Indian Journal of BiotechnologyVol 2. January 2003. pp 65-75

Biotechnological Importance of Piriformospora indica Verma et al-A NovelSymbiotic Mycorrhiza-like Fungus: An Overview

Anjana Singh I, Archana Singh2, Meera Kumari', Mahendra K Rai3 and Ajit Varma'",

I School of Life Sciences, Jawaharlal Nehru University, ew Delhi 110067, India"Department of Biological Sciences. University of Alabama, Huntsville. AL 35899. USA

3 Department of Biotechnology, Amravati University, Amravati 444 602, India

Piriforll/ospvra indica Verma et ai, a newly discovered root colonizing, AM fungi-like fungus, showed prominentpositive influence on a wide range of plants of agriculture, forestry and flori-horticultural importance. Interestingly,P. indica has a wide host range of monocots and dicots including legumes, terrestrial o,~chids (Dactylorhiza lIIaculata)and members of the bryophytes (Aneura pinguis). The fungus showed potential as an agent for biological control ofdisease against soil-borne root pathogens. 32p experiments suggest that this fungus is important for phosphorusacquisition by the roots, especially in the arid and semi-arid regions. Mycelium could utilize a wide variety ofinorganic and organic phosphate chemicals and produced acid phosphatases at the tip of the hyphae. The fungus wasfound to act as an excellent tool for biological hardening of tissue culture raised plants (tool for biological hardening).Fungus can be axenically grown on a wide range of synthetic simple and complex media with sucrose or glucose ascarbon energy source. Mass cultivation of the fungus can be easily achieved on simplified broth culture. The growthis best obtained between 25-35°C and pH 5.8. The fungus was discovered from the rhizospheric soils of desert plants,Prosopis chilensis Stuntz and Ziziphus /lulI/lIIlllaria Burm. f. in the sandy desert of Rajasthan, North-west India. Forits characteristic spore structure the isolate was named Piriforlllospora indica. Electron microscopy revealed thepresence of typical doli pore septum with continuous parenthosomes, which indicated that the fungus belongs to theHymenom)'cetes (Basidiomycota). Sequences of 188 rRNA and 28S rRNA indicate that P. indica is related to theRhizocto/lia group and the family Sebacinaceae (Basidiomycetes). Immunofluorescence, ELISA, western blot andimmuno-gold characterization indicated affinity of P. indica with the members of GlolI/eroll/ycota, namelyGlolllerales, Diversisporales and Archeaosporales.

IntroductionMost terrestrial plants on earth have a symbiotic

association in their roots with soil fungi, known asmycorrhizae, which are beneficial to the growth andhealth of plants and soil (Cruz et aI, 2002; Hodge etaI, 2001; Jeffries & Barea, 2001; Rausch et aI, 2001).The following six different types of associations ofplants with mycorrhizae have been recognized: (i)Yesicular-arbuscular mycorrhizae (YAM or AM)(Smith, 1995; Walker, 1995), (ii) Ectomycorrhizae(ECM), (iii) Ectendo-, arbutoid-and monotropoidmycorrhizal associations, (iv) Orchid mycorrhizae,

*Author for correspondence:Tel: 26704511. 26107676 Ext-45 I I; Fax: 26187338, 26198234E-mail: ajitvarma73@hotmail.com.ajitvarma73@mail.jnu.ac.inAbbreviations:AM: arbuscular mycorrhiza; cDNA: complementarydeoxyribonucleic acid; ECM: ectomycorrhizae; gmPGPRs:gene'ically modified plant growth promoting rhizobacteria;PGPRs: plant growth promoting rhizobacteria; Pitefl: planttranslation elongation factor; rRNA: rhibosomal ribonucleic acid;Ri T'-DNA: root inducing transfer DNA; YAM: vasculararbuscular mycorrhiza.

(v) Ericoid mycorrhizae and (vi) The Australian lilyThysanotus (Malia et aI, 2002). Here currenthypotheses of phylogenetic relationships withinheterobasidiomycetous- Hymenomycetes wi th pal1icu larreference to Auriculariales is presented. The familySebacinaceae contained two genera, namelyPiriformospora and Sebacina (Weiss et aI, 2002).

AM fungi are the most widespread and probablymost ancient symbionts in the world, found inmostbiomes and with most plant species. The co-evolutionof the symbionts in this intimate relationship since350 million years has involved a multitude ofecological, physiological and molecular interactionsenabling the formation of a partnership of mutualbenefit (Franken et aI, 2000; Kaldorf et aI, 1998). Thepartners in this association are members ofBasidiomycetes, Ascomycetes, Zygomycetes and thesecolonize most vascular plants belonging toCryptogams, Gymnosperms and Angiosperms (Read.1999; Smith & Read, 1997). Mycorrhizal associationsinvolve 3-way interactions between host plants.mutualistic fungi and soil factors (Declerck et aI,

2000; Franken & Requena, 2001; Morton & Bruns,2000; Morton & Redecker, 2001; Schuessler &Kluge, 2001).

The characteristic features of mycorrhizalassociations are summarized in Table 1. It ispostulated that about 1.5 million fungi exist in nature,however, only 0.7 million have been described to ataxonomical status. Among them about 6000 \mycorrhizal species have been reported (Sutton, 1996;Lilleskov et ai, 2002).

AM fungi are ubiquitous, important for terrestrialecosystems and are obligate biotrophs (Harrison,1999) exhibiting little host specificity (Bonfante,2001). The colonization of plant roots by AM fungican greatly affect the pla:1t uptake of mineralnutrients. It may also protect plants from harmfulelements in soil (Rufyikiri et ai, 2000). The potentialof AM fungi for growth promotion of plants has beenwell established (Azcon-Aguilar et ai, 1994; Bagyaraj& Varma, 1995; Morte et ai, 1996; Varma, 1995,1998, 1999a; Varma & Schuepp, 1995). Mosse &Hepper (1975) were the first to produce a simplifiedin vitro system for the study of AM developmentusing excised roots in place of whole plants. Mugnier& Mosse (1987) modified the technique by using RiT-DNA transformed roots (hairy roots) as host tissue.Becard & Piche (1992) presented an in-depthevaluation of the root organ culture method andimproved the procedures so that typical vesicular-arbuscular mycorrhiza can now be obtained on

transformed as well as non-transformed roots, leadingto complete control of the life cycle of a few speciesof AM fungi. There are also some reports of theenhancement of growth by in vitro culturableendophytes (Addy et ai, 2000; Dix & Webster, 1995;Froehlich et ai, 2000; Schulthess & Faeth, 1998). Innature, individual species infect plant speciesbelonging to different genera, families, orders andclasses (Schuessler & Kluge, 2001). However. theydo not establish symbiotic relationships with thespecies of some plant families, such as Brassicaceae,Chenopodiaceae, Cyperaceae, ]unceaceae, Proteaceaeor with Lupinus spp (Gianinazzi-Pearson et ai, 1996;Gollotte et ai, 1996). Non-mycorrhizal species andgenera have also been reported. in mycorrhizalfamilies (Hirrel et ai, 1978; Trappe, 1987). Tester etal (1987) have given the details of the occurrence ofmycorrhizae in non-mycorrhizal families.

Inoculum production of AM fungi presents a verydifficult problem. These fungi do not grow like anyother fungi, apart from with their hosts. Obligatesymbiotic mode of growth, non-availability of pureculture and expensive means of production and theirunreliability for the beneficial effects have greatlyjeopardized/undermined the mycorrhizal science.Non-availability of authentic pure cultures oncommercial scale is the greatest bottleneck in theapplication of AM fungi in plant biotechnology.However, mass production of several thousand viablepropagules of these fungi and their entrapment in

Table I-Types of mycorrhizal associations

AM ECM Ectendo- Arbutoid Monotropoid Ericoid Orchid

Root structures

Septate hyphae -(+) +- +- + + + +

Hyphae in cells + -(+) + + + + +

Hypha1 coils +- + + + + +

Arbuscules +

Fungal sheath -(+) -(+) + +

Hartig net + + + +

Vesicles +-

Host plants Vascular plants Gymnosperms Ericales Monotropaceae Ericales Orchidaceae& Angiosperms

Plant has + + + +- + +-chlorophyll

Fungi Zygo-Glomales Most Basid-, but some Asco- and Zygo- Asco-(Basid- ) Basid-

Note:- = absent, + = present, (+) = sqmetimes present, (-) = sometimes absent, +- = present or absent, Basid- = Basidiomycetes. Asco- =Ascomycetes, Zygo = Zygomycetes. c. f., Brundrett et aI, 1996.

alginate beads has shown promise of large-scaleapplication of AM fungi (Declerck et aI, 1996a,b,1998, 2000).

Piriformospora indica-AM-like fungusVerma et al (1998) have discovered a new plant

growth promoting fungus, Piriformospora indicafrom the desert soils of North-west India. The fungusgrows on a wide range of synthetic and complexmedia, e.g., minimal media, MM1, MM2, Moser Band Aspergillus (Kaefer, 1977) with 2% sucrose orglucose as a carbon and energy source. Youngmycelia are white and almost hyaline, butconspicuous zonations (rhythmic growth) areobserved in older cultures (Fig. 1a). The mycelium ismostly flat and submerged into the substratum.Hyphae are thin walled and of different diameterranging 0.7-3.5 flm. They often intertwine andoverlap each other. H-connections are often seen. Inolder cultures and on the root surface, hyphae areoften inegularly inflated, showing a nodose tocoralloid-shaped structures, and granulated densebodies. Hyphae sometimes show anastomosis and areirregularly septated. Chlamydospores appear singly orin clusters and are distinctive due to their pear-shapedstructure (Fig. Ib). They measure (14-) 16-25 (-33)flm in length and (9-) 10-17 (-20) flm in width. Youngspores have thin, hyaline walls. At maturity, thesewalls are up to 1.5 flm thick, which appear twolayered, smooth and pale yellow. Very strongautoflorescence is emitted from the wall of the sporesunder UV and blue light. Function of these pigmeritsis not yet established. The cytoplasm of thechlamydospores is densely packed with granular '0'

materials and usually contained 6-25 nuclei. Neitherclamp connections nor sexual structures are observed.

Ultrastructure studies of the septal pore and the cellwall have shown that the cell walls are very thin andhave multilayered structures. The septal pores consistof dolipoi'es with continuous parenthosomes (Fig. 2).The doli pores are prominent, with a multilayeredcross wall and a median swelling mainly consisting ofelectron-transparent material. The electron-transparent layers of the cross walls extend deep intothe median swellings but do not fan out. In the mediansections of the septal pores, the parenthosomes arealways straight and have the same diameter as theconesponding dolipore. Parenthosomes are flat discswithout any detectable perforation, and consist of anelectron dense outer layer and a less dense inner layer(Verma et aI, 1998).

In order to get a more precise idea about the closerrelati ves of P. indica, a part of 18S rRNA wasamplified, sequenced and compared withcorresponding data on a number of differentBasidiomycota from GenBank. Sclerotinia sclerotia(Ascomycota) and Glomus mosseae (Zygomycota)were used as outgroups. Based on the results. adendogram of the molecular phylogeny wasconstructed (Fig. 3), which indicated the lowestevolutionary distance of the 18S rRNA sequence ofthe new fungus to members of the Rhizoctonia group(Ceratobasidiales), namely Rhizoctonia solani Kuhn

Fig. l--{a) Piriformospora indica growth on aspergillus agarmedium (Kaefer. 1977). Black arrow shows the origin of theinoculum and the white arrows indicate the rhythmic zonation. (b)typical pear-shaped chlamydospores stained with trypan blue asseen under light microscope (x 320). Inset shows the magnified.view of a spore with granulated cytoplasm.

and Thanatephorus praticoia (Kotila) Talbot. Thesignificance of a common branch shared by thesefungi and P. indica in this reconstructed phylogeny isindicated by the bootstrap value of 61%. When thesame analysis was carried out without the Rhi-;,octoniagroup, the new fungus did not match up with anyother species (data not shown), but occupied its ownbranch. 28S rRNA analysis was completed and thisdid not alter the taxonomic status of the fungus(Weiss et ai, 2002).

The important feature of P. indica is that it is acultivable mycorrhiza-like fungus, responsible forphosphate transport to the host plants. P. indica alsoshows bio-control activity against some rootpathogenic fungi (Varma et aI, 1999, 2001). Thefungus also helps in better establishment anddevelopment of tissue culture raised plants (Varma etaI, 1999) including members of terrestrial orchids(Sahay & Varma, 1999,2000; Singh & Varma 2000:Singh et aI, 2000; Singh et aI, 2001).

/

Fig. 2-Dolipore and parenthosomes of P. indica. Sections ofy' hyphae were observed by TEM. Arrows indicate the dolipore (1)

and the continuous parenthosomes (2). This septal pore type istypical for Hymenomycetes.

Agaricus bisporus L36658

Cyathus striatus AF026617

P/uteus petasatus AF026634A/batrel/us syringae AF026632

65 Spongipellis unic%r M5976098 Ph/ebia radiata AF026606

53 G/oeophyllum sepiarium AF026608

100 Rhizoctonia so/ani E17097Rhizoctonia so/ani 085630

87 Rhizoctonia so/ani 085636

Rhizoctonia so/ani 085641100 Piriformospora indica •••••_----

Rhizoctonia zea e 085647

Geastrum saccatum AF026620

96 Pseudoco/us fusiform is AF026623Dacrymyces chrysospermus L22257

Heterotextus a/pinus L22259Fi/obasidiella neoformans 012804

Tremel/a moriformis T00977100 Trichosporon /aibachii AB001760

Leucosporidium scottii X5349991 Cronartium ribico/a M94338

Colletotrichum g/oeosporioides M55640Leucostoma persoonii M83259Eremascus a/bus M83258

Ta/aromyces flavus M83262

Saccharomyces cerevisiae T1040998 Candida a/bicans M60302

G /om us intraradices X58725

Gigaspora margarita X58726Acau/ospora rugosa Z14005

Basidiomycota

Ascomycota

I Zygomycota0.05su bstitutions/site

Fig. 3--Maximum-likelihood tree estimated by the quartet puzzling method (Strimmer & Haeseler 1996) as implemented by PAUP-tOb2a (Swofford. 1998) on 18S I'D A sequences showing phylogenetic relationships between Piri(orlnospora indica and otherrepresentatives of the Basidiomycetes. Branches with support values below 55% were collapsed. Puzzeling support indices are shown ateach branch.

P. indica resembles AM fungi in several functionaland physiological characteristics (Singh et ai, 2000;Varma et ai, 1999,2001,2002). It improves the planthealth and biomass of a wide host range and is anefficient phosphate solubilizer and transporter (Sudhaef ai, 1999; Varma et ai, 2001). Like AM fungi, P.indica does not colonize the members of Crucifereaeor the myc' mutants of soybean, Glycine max and pea,PiSU111safivul11 (Singh, 2001). More than 90% of themicropropagated plantlets of tested hosts treated withthe fungus survive transfer from laboratory to openenvironmental conditions (Sahay & Varma, 1999,2000). It also protects them from potent rootpathogens (Varma ef aI, 2001). The similar host rangeof P. indica and AM fungi suggests that thisphenomenon may be connected with some identicalfunctional aspects as indicated by the serological data(ELISA, western blotting, immunofluorescence andimmuno-gold labelling). One striking difference isthat unlike AM fungi, the host range of P. indica alsoincludes terrestrial orchids, Dactylorhiza purpurella(Steph's) Soo, D. incamata (Linn.) Soo, D. majalis(Rchb.f.) Hunt & Summerh and D. fuchsii (Druce)Soo (Blechert ef ai, 1999; Singh & Varma, 2000;Singh ef aI, 2001; Varma et ai, 2001). It would beuseful to assess the non-hosts of AM fungi withrespect to their interaction with P. indica for itsfurther functional characterization. The host and non-host spectrum is given in Table 2. The mechanismswhich determine the non-host nature of plant species,preventing the establishment of a functional AMsymbiosis, are not known at the genetic level.Comparison of salient features of P. indica with AMfungi is given in Table 3. Present knowledge of thesequence of fungal development leading toestablishment of functional AM symbiosis suggeststhat non-host nature of plants lies in their inability totrigger expression of fungal genes involved in hyphalcommitment to the symbiotic status. In order to obtaina tool for molecular studies on P. indica, Piteflencoding the translation elongation factor EF-la. in P.indica has been cloned and analyzed. Comparison ofthe genomic and cDNA sequence reveals the presenceof seven entrons in the coding part of the gene and atleast one in the 5' untranslated region of Pitefl is onlypresent as one copy in the genome, as determined bySouthern blot analysis. Interaction with roots of Zeamays in a time course experiment was analyzed inrelation to hyphal development and RNAaccumulation showing high expression of the gene(Buetehorn ef ai, 2000). The Pitefl promotor should

Adhatoda zeylanica Medic. syn. Beta vulgaris Linn.A vasica NeesAneUl"apinguis (Linn.) Dumort. Brassica oleracea Linn. val'.

botrytis Linn.Brassica napus Linn.Arabidopsis thaliana (Linn.)

Heynh.Artemisia annua Linn.

Azadirachta indica A. Juss.(neem)Bacopa lIIonnieri (Linn.)Wettst.Chlorophytum borivil/ianl/lnBaker (musli)C. IlIberosum Baker

Cicer arietinllln Linn. (chickpea)Dact)'lorhi~afuchsi (Druce)Soo'D. incamara (Linn.) Soo'.

D. lIIaculara (Linn.) Verno

D majalis (Rchb. f.) Hunt &SummerllD. purpurel/a ( Steph's) Soo'

Dalbergia sissoo Roxb.

FlInaria hygrOllletrica Hedw.

Glycine max (Linn.) Merrill(soybean)Nicotiana tabaccllm Linn.(tobacco)N. attenllata Linn.

Oryza sativa Linn.Petroselinllm crispllln (Mill.)Airy-ShawPiSll1llsativlllII Linn. (pea)

Popllius tremllia Linn.Prosopis chilensis Stuntz syn. Pjlllijlora DC.Quercus sp Linn. (oak)

Setaria italica Linn.

Solanlllllllleiongena Linn.SorghulII vulgare Linn.Spilanthes calva DC.Tectona grandis Linn. f.

Terminalia Gljllna Linn.Withania somnifera (Linn.)DunalZea ma),s val' White (maize)Zi~iphlls IIII/II/nlliaria Burm. f.

Dianthus carvophvlllls Linn.(carnation)Eruca sativa Mill. (Rocketsalad)myc' Glycine lIIax cv. Frisson(two strains)myc' Pisllll/ sariVll1IILinn.

Nastllrtilllll officinale R. Br..

Spinacea oleracea Linn.(spinach)

Data is based on the root colonization analysis in vivo and invitro (c.f. Varma et ai, 2001).

be a good tool to construct vectors for thedevelopment of a transformation system for P. indica.The gene Pitefl might in addition be useful forestimating the amount of active mycelium during inplanta development and for the calibration of RNAaccumulation analysis of differentially expressedgenes. Like AM fungi, P. indica functions asbioregulator, biofertilizer and bioprotectant againstroot pathogens, overcomes the water stress(dehydration), delays the wilting of the leaves,prolongs ageing and tissue lysis (Abdalla & Gamal,2000; AI-Karaki 2000a, b, c; Calvet, 2001; Elsen,

Table 3-Comparison of salient features of P. indica with AMfungi

P. indica AM fungi

Geographical India, Pakistan, ubiquitousdisiriblliion Philippines,

AustraliaA.renic cllllllre yes noHyphal strains often undulating straightHyphal diameter 0.7-3.5 )..UTI 10-20 ).UTI

Spore shape pear shaped globoseNo. of nuclei/spores 8-25 >1000Dolipore present absentParenthosomes present absentExtramatrical hyphae present presentAppressorium present presentVesicle yes yesArbuscule arbuscule like yes

structuresAcid phosphatase detected detectedAlkaline phosphatase detected detectedNitrate reductase detected detectedChitinase detected detectedCellulase detected detectedCatalase no detectedMonooxygenase detected not knownGlucanase detected noi knownFerulase detected not known

Laccase detected not knownTyrosinase detected not knownAmylase detected not knownProteinase detected detectedPolyphenoloxidase no detectedPolymethy Igalacturonase no detectedPhI/II prol1lolional effeci yes yesBiocontrol agel}t forplant disease (s) yes yesCrucifers coloni~alion no noGlycine l1Iax Myc' absent absentPiSlI1Il salil'/Iln Myc absent absent

Biological hardeningagent for micropropaga-ted plants positive positiveOrchid mycorrhiza yes no

c.f. Varma el al. 2001, 2002.

2001; Ghazi, 2001; Kranner, 1998; Varma. 1999b:Varma et ai, 2001). The properties of P. indica havebeen patented (Varma & Franken P, 1997, EuropeanPatent Office, Muenchen, Germany. Patent No.97121440.8-2105, Nov. 1998). The culture has beendeposited at Braunsweich, Germany (OMS No.1 1827)and 18S rDNA fragment deposited with GenBank.Bethesda, USA, AF 014929.

Growth pattern of P. indicaCircular agar disc (about 4 mm in diameter)

infested with spores and actively growing hyphae ofP. indica was placed onto petri-dishes (90 mm.disposable, Tarson) containing solidified Aspergillusmedium (Kaefer, 1977). Inoculated petri-dishes wereincubated in an inverted position for 7d at 30±2°C indark. Within seven days, the mycelia completelycover the surface of the agar medium with severalrhythmic zonations (Fig. 1a). The rhythm indicatesthe production of the spores and their re-germination.The physiological and metabolic regulation leading torhythmic growth is not clear. Spores are producedsingly and/or in chains (Fig. 1b).

Influence of P. indica on Plant GrowthIn vitro cultivation of young seedlings of Withania

somnifera (Linn.) Dunal, Zea mays var. White andSpilanthus calva DC on interaction with fungusresulted in profused root proliferation (Fig. 4a.d).After the biological hardening of micro propagatedplantlets of W. somnifera and S. calva in a mistchamber, the plants were transfened for a large-scalefield trial. A significant increase in growth and yieldof both plant species was recorded relative tountreated controls (Fig. 5, Table 4).

The differences in growth observed betweeninoculated and control plants may have been causedby greater absorption of water and mineral nutrientsdue to extensive colonization of root by P. indica. Theability of P. indica to continue improving growth of S.calva and W. somnifera even during the hot March-June summer season (day temperature above 40°C)suggests that the fungus may improve droughttolerance. Positive influence of this fungus on plantgrowth clearly indicates the commercial potential forlarge-scale cultivation of medicinal plants in gener ••1and S. calva and W. somnifera in particular (Rai et al.2001; Rai & Varma, 2002).

Like the medicinal plants, the tissue culture raisedAdhatoda zeylanica Linn. and Nicotiana tabacUiIlLinn.were also allowed biological hardening under strict

Fig. 4--Surface sterile seeds of Withania sOll1nifera and Zea maysvar White were pre-germinated on water agar. About 3 cm longyoung seedlings were placed on MS agar slants. One agar disc 4mm in diam infested with spores and hyphae were placed by theside of the radicle (a, d). Test tubes were incubated in a tissueculture laboratory maintained at 25±2°C, 1000 lux and 70%humidity. Roots proliferations were photographed after 6 days.Black arrows show the extensive root proliferation as a result ofinteraction with fungus. Control plants did not receive anyfungus (b, c).

Fig. 5--Young Withal/ia somnifera seedlings allowed to colonizein the tissue culture laboratory as described in Fig. 4. Treated anduntreated plants were transferred to small clean plastic pots filledwith sterile substratum (see Sahay & Varma, 1999. 2000). Theywere allowed for physical, chemical and biological hardening fora period of 4 weeks in a mist chamber (Varma & Schuepp. 1995).These plants were transferred to the field as per the experimentaldesign described by Rai et ai, 2001. (a) left is control and (b) righttreated with P. il/dica. (c, d) show an overall view of the plamsgrown in a field trial near Chhindawara, Madhya Pradesh. (c)control, (d) treated with fungus. Wilting apparent in control plants(e) an enlarged inflorescence and leaves proliferation seen intreated plants (f).

control conditions by P. indica. Pot cultureexperiments conducted in an environmentallycontrolled green house also showed a pronouncedphyto-promotional growth (Table 5) (Rai & Varma.2002).

Neem plants (Azadirachta indica) treated with twoAM fungi, Glomus mosseae and Scute//osporagilmorei and P. indica, and grown on un-autoclavedand sterile autoclaved potted soils for twelve months.Plants inoculated with P. indica were found to have abetter growth as compared to those inoculated with G.mosseae and S. gilmorei (Table 6).

Culture filtrate of P. indica initially showed asignificant increase in neem and maize plant growth

Table 4--Influence of P. indica on biomass

Hosts Treatment Fresh wt (gm) Dry wt (gm) NPP EDAGP UGP AGP UGP gm/plant/day

Spilanlhes calva Fungus 74.74±0.65 9.26±0.15 14.76±0.11 2.13±0.23 0.06 211.13

Control 8.74±0.55 6.26±0.35 6.54±0.06 1.46±0.06 0.02Wilhania Fungus 152.53±0.76 1O.70±0.26 63.03±0.15 4.63±0.15 0.23 671.90sOll1/l1fera Control 19.07±1.l 3.77±0.25 8.67±0.35 IAO±0.36 0.12

The control plants were treated with equal amount of autoclaved fungus biomass. NPP, Net Primary Productivity: AGP, Above GroundParts: UGP. Under Ground Parts; ED, Endophyte Dependency. c.f. Rai el al. 2001; Rai & Varma 2002.

9.00±0.25

18.56±OAI

15.06±0.35

23.10±OAI

18.2±OAO

24.0±0.35

Data represents the total plant height (em/per plant). Value represents the average for ten replicates. Experimentswere conducted in an environmentally controlled green house. c.f. Rai & Varma 2002.

Table 6-A comparative evaluation of biomass (g) of Azadirachla indica treated with AM fungi or P. indica

a. aerial portionMycobionts Fresh wt Per cent increase over the Dry wt Per cent increase over the

control control

Control I.99±0.35 0.80±0.28

P. indica 2.55±0.05 28.14 1.17±0.29 46.25,C. mosseae 2.27±OA5 14.07 0.99±0.15 21.25

S. gilll10rei 2.34±0.36 17.56 0.99±0.20 23.75

b. underground portionMycobionts Fresh wt Per cent increase over the Dry wt Per cent increase over the

control control

P. indica 1.14±OA6 31.03 0.65±0.31 66.66

C. mosseae 0.96±0.28 14.34 OA7:t0.30 20.51

S. gilmorei 1.00±OA6 14.94 0.52±0.27 33.33

The control plants were treated with an equal amount of autoclaved mycelium. Data represents mean ± standard deviation of 15replicates. Mean values are significantly different at P<0.05 and P<0.051 in (a) and (b), respectively (Kumari, 2002).

and development over the control, however, theimpact was steadily slowed down over the period ofone year. Nevertheless, the plant height wasinvariably higher than those received AM fungi ornone (Kumari, 2002).

ConclusionAM fungi and P. indica act as bioprotective agents

against root pathogens (bacteria, fungi andnematodes). These playa key role by increasing thetolerance of the host roots to soil-borne pathogens.

Further, these fungi confer increased protectionagainst nematodes and suppressing nematodereproduction and infection. However, themechanism(s) by which these fungi induce resistancein their hosts and the environmental conditionsrequired for enhancing disease resistance need criticalevaluation and examination.

The concepts used In the taxonomy ofGlomeromycota are based on the spore morphologywith the main criteria used for species delimitation

being spore size, shape, colour, basal structure,ornamentation and wall structure. The use of wallstructures is likely to be of increasing value forseparating taxa at the supra-specific level, since theyare useful ontogenetic studies, which should help indetermining the different nature of spores. Theintroduction of molecular characters has been useful.The study of bio-diversity requires tools, whichprovide criteria for defining and resolving biologicalgroups at different taxonomic levels, and differenttechniques can be applied to analyse genetic diversitydepending on the level to be considered. In the case ofanamorphic, mitotically reproducing fungi that do notundergo sexual reproduction, like AM fungi andP. indica, molecular characters offer extremelyinteresting possibilities for problems of diversity,complexity and phylogeneticity. There is clearevidence that progress with polymerase chain reactionis being made, both inter- and intra-specific taxaapparently been identifiable through DNApolymorphisms, detected through the use of shortarbitrary primers. The species definition will notusually allow generalization of biological behaviourto be made (though some instances might), but it doesallow for the comparative species and the consequentgradual accumulation of knowledge that might laterbe used in a phylogenetic classification.

Studies of mycorrhiza are also beginning to takeinto account multi-trophic interactions. This, coupledwith a trend to consider these issues in the context ofnatural environment is a healthy development.Mycorrhizae are the structures of biological interest intheir own right. But the wider importance lies in theircontribution to ecosystem as components of plant andmicrobial communities. The first century ofmycorrhizal research has enabled us to appreciate themain structural characteristics of mycorrhiza and theirdistribution in nature. The new millennium holds thepromise that we shall be able to identify the fullerimpacts of the symbiosis on fitness of both partnersand to understand the place of symbiosis in thecontext of ecosystem function. Discovery of asymbiotic and cultivable fungus, P. indica is anunique landmark of the millennium, which promisesimmense biotechnological applications.

AcknowledgementThe authors are thankful to UGC, DBT, CSIR,

DST, Government of India and Indo-German BilateralCollaboration for partial financial support.

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