–arbuscularmycorrhizal fungi propagulesMycorrhiza 1(2): 79–82

Vogelei A and Rothe G M. 1988The effects of acid and aluminium ions on thenutrient contents and histological condition ofnon-mycorrhizal Norway spruce (Picea abies)rootsForstwissenschaftliches Centralblatt 107(6): 348–357

Wilkins D A. 1991The influence of sheathing (ecto-)mycorrhizas oftrees on the uptake and toxicity of metalsAgriculture, Ecosystem and Environment 35(2–3): 245–260

Wilkins D A and Hodson M J. 1989The effects of aluminium and Paxillus involutusFr. on the growth of Norway spruce (Picea abies(L.) Karst.)The New Phytologist 113(2): 225–232

Yamasaki S H, Fyles J W, Egger K N, Titus B D. 1998The effect of Kalmia angustifolia on the growth,nutrition, and ectomycorrhizal symbiontcommunity of black spruceForest Ecology and Management 105(1–3): 197–207

Yang C S, Schaedle M, and Tepper H B. 1989Phytotoxicity of scandium in solution culture ofloblolly pine (Pinus taeda L.) and honey locust(Gleditsia triacanthos L.)Environmental and Experimental Botany 29(2): 155–164

Research findings

Effect of VAM on root induction in in vitro sugarcane (Saccharum officinarum L.)seedlings � a new technique

Muniyamma M, Bharati B K, Reddy C NDepartment of Botany, Gulbarga University, Gulbarga – 585 106, India

IntroductionTissue culture technique has been a novelapproach for large-scale production of qualityplants, establishment of hybrids, and developmentof varieties resistant to various stresses and dis-eases. However, one of the constraints still is goodrooting in the differentiated shoots of in vitrosugarcane (Islam, Begum, and Haque 1982). Themodified MS (Murashige and Skoog) liquid me-dium containing 5 mg/litre NAA (naphthalene ace-tic acid) is widely used for root induction insugarcane (Chen et al. 1988). The plants thusformed have feeble root formation and do notquickly acclimatize to field conditions.

In many instances, VAM (vesicular–arbuscularmycorrhizal) inoculations have been successfullyused to increase the survival capacity ofmicropropagated seedlings (Adholeya and Cheema1990; Jamaluddin and Chaturvedi 1996). TheVAM fungal association has been reported tostimulate rooting (Barrows and Roncadori 1977)and enhance root growth by hormone secretion(Allen, Moore, and Christensen 1980; Powell andBagyaraj 1986; Whipps and Lumsden 1994).Hence, in this investigation, VAM fungal sporeextract and VAM root extract were tested as substi-

tutes to the rooting hormone NAA in MS liquidmedium to enhance root induction.

Materials and methods

Source of explantA high-yielding locally grown sugarcane varietyCo. 419 was selected and 3–4 month old sugarcaneseedlings were used as the source of explant.

Callus inductionThe leaf sheaths of the sugarcane seedlings werecarefully peeled up to the first node and the fewinnermost leaf whorls used. The leaf sheath wasexcised up to 4–6 cm from the shoot tip and thesurface sterilized with a liquid soap solutionfollowed by rinsing thrice in sterile distilled water.Further inoculation procedures were carried outunder aseptic conditions (inside laminar airflow).Leaf explants were treated with aqueous mercuricchloride (0.1%) followed by rinsing thrice in steriledistilled water. The innermost whorl was carefullypeeled and chopped into small pieces (approxi-mately 0.5 mm) and immediately inoculated inMS medium supplemented with 3mg/litre 2–4 D(2–4 dichlorophenoxyacetic acid) and 10% coconut


Table 1 Effect of NAAa, VAMb fungal spore extract, VAM rootextracts, and non-mycorrhizal root extract as supplements inMSc liquid media on rooting response of sugarcane tissueculture shoots

Length of roots* (cm)Days taken Number offor root roots per after after

Treatment initiation shoot 20 days 40 days

NAAa 8�12 10�15 0.4�0.6 1�2VAMb fungal spore

extract 3�6 3�12 1.5�3 3�4VAMb root extract 2�6 30�50 0.5�1 1.5�3Non-mycorrhizal

root extract No rooting Nil � �

aNAA � naphthalene acetic acid; bVAM � vesicular�arbuscular mycorrhiza;cMS � Murashige and Skoog*Average length of 10 roots selected randomly

Figure 1 Transferable shoots of sugarcane variety Co. 419for rooting response

milk (Heinz and Mee 1969; Liu, Huang, and Shib1972) for callus induction. Further subculturing ofthe callus was done on the same medium to obtainluxuriant growth of friable callus. All the culturetubes were incubated in the dark at 25 + 2 °C.

MorphogenesisThe friable sugarcane callus was subcultured ontothe MS medium supplemented with 2 mg/litrekinetin and 10% coconut milk for shoot induction.The flasks were incubated at 25 + 2 °C underfluorescent light maintaining a 12-hour photope-riod. The shoots were subcultured on the samemedium for profuse tillering (Figure 1). Then re-generator shoots were transferred to MS liquidmedium using the following alternatives for rootinduction.1 MS medium supplemented with NAA (5 mg/

litre) along with 10% coconut milk (Chen et al.1988)

2 MS medium supplemented with VAM fungalspore (Glomus aggregatum) extract (2500 spores/litre) and 10% coconut milk

3 MS medium supplemented with VAM rootextract (10 g/litre) and 10% coconut milk

4 MS medium supplemented with non-mycorrhizal root extract (10 g/litre) and 10%coconut milk

Fifteen tubes were used for each treatment andthe experiment was done twice.

Preparation of spore and root extractsThe root extract was prepared by homogenizing10 g of fresh, surface sterilized mycorrhizal or non-mycorrhizal roots of sugarcane in 25 ml distilledwater. The homogenate was filtered throughWhatman No. 2 filter paper and the filtrate wasadded to the medium before sterilization.

Spore extract was prepared by harvestingspores of Glomus aggregatum from pure soilinoculum using the wet sieving and decantingmethod (Gerdemann and Nicolson 1963); aftersurface sterilization it was treated with 10% Chlo-ramine-T for 10 minutes followed by washing insterile distilled water; and later treated with 200ppm streptomycin sulphate and again washed insterile distilled water and crushed in a cavity glasswith the help of a glass rod in distilled water. Thehomogenized mixture was then added to themedium. The pH of the medium was maintained at5.6–5.8.

ResultsCallus induction was observed on explants afterthree weeks of inoculation and a well-developedfriable callus was obtained within two weeks aftersubculturing. The callus when subcultured onshooting medium produced multiple shoots in3–4 weeks after incubation. Further subculturing

resulted in proliferation of transferable shoots(Figure 1).

Well-developed, healthy, transferable shootswere excised and transferred into modified MSliquid medium supplemented with four alternativesof rooting stimuli (vide materials and methods) andincubated under controlled conditions of light andtemperature. The response of shoots for rootingvaried from one treatment to another and moreclear measurable variation was obtained after 40days of transfer (Table 1, Figure 2).

In the shoots transferred in MS liquid mediumsupplemented with NAA (control), root initiationwas observed within 8–12 days of incubation.However, clear root formation and growth


(0.4–0.6 cm) could be observed only after 20 daysof continuous incubation and 1–2 cm long rootswere observed after 40 days (Table 1, Figure 2a).The total number of roots formed were in therange 10–15 per shoot.

In the shoots transferred to MS liquid mediumsupplemented with VAM spore extract, root initia-tion was observed within 3–6 days of incubationand the growth of the roots was faster as comparedto that in the NAA supplement. The roots were1.5–3 cm and 3–4 cm long after 20 and 40 days ofincubation, respectively (Table 1, Figure 2b).However, fewer roots were produced, 3–12 per shoot.

Similarly, in the shoots transferred to VAMroot extract supplemented medium, the rootingresponse was observed within 2–6 days of incuba-tion. The growth and proliferation of roots werealso high, 0.5–1 cm and 1.5–3 cm long roots after20 and 40 days of incubation, respectively(Table 1, Figure 2c). Also, 30–50 roots per shootwere produced.

The shoots transferred to the medium supple-mented with non-mycorrhizal root extract did notshow rooting response even after 40 days ofcontinuous incubation (Table 1, Figure 2d).

DiscussionIn this study, the meristem leaf explants ofsugarcane variety Co. 419 when cultured on modi-fied MS medium produced profuse callus andshoot formations. This is in accordance with theearlier reports by Heinz and Mee (1969), Liu,Huang, and Shib (1972), Hendre et al. (1983),

Pant and Hapase (1987), and Burner and Grisham(1995), in which the same combination of mediafor callus induction and shoot formations insugarcane were used. Islam, Begum, and Haque(1982) have observed that despite rooting in differ-entiated sugarcane seedlings with NAA, subse-quent adaptability upon transfer to pots seems tobe difficult. As an alternative to NAA, VAM fungalspore extract or VAM root extract showed betterrooting response. These substitutes not only has-tened the rooting initiation but also appreciablyenhanced root growth and proliferation. This in-creased response is attributed to VAM fungal influ-ence since non-mycorrhizal root extract failed toinduce rooting. Barrows and Roncadori (1977)have observed the stimulation of rooting due to theassociation and synthesis of a hormone by VAMfungus Gigaspora margarita in Poinsettia.

The enhanced rooting response in sugarcanewith either VAM fungal spore extract or VAM rootextract substitutes in MS liquid medium couldmake them a better solution than the NAA supple-ment. These seedlings on transfer to pots have alsoshown increased vigour as compared to control plants.

ReferencesAdholeya A and Cheema G S. 1990Evaluation of VA mycorrhizal inoculation inmicropropagated Populus deltoides Marsh clonesCurrent Science 59(23): 1245–1247

Allen M F, Moore T S, and Christensen M. 1980Phytohormone changes in Bouteloua gracilisinfected by vesicular–arbuscular mycorrhizae. I.Cytokinin increases in the host plantCanadian Journal of Botany 58: 371–374

Barrows J B and Roncadori R W. 1977Endomycorrhizal synthesis by Gigasporamargarita in PoinsettiaMycologia 69: 1173–1184

Burner D M and Grisham M P. 1995Induction and stability of phenotypic variation insugarcane as affected by propagation procedureCrop Science 33(3): 875–880

Chen W H, Davey M R, Power J B, Cooking E C. 1988Control and maintenance of plant regeneration insugarcane callus cultureJournal of Experimental Biology 39: 251–261

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

Heinz D S and Mee G W P. 1969Plant differentiation from callus tissue ofSaccharum sp.Crop Science 9: 336–348

Figure 2 Rooting response and root proliferation in(a) MS + 5 mg/litre NAA; (b) MS + VAM fungal spore extract;(c) MS + VAM root extract; and (d) MS + non-mycorrhizal rootextract in liquid medium

(a) (b) (c) (d)


Distribution of an ectomycorrhizal fungus (Pisolithus tinctorius) in associationwith different forest tree species in Andhra Pradesh, southern India

Vijaya Kumar R, Prasada Reddy B V, Mohan V*Biotechnology Research Centre, PO Box 10, State Forest Department, Tirupathi – 517 507, India

* Division of Forest Protection, Institute of Forest Genetics and Tree Breeding, Forest Campus, Coimbatore – 641 002, India

Hendre R R, Iyer R S, Kotwal M, Khuspe S S,Mascarenhas A F. 1983Rapid multiplication of sugarcane by tissue cultureSugarcane 1: 5–8

Islam A S, Begum H A, and Haque M M. 1982Studies on generated Saccharum officinarum fordisease resistant varietiesPlant Tissue Culture 709–710

Jamaluddin and Chaturvedi P. 1996Colonization of VAM in Dendrocalamus asperMycorrhiza News 8(3): 10–12

Liu M C, Huang Y J, and Shib S C. 1972Journal of Agriculture Association China 77: 52

Pant N M and Hapase D G. 1987Raising of tissue cultured sugarcane plants outside thelaboratory without aid of glass house/green houseDeccan Sugarcane Technologists Association 37A: 81–85

Powell C L and Bagyaraj D J. 1986Why all the interest?In VA Mycorrhizae edited by C L Powell andD J Bagyaraj, pp. 1–3Florida: CRC Press. 234 pp.

Whipps J M and Lumsden R D. 1994Biotechnology of fungi for improving plant growthMycorrhiza News 6(1): 12–13

IntroductionMycorrhizal association is essential to forest trees.Among the different mycorrhizae, ectomycorrhizalfungi occur in 10% of the world’s flora, whichinclude most of the gymnosperms (especiallyconifers) and few angiosperms. Many species ofacacia viz. Acacia auriculiformis, A. holosericea, andA. mangium, and eucalypti viz. Eucalyptuscamaldulensis and E. tereticornis, native to Australia,have been successfully introduced in different partsof India by the state forest departments. These treespecies grow on a variety of soils such as red soils,laterite soils, gravely brown soils, sand dunes,shallow to deep, infertile to fertile soils, and from acidto highly alkaline soils and can tolerate the periodicwaterlogging in Andhra Pradesh (India). These treesare useful for pulpwood, poles, cheap timber, etc.

Reports on the association of ectomycorrhizalfungi in tropical angiosperm tree species is scanty;hence, the present survey for collection, isolation,and identification of different ectomycorrhizalfungi in association with Acacia spp. and Eucalyptusspp. The distribution of the ectomycorrhizalfungus Pt (Pisolithus tinctorius) in association withdifferent tree species in 10 different plantations ofthe state forest department in three districts ofAndhra Pradesh during the period 1994–96 ispresented. This ectomycorrhizal fungus is beingreported for the first time in association with differ-ent tree species of acacia and eucalyptus planta-tions in low fertile soils of Andhra Pradesh.

Materials and methodsPeriodical surveys were undertaken to study thenatural occurrence of the ectomycorrhizal fungusPisolithus tinctorius in association with differenteconomically important forest tree species such asAcacia auriculiformis, A. holosericea, A. mangium,Eucalyptus camaldulensis, and E. tereticornis in10 different plantations of three districts ofAndhra Pradesh. Basidiomata of Pt were recordedfrom the tree base in all the plantations (3–11years old).

Root and rhizosphere soil samples werecollected from underneath the fungal basidiomata.Soil samples were analysed for pH, carbon, andavailable phosphorus and potassium. Root sampleswere processed for identifying ectomycorrhizalassociation with the tree species. Pure culture ofthe ectomycorrhizal fungus was attempted fromyoung, fresh, unopened basidiomata.

Results and discussionBasidiomata of Pt emerged within a fortnight of thefirst showers during June and July and continuedappearing until October/November. Basidiomatawere distributed in irregular patterns at varyingdistances from the tree base in plantations with orwithout leaf litter. The frequency distribution ofthe ectomycorrhizal fungus (Pt) collected and iden-tified from various tree plantations is presented inTable 1. The soil nutrient status of each plantationsite is given in Table 2.


association in the roots showing fungal mantle andHartig net structures.

ConclusionThe acreage under Acacia spp. and Eucalyptus spp.is increasing both in forest plantations andagricultural lands. Mycorrhization of forest cropshas attracted considerable attention over the lastfew years because of their role as biofertilizer,improving host growth as well as contributing todisease suppression (Marx 1969 and 1973;Sampangiramaiah and Bagyaraj 1989). Hence, thenatural association of Pt with acacia and eucalyptiis significant and needs further study and exploita-tion in the nursery and the field.

Table 1 Frequency distribution of Pisolithus tinctorius in different plantations at various sites in Andhra Pradesh

Chittoor district

Cuddapah Nellore district Site

Kalyani district frequency

Name of tree species Tirupathi Tirumala Dam Punganur Sathyavedu Settigunta Gadela Dakkili Dagadarthi Kavali (%)

Acacia auriculiformis � + � + � � � + � � 30

Acacia holosericea + + � � � + + + � � 50

Acacia mangium � � � � � � � + � � 10

Eucalyptus camaldulensis + + + + + + + + + + 100

Eucalyptus tereticornis + + + + + + + + + + 100

Figure 1 Basidiomata of Pisolithus tinctorius near the treebase of Acacia holosericea (close-up of the basidiomata)

Table 2 Soil nutrient status of different plantation sites inAndhra Pradesh

Nutrient status

Organic Phosphorus PotassiumSites pH carbon (%) (ppm) (ppm)

Tirupathi 6.5�9.0 17 22 150Tirumala 6.0�8.2 17 7 550Kalyani Dam 6.7�8.6 Low Trace 396Punganur 6.5�9.5 8 9 170Sathyavedu 6.5�9.0 12 14 360Settigunta 6.9�9.0 17 4 550Gadela 7.0�9.0 12 4 330Dakkili 7.0�10.0 12 22 97Dagadarthi 6.5�9.0 18 14 400Kavali 7.0�9.0 13 16 370

The ectomycorrhizal fungus Pt was welldistributed in all the plantations (3–11 years old)(Table 1; Figure 1). Maximum distribution was re-corded in E. camaldulensis (100%), E. tereticornis(100%), and A. holosericea (50%), and minimum inA. auriculiformis (30%) and A. mangium (10%) (whileA. mangium plantation was grown in one site only).

The fungus has been earlier reported inassociation with Pinus spp. (Marx 1977) in theUnited States; Hopea odorata and Shorea spp.(Suhardi and Tasimin 1988) in Indonesia; Pinuskesiya, P. merkusii, and P. caribaeae(Nopamornbodi 1988) in Thailand; Eucalyptustereticornis (Natarajan, Mohan, and Kaviyarasan1988) in India; and Acacia auriculiformis(Sampangiramaiah and Bhatta 1996) in India. Thisfungus is reported from India for the first time inassociation with Acacia mangium, A. holosericea,and Eucalyptus camaldulensis tree species in lowfertile soils of Andhra Pradesh.

Root samples collected from underneath thebasidiomata of Pt in association with different treespecies were processed for identification of fungalassociation. All the samples had ectomycorrhizal


IntroductionResearch in the field of mycorrhiza requires pureand viable inoculum for investigations at thelaboratory level or field level. Reliable storage anddelivery materials are required to promote the useof mycorrhiza as a biofertilizer. Germplasm needsto be conserved for a longer duration not only topreserve the culture but also because its deliveryinto the field along with base or carrier materialrequires a suitable medium. Ectomycorrhizal fungiwhich grow as mycelial filaments in vitro arecommonly maintained for long durations byconventional storage methods like parafilm oillayering; storage in cold water, silicon gel, hydro-gel, and sterile water; freeze-drying or lyophiliza-tion. Conventional methods have certaindisadvantages like loss of infectivity, contamination,

Long-term preservation and delivery of ectomycorrhizal fungal inoculum

Patel C R, Kothari I LDepartment of Biosciences, Sardar Patel University, Vallabh Vidyanagar – 388 120, India

structural damage, variation, and high cost(Anonymous 1997).

Various carrier materials have been triedand tested for delivery of the mycelium into thefields. These include charcoal or lignite-starch,cereal grains, vermiculite, and mycobeads oralginate beads. Cereal grains and vermiculite oftencontain undesirable levels of residual carbohydratesthat can adversely affect the success of theinoculum and also invite contamination(Anonymous 1992).

In this study, base material was screened andused for the ectomycorrhizal inoculum to overcomethese problems. Softone, 2MgSi

2O

5 .Mg(OH)2−,

was

tried as storage as well as carrier material. Alsoknown as talc, it consists of Si2O5

2− layers withMg2+ and OH1- ions sandwiched in between; the

ReferencesMarx D H. 1969The influence of ectotrophic mycorrhizal fungi onthe resistance of pine roots to pathogenic infections.I. Antagonism of mycorrhizal fungi to root patho-genic fungi and soil bacteriaPhytopathology 59: 158–163

Marx D H. 1973Mycorrhizae and feeder root diseasesIn Ectomycorrhizae – their ecology and physiology, editedby G C Marks and T T Kozlowski, pp. 351–382London: Academic Press. 444 pp.

Marx D H. 1977Tree host range and world distribution of theectomycorrhizal fungus Pisolithus tinctoriusCanadian Journal of Microbiology 23: 217–223

Natarajan K, Mohan V, and Kaviyarasan V. 1988On some ectomycorrhizal fungi occurring inSouthern IndiaKavaka 16: 1–7

Nopamornbodi O. 1988Status of mycorrhizal research in ThailandIn Mycorrhizae for Green Asia, edited by A Mahadevan,N Raman, and K Natarajan, pp. 27–28Madras: University of Madras[Proceedings of the 1st Asian Conference on Mycorrhizae,29–31 January 1988, Madras, India]

Sampangiramaiah K and Bagyaraj D J. 1989Root diseases and mycorrhizae – a reviewJournal of Phytopathological Research 2: 1–6

Sampangiramaiah K and Bhatta S M G. 1996Natural association of Pisolithus tinctorius withAcacia auriculiformis in IndiaIn Mycorrhizae: Biofertilizers for the future, edited byA Adholeya and S Singh, pp. 65–66New Delhi: TERI. 548 pp.[Third National Conference on Mycorrhizae organizedby TERI, 13–15 March 1995, New Delhi, India]

Suhardi I and Tasimin Y F. 1988Status of mycorrhizal research in IndonesiaIn Mycorrhiza for Green Asia, edited by A Mahadevan,N Raman, and K Natarajan, pp.14–15Madras: University of Madras[Proceedings of the 1st Asian Conference onMycorrhizae, 29–31 January 1988, Madras, India]


Molecular tools for genericdifferentiation in GlomalesA technique involving PCR (polymerase chainreaction) and restriction fragment length polymor-phism analysis was used by Redecker D,Thierfelder H, Walker C, and Werner D (1997) togenerate specific DNA fragment patterns fromspore extracts of AM (arbuscular mycorrhizal)fungi (Applied and Environmental Microbiology63(5): 1756–1761). DNA fragments that wereapproximately 500 bp long were amplified fromspecies of Scutellospora and Gigaspora with theuniversal primers ITS 1 and ITS 4.

The apparent lengths of the correspondingfragments from Glomus spp. varied between580 and 600 bp. In the genus Glomus, Mbo I, HinfI, and Taq I restriction enzymes were useful fordistinguishing species. Depending on the restric-tion enzyme used, groups of species with commonfragment patterns could be found. Five tropicaland subtropical isolates identified as Glomusmanihotis and G. clarum could not be distinguishedby their restriction patterns, corresponding to mor-phological similarity of the spores. The variation ofinternal transcribed spacer sequences among theGigaspora spp. under study was low. Fragment

patterns of Scutellospora spp. indicated aphylogenetic relationship with Gigaspora andrevealed only a slightly higher degree of variation.

An improved procedure for mycorrhizalroot surface disinfectionA disinfection procedure for mycorrhizal rootsegment surface was improved by Gryndler M,Hrselova H, and Chvatalova I (1997) by usingneomycin and polymyxin B besides sodiumhypochlorite, streptomycin, and penicillin G toobtain clean material for observation of prolifera-tion of hyphae of AM (arbuscular mycorrhizal)fungi (Folia Microbiologica 42(5): 489–494). Thisprocedure is more efficient in terms of incidence ofcontamination compared to the original procedureinvolving only sodium hypochlorite, streptomycin,and penicillin G. Visually detectable contaminationcan be further decreased using bacteriostatic con-centrations of rolitetracycline which reduces hyphalgrowth of the AM fungus, Glomus fistulosum. Thistechnique was used for studying the uptake ofU-C-14 glucose by proliferating hyphae. An activeuptake of glucose occurred even in the presence ofall the four antibiotics at concentrations of 500 mgper litre.

New approaches

whole sandwich being electrically neutral.Therefore, talc is very soft; in fact, one of the soft-est minerals known (Sienko and Plane 1979).

Materials and methodsSoftone in fine powder form is used as storage andcarrier material. Ectomycorrhizal biomass ofpapaya (250 g) was homogenized in a blender andmixed vigorously with 750 g of softone underaseptic conditions. This was water saturated usingsterile distilled water and stored at 20 oC in a glassbeaker. The mixture was tested for viability ofmycelium in both liquid as well as solid agarmedium at monthly intervals for three months.Inoculated base material was streaked on a PDA(potato dextrose agar) plate as well as an MMN’s(Modified Melin Norkran’s) liquid medium andkept for further growth at room temperature.

Results and discussionEctomycorrhizal association in papaya wasconfirmed by observing Hartig net and mantle inthe short roots. Mycelial growth could be seen inabout 4 cm diameter of colony in solid medium

and about 0.3 g biomass in 250 ml of MMN liquidmedium was generated in 35 days. The myceliumshowed viability even after three months; thoughsoftone does not contain easily available nutrients,it can hold enough water to sustain mycelial life fora long time without any deleterious effects. Inocu-lum mixture can turn into brittle cake after sometime, which can be broken down into granularform to serve as an easy delivery system. The datafavourably suggest the use of softone as storage andcarrier material for mycorrhizal biofertilizer.

ReferencesAnonymous. 1992Hydrogel bead inocula for ectomycorrhizaeMycorrhiza News 4(3): 9

Anonymous. 1997Lyophilization: a method for long term preservationof mycorrhizal fungiMycorrhiza News 9(1): 18

Sienko M J and Plane R A. 1979Chemistry: Principles and Applications[s.l.] McGraw-Hill Book Company. 591 pp.


Australasian truffle-like fungi. IX. History and current trends in the study ofthe taxonomy of sequestrate macrofungi from Australia and New ZealandAustralian Systematic Botany 12(6): 803–817[*Oregon State University, Department of Botany and Plant Pathology, Corvallis,OR 97331, USA]

Arbuscular mycorrhizal fungi respond to rhizobial inoculation and croppingsystems in farmers’ fields in the Guinea savannaBiology and Fertility of Soils 30(3): 179–186[*LW Lambourn and Company, Carolyn House, 26 Dingwall Road,Croydon CR9 3EE, England]

Community structure of ectomycorrhizal fungi in a Pinus muricata forest:minimal overlap between the mature forest and resistant propagule communitiesMolecular Ecology 8(11): 1837–1850[*University of California, Santa Barbara, Department of Ecology, Evolution andMarine Biology, Santa Barbara, CA 93106, USA]

Glomus eburneum and G-luteum, two new species of arbuscular mycorrhizalfungi, with emendation of G-spurcumMycologia 91(6): 1083–1093[*Arizona State University, Department of Plant Biology, PO Box 1601-87, Tempe,AZ 85287, USA]

Intra- and interspecific variation in patterns of organic and inorganic nitrogenutilization by three Australian Pisolithus speciesMycological Research 103: 1579–1587[*University of Western Sydney, Nepean School of Science, Mycorrhiza ResearchGroup, PO Box 10, Kingswood, NSW 2747, Australia]

Morphological and molecular variation between Australian isolates of PucciniamenthaeMycological Research 103: 1505–1514[*University of Melbourne, Institute of Land and Food Resources, Parkville,VIC 3052, Australia]

In vitro propagation and life cycle of the arbuscular mycorrhizal fungusGlomus etunicatumMycological Research 103: 1549–1556[*University of Minnesota, Department of Plant Biology, Biological Science Centre220, 1445 Gortner Avenue, St Paul, MN 55108, USA]

Strain differences in the mycelium of the ectomycorrhizal Tuber borchiiMycological Research 103: 1524–1528[*University of Urbino, Ist Chemical Biology of Giorgio Fornaini, Via Saffi 2,I-61029 Urbino, Italy]

Visualisation of mycorrhizal fungal structures and quantification of theirsurface area and volume using laser scanning confocal microscopyMycorrhiza 9(4): 205–213[*Department of Soil and Water and the Centre for Plant Root Symbioses,The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, South Australia5064, Australia]<[email protected]> Tel. +61 8 83036530 Fax +61 8 83036511

Recent referencesName of the author(s) and yearof publication

Title of the article, name of the journal, volume no., issue no., page nos[*address of the corresponding author]

Lebel T*, Castellano M A.1999

Sanginga N*, Carsky R J,Dashiell K. 1999

Taylor D L*, Bruns T D. 1999

Kennedy L J, Stutz J C*,Morton J B. 1999

Anderson I C, Chambers S M,Cairney J W G*. 1999

Edwards J*, Ades P K,Parbery D G, Halloran G M,Taylor P W J. 1999

Pawlowska T E, Douds D D,Charvat I*. 1999

Saltarelli R, Ceccaroli P,Cesari P, Zeppa S, Potenza L,Stocchi V*. 1999

Dickson S*, Kolesik P. 1999


Mycorrhizal effects on the acclimatization, survival, growth and chlorophyllof micropropagated Syngonium and Draceana inoculated at weaning andhardening stagesMycorrhiza 9(4): 215–219[*Centre for Mycorrhizal Research, Tata Energy Research Institute, Darbari SethBlock, Habitat Place, Lodhi Road, New Delhi – 110 003, India]<[email protected]> Fax 91 11 462 1770

Tricholoma matsutake – an assessment of in situ and in vitro infection byobserving cleared and stained whole rootsMycorrhiza 9(4): 227–231[*Laboratory of Forest Botany, Graduate School of Agricultural and Life Sciences,The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan]< [email protected]> Fax +81 3 5841 7554

Influence of soil impoverishment on the interaction between Glomus mosseaeand saprobe fungiMycorrhiza 9(4): 185–189[*Department Ciencias Biológicas, 40 II Pabellón, Universidad de Buenos Aires,1428 Buenos Aires, Argentina]

The mycorrhizal colonization of six wetland plant species at sites differingin land use historyMycorrhiza 9(4): 233–235[*Department of Forest Science, Oregon State University, Corvallis, OR 97331, USA]<[email protected]> Fax +1 541 752 5142

The relationship between density of Glomus mosseae propagules and theinitiation and spread of arbuscular mycorrhizas in cotton rootsMycorrhiza 9(4): 221–225[*School of Biological Sciences, University of Sydney, NSW 2006, Australia]<[email protected]> Fax +61 02 9351 4771

Pisolithus arhizus ectomycorrhiza affects plant competition for phosphorusbetween Pinus elliottii and Panicum chamaeloncheMycorrhiza 9(4): 199–204[*DuPont Agricultural Products, Stine-Haskell Research Center, PO Box 30, Newark,DE 19714, USA]

Genes involved in resistance to powdery mildew in barley differentiallymodulate root colonization by the mycorrhizal fungus Glomus mosseaeMycorrhiza 9(4): 237–240[*Laboratoire de Phytoparasitologie, INRA (Institut National de la RechercheAgronomique) – CNRS (Centre National de Recherches Scientifique), CMSE-INRA,BV 1540, 21034 Dijon Cedex, France]<[email protected]> Fax +33 380 693 263

The mycorrhizal status of an emergent aquatic, Lythrum salicaria L., atdifferent levels of phosphorus availabilityMycorrhiza 9(4): 191–197[*University of Minnesota, Plant Biology Department, 220 Biological Sciences Center,1445 Gortner Avenue, St Paul, MN 55108, USA]<[email protected]> Fax +1 612 625 1738

Gaur A, Adholeya A*. 1999

Gill W M*, Lapeyrie F,Gomi T, Suzuki K. 1999

Godeas A, Fracchia S, MujicaM T, Ocampo J A*. 1999

Ingham E R*, Wilson M V.1999

McGee P A*, Torrisi V,Pattinson G S. 1999

Pedersen C T*, Sylvia D M,Shilling D G. 1999

Ruiz-Lozano J M, Gianinazzi S,Gianinazzi-Pearson V*. 1999

White J A, Charvat I*. 1999

Name of the author(s) and yearof publication



Effect of mycorrhizal colonization and phosphorus on ethylene production bysnapdragon (Antirrhinum majus L.) flowersMycorrhiza 9(3): 161–166[*The Pennsylvania State University, Department of Horticulture, University Park,PA 16802, USA]<[email protected]> Fax +1 814 863 6139

Intraspecific physiological variation: implications for understanding functionaldiversity in ectomycorrhizal fungiMycorrhiza 9(3): 125–135[Mycorrhiza Research Group, Nepean School of Science, University of Western Sydney,PO Box 10, Kingswood, NSW 2747, Australia]<[email protected]>

Effects of mycorrhizal fungus isolates on mineral acquisition by Panicumvirgatum in acidic soilMycorrhiza 9(3): 167–176[*Agricultural Research Service, US Department of Agriculture, Appalachian FarmingSystems Research Centre, 1224 Airport Road, Beaver, WV 25813-9423, USA]<[email protected]> Fax +1 304 256 2921

Exudation–reabsorption in a mycorrhizal fungus, the dynamic interface forinteraction with soil and soil microorganismsMycorrhiza 9(3): 137–144[*Swedish University of Agriculture Science, Department of Forest Mycology and Pa-thology, PO Box 7026, S-75007 Uppsala, Sweden]<[email protected]>

In vitro ectomycorrhiza formation between Abies firma and Pisolithus tinctoriusMycorrhiza 9(3): 177–183[*The University of Tokyo, Graduate School of Agricultural and Life Science,Laboratory of Forest Botany, Bunkyo-Ku, Yayoi 1-1-1, Tokyo 113-8657, Japan]

Competition between ectomycorrhizal fungi colonizing Pinus densifloraMycorrhiza 9(3): 151–159[*Research Unit for Symbiotic Function, Asian National Environmental Science Centre,The University of Tokyo, Midorimachi 1-1-8, Tanashi, Tokyo 188-0002, Japan]<[email protected]> Fax +81 426 65 5601

Organic nitrogen use by salal ericoid mycorrhizal fungi from northernVancouver Island and impacts on growth in vitro of Gaultheria shallonMycorrhiza 9(3): 145–149[*Ministry of Forests, Glyn Road Research Station, PO Box 9536 Stn. Prov. Govt.,Victoria, BC V8W 9C4, Canada]<[email protected]>

Monitoring plant physiological characteristics to evaluate mine siterevegetation: a case study from the wet–dry tropics of northern AustraliaPlant and Soil 215(1): 73–84[*University of Queensland, Department of Botany, Brisbane, Queensland 4072, Australia]

Occurrence of Tuber melanosporum in relation to soil surface layer propertiesand soil differentiationPlant and Soil 214(1–2): 85–92[*Ist Sperimentale Studio and Difesa Suolo, Piazza Dazeglio 30, I-50121 Florence, Italy]

Effects of earthworms on biomass production, nitrogen allocation and nitrogentransfer in wheat–clover intercropping model systemsPlant and Soil 214(1–2): 187–198[*University Coll Dublin, Faculty of Agriculture, Department of Environment ResourceManagement, Dublin 4, Ireland]

Besmer Y L, Koide R T*. 1999

Cairney JWG. 1999

Clark R B*, Zobel R W,Zeto S K. 1999

Sun Y P, Unestam T*,Lucas S D, Johanson K J,Kenne L, Finlay R. 1999

Vaario L M*, Tanaka M,Ide Y J, Gill W M, Suzuki K.1999

Wu B*, Nara K, Hogetsu T.1999

Xiao G P, Berch S M*. 1999

Schmidt S*, Stewart G R,Ashwath N. 1999

Lulli L, Bragato G *, Gardin L.1999

Schmidt O*, Curry J P. 1999

Name of the author(s) and yearof publication



Forthcoming eventsConferences, congresses, seminars, symposiums, andworkshops

52nd International Symposium on Crop ProtectionProf. Dr P De Clercq

Fax +32 0 9 264 62 39 • Tel. +32 0 9 264 61 58E-mail [email protected] site http://allserv.rug.ac.be/~hvanbost/symposium or

http://allserv.rug.ac.be/~hvanbost/symposium/index.html

Forest Sustainability – Beyond 2000Frances Bennett-Sutton, Conference Coordinator

Fax +1 807 622 4353 • Tel. +1 807 622 8228E-mail [email protected]

Ed Iwachewski, Program Chair

Fax +1 807 343 4001 • Tel. +1 807 343 4106E-mail [email protected]

World Congress for Soilless Culture on ‘Agriculture in the Coming Millennium’Congress Secretariat, ORTRA Ltd, 1 Nirim Street, PO Box 9352,Tel-Aviv 61092, Israel

Fax +972 3 6384455 • Tel. + 972 3 6384444E-mail [email protected] • Web site www.agnic.org/mtg/2000/wcsc.html

3rd Agricultural Biotechnology International Conference (ABIC 2000)Sharon Murray, ABIC Conference Coordinator, C/o The Signature Group Inc.,489 Second Avenue, North Saskatoon, SK, Canada S7K 2C1

Fax +1 877 333 2242 (North America) or 306 664 6615Tel. +1 877 925 2242 (North America) or 306 934 1772E-mail [email protected] • Web site www.abic.net/

8th International Symposium on Society and Resource ManagementWestern Washington University

John C Miles E-mail [email protected] J Burdge E-mail [email protected]

6th Congress of International Society for Plant Molecular BiologyInternational Society for Plant Molecular Biology Quebec 2000, C/o AgoraCommunication Inc. 2600, Boulevard Laurier (suite 2680), Sainte-Foy, QC GIV4M6, Canada

Tel. +1 418 658 6755 • Fax +1 418 658 8850E-mail [email protected] • Web site www.ISPMB-2000.org

21st IUFRO World CongressCongress Scientific Committee, IUFRO Secretariat, C/o Federal Forest ResearchCentre, Seckendorff-Gudent-Weg 8, A-1131 Vienna, Austria

Fax +43 1 8779355 • Tel. +43 1 8770151E-mail [email protected]

3rd Workshop on Disturbance Dynamics in Boreal ForestsWorkshop on Disturbance Dynamics, Department of Forest Ecology,PO Box 24, FIN-00014, University of Helsinki, Finland

Fax +358 9 191 7605 • E-mail [email protected] site http://honeybee.helsinki.fi/dist2000/

Gent, Belgium9 May 2000

Thunder Bay, Ontario, Canada14�18 May 2000

Ma�ale Ha�chamisha Kibbutz, Israel14�18 May 2000

Toronto, Ontario, Canada5�8 June 2000

Bellingham, Washington17�22 June 2000

Québec, Canada18�24 June 2000

Kuala Lumpur, Malaysia7�12 August 2000

Kuhmo, Finland21�25 August 2000


ISSN 0970-695X Regd No. 49170/89

Printed and published by Dr R K Pachauri on behalf of the Tata Energy Research Institute, Darbari Seth Block, Habitat Place, Lodhi Road,New Delhi – 110 003, and printed at Multiplexus (India), 94 B D Estate, Timarpur, Delhi – 110 054.

Editor Alok Adholeya Associate Editor Subrata Deb Assistant Editor Pooja Mehrotra

FORM IV(as per Rule 8)

Statements regarding ownership and other particulars about Newsletter: Mycorrhiza News

1. Place of publication New Delhi

2. Periodicity of publication Quarterly

3. Printer’s name Dr R K PachauriWhether citizen of India YesAddress Tata Energy Research Institute,

Darbari Seth Block, Habitat Place,Lodhi Road, New Delhi- 110 003

4. Publisher’s name Dr R K PachauriWhether citizen of India YesAddress Tata Energy Research Institute,


5. Editor’s name Dr Alok AdholeyaWhether citizen of India YesAddress Tata Energy Research Institute,


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

Signature of Publisher(Sd/-)

Date: April 2000 Dr R K Pachauri

List of cultures available with the Centre forMycorrhizal Culture Collection as on 23 March 2000

S No. Bank code Name of the fungus Host

1 EM-1243 Suillus luteus –2 EM-1245 Suillus variegatus Sous resineux3 EM-1239 Suillus bovinus Sous resineux4 EM-1246 Suillus variegatus Sous resineux5 EM-1136 Suillus luteus Pinus6 EM-1137 Suillus luteus Picea7 EM-1087 Suillus subluteous Pinus patula8 EM-1237 Suillus bellini –9 EM-1238 Suillus bovinoides Pinus sylvestris

10 EM-1236 Suillus bellini –

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