Biotechnological Importance of Piriformospora indica Verma et al-a Novel Symbiotic Mycorrhiza-like Fungus: An Overview

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1 Indian Journal of Biotechnology Vol 2. January pp Biotechnological Importance of Piriformospora indica Verma et al-a Novel Symbiotic Mycorrhiza-like Fungus: An Overview Anjana Singh I, Archana Singh 2, Meera Kumari', Mahendra K Rai 3 and Ajit Varma'", I School of Life Sciences, Jawaharlal Nehru University, ew Delhi , India "Department of Biological Sciences. University of Alabama, Huntsville. AL USA 3 Department of Biotechnology, Amravati University, Amravati , India Piriforll/ospvra indica Verma et ai, a newly discovered root colonizing, AM fungi-like fungus, showed prominent positive 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 of disease against soil-borne root pathogens. 32p experiments suggest that this fungus is important for phosphorus acquisition by the roots, especially in the arid and semi-arid regions. Mycelium could utilize a wide variety of inorganic and organic phosphate chemicals and produced acid phosphatases at the tip of the hyphae. The fungus was found 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 as carbon energy source. Mass cultivation of the fungus can be easily achieved on simplified broth culture. The growth is best obtained between 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. For its characteristic spore structure the isolate was named Piriforlllospora indica. Electron microscopy revealed the presence of typical doli pore septum with continuous parenthosomes, which indicated that the fungus belongs to the Hymenom)'cetes (Basidiomycota). Sequences of 188 rrna and 28S rrna indicate that P. indica is related to the Rhizocto/lia group and the family Sebacinaceae (Basidiomycetes). Immunofluorescence, ELISA, western blot and immuno-gold characterization indicated affinity of P. indica with the members of GlolI/eroll/ycota, namely Glolllerales, Diversisporales and Archeaosporales. Introduction Most terrestrial plants on earth have a symbiotic association in their roots with soil fungi, known as mycorrhizae, which are beneficial to the growth and health of plants and soil (Cruz et ai, 2002; Hodge et ai, 2001; Jeffries & Barea, 2001; Rausch et ai, 2001). The following six different types of associations of plants with mycorrhizae have been recognized: (i) Yesicular-arbuscular mycorrhizae (YAM or AM) (Smith, 1995; Walker, 1995), (ii) Ectomycorrhizae (ECM), (iii) Ectendo-, arbutoid-and monotropoid mycorrhizal associations, (iv) Orchid mycorrhizae, *Author for correspondence: Tel: Ext-45 I I; Fax: , ajitvarma73@hotmail.com.ajitvarma73@mail.jnu.ac.in Abbreviations: AM: arbuscular mycorrhiza; cdna: complementary deoxyribonucleic acid; ECM: ectomycorrhizae; gmpgprs: gene'ically modified plant growth promoting rhizobacteria; PGPRs: plant growth promoting rhizobacteria; Pitefl: plant translation elongation factor; rrna: rhibosomal ribonucleic acid; Ri T'-DNA: root inducing transfer DNA; YAM: vascular arbuscular mycorrhiza. (v) Ericoid mycorrhizae and (vi) The Australian lily Thysanotus (Malia et ai, 2002). Here current hypotheses of phylogenetic relationships within heterobasidiomycetous- Hymenomycetes wi th pal1icu lar reference to Auriculariales is presented. The family Sebacinaceae contained two genera, namely Piriformospora and Sebacina (Weiss et ai, 2002). AM fungi are the most widespread and probably most ancient symbionts in the world, found inmost biomes and with most plant species. The co-evolution of the symbionts in this intimate relationship since 350 million years has involved a multitude of ecological, physiological and molecular interactions enabling the formation of a partnership of mutual benefit (Franken et ai, 2000; Kaldorf et ai, 1998). The partners in this association are members of Basidiomycetes, Ascomycetes, Zygomycetes and these colonize most vascular plants belonging to Cryptogams, Gymnosperms and Angiosperms (Read. 1999; Smith & Read, 1997). Mycorrhizal associations involve 3-way interactions between host plants. mutualistic fungi and soil factors (Declerck et ai,

2 2000; Franken & Requena, 2001; Morton & Bruns, 2000; Morton & Redecker, 2001; Schuessler & Kluge, 2001). The characteristic features of mycorrhizal associations are summarized in Table 1. It is postulated that about 1.5 million fungi exist in nature, however, only 0.7 million have been described to a taxonomical status. Among them about 6000 \ mycorrhizal species have been reported (Sutton, 1996; Lilleskov et ai, 2002). AM fungi are ubiquitous, important for terrestrial ecosystems and are obligate biotrophs (Harrison, 1999) exhibiting little host specificity (Bonfante, 2001). The colonization of plant roots by AM fungi can greatly affect the pla:1t uptake of mineral nutrients. It may also protect plants from harmful elements in soil (Rufyikiri et ai, 2000). The potential of AM fungi for growth promotion of plants has been well 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 simplified in vitro system for the study of AM development using excised roots in place of whole plants. Mugnier & Mosse (1987) modified the technique by using Ri T-DNA transformed roots (hairy roots) as host tissue. Becard & Piche (1992) presented an in-depth evaluation of the root organ culture method and improved the procedures so that typical vesiculararbuscular mycorrhiza can now be obtained on transformed as well as non-transformed roots, leading to complete control of the life cycle of a few species of AM fungi. There are also some reports of the enhancement of growth by in vitro culturable endophytes (Addy et ai, 2000; Dix & Webster, 1995; Froehlich et ai, 2000; Schulthess & Faeth, 1998). In nature, individual species infect plant species belonging to different genera, families, orders and classes (Schuessler & Kluge, 2001). However. they do not establish symbiotic relationships with the species of some plant families, such as Brassicaceae, Chenopodiaceae, Cyperaceae, ]unceaceae, Proteaceae or with Lupinus spp (Gianinazzi-Pearson et ai, 1996; Gollotte et ai, 1996). Non-mycorrhizal species and genera have also been reported. in mycorrhizal families (Hirrel et ai, 1978; Trappe, 1987). Tester et al (1987) have given the details of the occurrence of mycorrhizae in non-mycorrhizal families. Inoculum production of AM fungi presents a very difficult problem. These fungi do not grow like any other fungi, apart from with their hosts. Obligate symbiotic mode of growth, non-availability of pure culture and expensive means of production and their unreliability for the beneficial effects have greatly jeopardized/undermined the mycorrhizal science. Non-availability of authentic pure cultures on commercial scale is the greatest bottleneck in the application of AM fungi in plant biotechnology. However, mass production of several thousand viable propagules 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.

3 alginate beads has shown promise of large-scale application of AM fungi (Declerck et ai, 1996a,b, 1998, 2000). Piriformospora indica-am-like fungus Verma et al (1998) have discovered a new plant growth promoting fungus, Piriformospora indica from the desert soils of North-west India. The fungus grows on a wide range of synthetic and complex media, e.g., minimal media, MM1, MM2, Moser B and Aspergillus (Kaefer, 1977) with 2% sucrose or glucose as a carbon and energy source. Young mycelia are white and almost hyaline, but conspicuous zonations (rhythmic growth) are observed in older cultures (Fig. 1a). The mycelium is mostly flat and submerged into the substratum. Hyphae are thin walled and of different diameter ranging flm. They often intertwine and overlap each other. H-connections are often seen. In older cultures and on the root surface, hyphae are often inegularly inflated, showing a nodose to coralloid-shaped structures, and granulated dense bodies. Hyphae sometimes show anastomosis and are irregularly septated. Chlamydospores appear singly or in clusters and are distinctive due to their pear-shaped structure (Fig. Ib). They measure (14-) (-33) flm in length and (9-) (-20) flm in width. Young spores have thin, hyaline walls. At maturity, these walls are up to 1.5 flm thick, which appear two layered, smooth and pale yellow. Very strong autoflorescence is emitted from the wall of the spores under UV and blue light. Function of these pigmerits is not yet established. The cytoplasm of the chlamydospores is densely packed with granular '0' materials and usually contained 6-25 nuclei. Neither clamp connections nor sexual structures are observed. Ultrastructure studies of the septal pore and the cell wall have shown that the cell walls are very thin and have multilayered structures. The septal pores consist of dolipoi'es with continuous parenthosomes (Fig. 2). The doli pores are prominent, with a multilayered cross wall and a median swelling mainly consisting of electron-transparent material. The electrontransparent layers of the cross walls extend deep into the median swellings but do not fan out. In the median sections of the septal pores, the parenthosomes are always straight and have the same diameter as the conesponding dolipore. Parenthosomes are flat discs without any detectable perforation, and consist of an electron dense outer layer and a less dense inner layer (Verma et ai, 1998). In order to get a more precise idea about the closer relati ves of P. indica, a part of 18S rrn A was amplified, sequenced and compared with corresponding data on a number of different Basidiomycota from GenBank. Sclerotinia sclerotia (Ascomycota) and Glomus mosseae (Zygomycota) were used as outgroups. Based on the results. a dendogram of the molecular phylogeny was constructed (Fig. 3), which indicated the lowest evolutionary distance of the 18S rrna sequence of the new fungus to members of the Rhizoctonia group (Ceratobasidiales), namely Rhizoctonia solani Kuhn Fig. l--{a) Piriformospora indica growth on aspergillus agar medium (Kaefer. 1977). Black arrow shows the origin of the inoculum and the white arrows indicate the rhythmic zonation. (b) typical pear-shaped chlamydospores stained with trypan blue as seen under light microscope (x 320). Inset shows the magnified. view of a spore with granulated cytoplasm.

4 / Fig. 2-Dolipore and parenthosomes of P. indica. Sections of y' hyphae were observed by TEM. Arrows indicate the dolipore (1) and the continuous parenthosomes (2). This septal pore type is typical for Hymenomycetes. and Thanatephorus praticoia (Kotila) Talbot. The significance of a common branch shared by these fungi and P. indica in this reconstructed phylogeny is indicated by the bootstrap value of 61 %. When the same analysis was carried out without the Rhi-;,octonia group, the new fungus did not match up with any other species (data not shown), but occupied its own branch. 28S rrna analysis was completed and this did not alter the taxonomic status of the fungus (Weiss et ai, 2002). The important feature of P. indica is that it is a cultivable mycorrhiza-like fungus, responsible for phosphate transport to the host plants. P. indica also shows bio-control activity against some root pathogenic fungi (Varma et ai, 1999, 2001). The fungus also helps in better establishment and development of tissue culture raised plants (Varma et ai, 1999) including members of terrestrial orchids (Sahay & Varma, 1999,2000; Singh & Varma 2000: Singh et ai, 2000; Singh et ai, 2001) su bstitutions/site Agaricus bisporus L36658 Cyathus striatus AF P/uteus petasatus AF A/batrel/us syringae AF Spongipellis unic%r M Ph/ebia radiata AF G/oeophyllum sepiarium AF Rhizoctonia so/ani E17097 Rhizoctonia so/ani Rhizoctonia so/ani Rhizoctonia so/ani Piriformospora indica _---- Rhizoctonia zea e Geastrum saccatum AF Pseudoco/us fusiform is AF Dacrymyces chrysospermus L22257 Heterotextus a/pinus L22259 Fi/obasidiella neoformans Tremel/a moriformis T Trichosporon /aibachii AB Leucosporidium scottii X Cronartium ribico/a M94338 Colletotrichum g/oeosporioides M55640 Leucostoma persoonii M83259 Eremascus a/bus M83258 Ta/aromyces flavus M83262 Saccharomyces cerevisiae T Candida a/bicans M60302 G /om us intraradices X58725 Gigaspora margarita X58726 Acau/ospora rugosa Z14005 Basidiomycota Ascomycota I Zygomycota 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 other representatives of the Basidiomycetes. Branches with support values below 55% were collapsed. Puzzeling support indices are shown at each branch.

5 P. indica resembles AM fungi in several functional and physiological characteristics (Singh et ai, 2000; Varma et ai, 1999,2001,2002). It improves the plant health and biomass of a wide host range and is an efficient phosphate solubilizer and transporter (Sudha ef ai, 1999; Varma et ai, 2001). Like AM fungi, P. indica does not colonize the members of Crucifereae or the myc' mutants of soybean, Glycine max and pea, PiSU111safivul11 (Singh, 2001). More than 90% of the micropropagated plantlets of tested hosts treated with the fungus survive transfer from laboratory to open environmental conditions (Sahay & Varma, 1999, 2000). It also protects them from potent root pathogens (Varma ef ai, 2001). The similar host range of P. indica and AM fungi suggests that this phenomenon may be connected with some identical functional aspects as indicated by the serological data (ELISA, western blotting, immunofluorescence and immuno-gold labelling). One striking difference is that unlike AM fungi, the host range of P. indica also includes 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 be useful to assess the non-hosts of AM fungi with respect to their interaction with P. indica for its further functional characterization. The host and nonhost spectrum is given in Table 2. The mechanisms which determine the non-host nature of plant species, preventing the establishment of a functional AM symbiosis, are not known at the genetic level. Comparison of salient features of P. indica with AM fungi is given in Table 3. Present knowledge of the sequence of fungal development leading to establishment of functional AM symbiosis suggests that non-host nature of plants lies in their inability to trigger expression of fungal genes involved in hyphal commitment to the symbiotic status. In order to obtain a tool for molecular studies on P. indica, Pitefl encoding the translation elongation factor EF-la. in P. indica has been cloned and analyzed. Comparison of the genomic and cdna sequence reveals the presence of seven entrons in the coding part of the gene and at least one in the 5' untranslated region of Pitefl is only present as one copy in the genome, as determined by Southern blot analysis. Interaction with roots of Zea mays in a time course experiment was analyzed in relation to hyphal development and RNA accumulation showing high expression of the gene (Buetehorn ef ai, 2000). The Pitefl promotor should Adhatoda zeylanica Medic. syn. Beta vulgaris Linn. A vasica Nees AneUl"apinguis (Linn.) Dumort. Brassica oleracea Linn. val'. botrytis Linn. Arabidopsis thaliana (Linn.) Brassica napus Linn. Heynh. Artemisia annua Linn. Dianthus carvophvlllls Linn. (carnation) Azadirachta indica A. Juss. Eruca sativa Mill. (Rocket (neem) salad) Bacopa liionnieri (Linn.) myc' Glycine liiax cv. Frisson Wettst. (two strains) Chlorophytum borivil/ianl/ln myc' Pisllll/ sarivll1iilinn. Baker (musli) C. IlIberosum Baker Nastllrtilllll officinale R. Br.. Cicer arietinllln Linn. (chick pea) Dact)'lorhi~afuchsi (Druce) Soo' D. incamara (Linn.) Soo'. D. liiaculara (Linn.) Verno D majalis (Rchb. f.) Hunt & Summerll D. 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-Shaw PiSll1llsativlllII Linn. (pea) Popllius tremllia Linn. Prosopis chilensis Stuntz syn. P jlllijlora DC. Quercus sp Linn. (oak) Setaria italica Linn. Solanlllllllleiongena SorghulII vulgare Linn. Spilanthes calva DC. Linn. Tectona grandis Linn. f. Terminalia Gljllna Linn. Withania somnifera (Linn.) Dunal Zea ma),s val' White (maize) Zi~iphlls IIII/II/nlliaria Burm. f. Spinacea oleracea (spinach) Linn. Data is based on the root colonization analysis in vivo and in vitro (c.f. Varma et ai, 2001).

6 be a good tool to construct vectors for the development of a transformation system for P. indica. The gene Pitefl might in addition be useful for estimating the amount of active mycelium during in planta development and for the calibration of RNA accumulation analysis of differentially expressed genes. Like AM fungi, P. indica functions as bioregulator, biofertilizer and bioprotectant against root 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 AM fungi P. indica AM fungi Geographical India, Pakistan, ubiquitous disiriblliion Philippines, Australia A.renic cllllllre yes no Hyphal strains often undulating straight Hyphal diameter )..UTI ).UTI Spore shape pear shaped globose No. of nuclei/spores 8-25 >1000 Dolipore present absent Parenthosomes present absent Extramatrical hyphae present present Appressorium present present Vesicle yes yes Arbuscule arbuscule like yes structures Acid phosphatase detected detected Alkaline phosphatase detected detected Nitrate reductase detected detected Chitinase detected detected Cellulase detected detected Catalase no detected Monooxygenase detected not known Glucanase detected noi known Ferulase detected not known Laccase detected not known Tyrosinase detected not known Amylase detected not known Proteinase detected detected Polyphenoloxidase no detected Polymethy Igalacturonase no detected PhI/II prol1lolional effeci yes yes Biocontrol agel}t for plant disease (s) yes yes Crucifers coloni~alion no no Glycine l1iax Myc' absent absent PiSlI1Il salil'/iln Myc absent absent Biological hardening agent for micropropagated plants positive positive Orchid mycorrhiza yes no c.f. Varma el al. 2001, ; Ghazi, 2001; Kranner, 1998; Varma. 1999b: Varma et ai, 2001). The properties of P. indica have been patented (Varma & Franken P, 1997, European Patent Office, Muenchen, Germany. Patent No , Nov. 1998). The culture has been deposited at Braunsweich, Germany (OMS No ) and 18S rdna fragment deposited with GenBank. Bethesda, USA, AF Growth pattern of P. indica Circular agar disc (about 4 mm in diameter) infested with spores and actively growing hyphae of P. indica was placed onto petri-dishes (90 mm. disposable, Tarson) containing solidified Aspergillus medium (Kaefer, 1977). Inoculated petri-dishes were incubated in an inverted position for 7d at 30±2 C in dark. Within seven days, the mycelia completely cover the surface of the agar medium with several rhythmic zonations (Fig. 1a). The rhythm indicates the production of the spores and their re-germination. The physiological and metabolic regulation leading to rhythmic growth is not clear. Spores are produced singly and/or in chains (Fig. 1b). Influence of P. indica on Plant Growth In vitro cultivation of young seedlings of Withania somnifera (Linn.) Dunal, Zea mays var. White and Spilanthus calva DC on interaction with fungus resulted in profused root proliferation (Fig. 4a.d). After the biological hardening of micro propagated plantlets of W. somnifera and S. calva in a mist chamber, the plants were transfened for a large-scale field trial. A significant increase in growth and yield of both plant species was recorded relative to untreated controls (Fig. 5, Table 4). The differences in growth observed between inoculated and control plants may have been caused by greater absorption of water and mineral nutrients due to extensive colonization of root by P. indica. The ability 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 drought tolerance. Positive influence of this fungus on plant growth clearly indicates the commercial potential for large-scale cultivation of medicinal plants in gener 1 and S. calva and W. somnifera in particular (Rai et al. 2001; Rai & Varma, 2002). Like the medicinal plants, the tissue culture raised Adhatoda zeylanica Linn. and Nicotiana tabacuiil Linn. were also allowed biological hardening under strict

7 Fig. 5--Young Withal/ia somnifera seedlings allowed to colonize in the tissue culture laboratory as described in Fig. 4. Treated and untreated plants were transferred to small clean plastic pots filled with sterile substratum (see Sahay & Varma, ). They were allowed for physical, chemical and biological hardening for a period of 4 weeks in a mist chamber (Varma & Schuepp. 1995). These plants were transferred to the field as per the experimental design described by Rai et ai, (a) left is control and (b) right treated with P. il/dica. (c, d) show an overall view of the plams grown 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 in treated plants (f). Fig. 4--Surface sterile seeds of Withania soll1nifera and Zea mays var White were pre-germinated on water agar. About 3 cm long young seedlings were placed on MS agar slants. One agar disc 4 mm in diam infested with spores and hyphae were placed by the side of the radicle (a, d). Test tubes were incubated in a tissue culture 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 of interaction with fungus. Control plants did not receive any fungus (b, c). control conditions by P. indica. Pot culture experiments conducted in an environmentally controlled green house also showed a pronounced phyto-promotional growth (Table 5) (Rai & Varma. 2002). Neem plants (Azadirachta indica) treated with two AM fungi, Glomus mosseae and Scute//ospora gilmorei and P. indica, and grown on un-autoclaved and sterile autoclaved potted soils for twelve months. Plants inoculated with P. indica were found to have a better growth as compared to those inoculated with G. mosseae and S. gilmorei (Table 6). Culture filtrate of P. indica initially showed a significant increase in neem and maize plant growth

8 Table 4--Influence of P. indica on biomass Hosts Treatment Fresh wt (gm) Dry wt (gm) NPP ED AGP UGP AGP UGP gm/plant/day Spilanlhes calva Fungus 74.74± ± ± ± Control 8.74± ± ± ± Wilhania Fungus ±0.76 1O.70± ± ± soll1/l1fera Control 19.07±1.l 3.77± ±0.35 IAO± The control plants were treated with equal amount of autoclaved fungus biomass. NPP, Net Primary Productivity: AGP, Above Ground Parts: UGP. Under Ground Parts; ED, Endophyte Dependency. c.f. Rai el al. 2001; Rai & Varma ± ±OAI 15.06± ±OAI 18.2±OAO 24.0±0.35 Data represents the total plant height (em/per plant). Value represents the average for ten replicates. Experiments were conducted in an environmentally controlled green house. c.f. Rai & Varma Table 6-A comparative evaluation of biomass (g) of Azadirachla indica treated with AM fungi or P. indica a. aerial portion Mycobionts Fresh wt Per cent increase over the Dry wt Per cent increase over the control control Control I.99± ±0.28 P. indica 2.55± ± , C. mosseae 2.27±OA ± S. gilll10rei 2.34± ± b. underground portion Mycobionts Fresh wt Per cent increase over the Dry wt Per cent increase over the control control P. indica 1.14±OA ± C. mosseae 0.96± OA7:t S. gilmorei 1.00±OA ± The control plants were treated with an equal amount of autoclaved mycelium. Data represents mean ± standard deviation of 15 replicates. 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, the impact was steadily slowed down over the period of one year. Nevertheless, the plant height was invariably higher than those received AM fungi or none (Kumari, 2002). Conclusion AM fungi and P. indica act as bioprotective agents against root pathogens (bacteria, fungi and nematodes). These playa key role by increasing the tolerance of the host roots to soil-borne pathogens. Further, these fungi confer increased protection against nematodes and suppressing nematode reproduction and infection. However, the mechanism(s) by which these fungi induce resistance in their hosts and the environmental conditions required for enhancing disease resistance need critical evaluation and examination. The concepts used In the taxonomy of Glomeromycota are based on the spore morphology with the main criteria used for species delimitation

9 being spore size, shape, colour, basal structure, ornamentation and wall structure. The use of wall structures is likely to be of increasing value for separating taxa at the supra-specific level, since they are useful ontogenetic studies, which should help in determining the different nature of spores. The introduction of molecular characters has been useful. The study of bio-diversity requires tools, which provide criteria for defining and resolving biological groups at different taxonomic levels, and different techniques can be applied to analyse genetic diversity depending on the level to be considered. In the case of anamorphic, mitotically reproducing fungi that do not undergo sexual reproduction, like AM fungi and P. indica, molecular characters offer extremely interesting possibilities for problems of diversity, complexity and phylogeneticity. There is clear evidence that progress with polymerase chain reaction is being made, both inter- and intra-specific taxa apparently been identifiable through DNA polymorphisms, detected through the use of short arbitrary primers. The species definition will not usually allow generalization of biological behaviour to be made (though some instances might), but it does allow for the comparative species and the consequent gradual accumulation of knowledge that might later be used in a phylogenetic classification. Studies of mycorrhiza are also beginning to take into account multi-trophic interactions. This, coupled with a trend to consider these issues in the context of natural environment is a healthy development. Mycorrhizae are the structures of biological interest in their own right. But the wider importance lies in their contribution to ecosystem as components of plant and microbial communities. The first century of mycorrhizal research has enabled us to appreciate the main structural characteristics of mycorrhiza and their distribution in nature. The new millennium holds the promise that we shall be able to identify the fuller impacts of the symbiosis on fitness of both partners and to understand the place of symbiosis in the context of ecosystem function. Discovery of a symbiotic and cultivable fungus, P. indica is an unique landmark of the millennium, which promises immense biotechnological applications. Acknowledgement The authors are thankful to UGC, DBT, CSIR, DST, Government of India and Indo-German Bilateral Collaboration for partial financial support. References Abdalla M E & Gamal M A-F, Influence of the endomycorrhizal fungus Glomus mosseae on the development of peanut pod rot disease in Egypt. 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Estaun V & Camprubi A, Field microplot performance of the peach-almond hybrid GF-677 after inoculation with arbuscular mycorrhizal fungi in a replant soil infested with root-knot nematodes. Mycorrhiza, 10, Cruz A F, Ishii T, Matsumoto I & Kadoya K, Network establishment of vesicular-arbuscular mycorrhizal hyphae in the rhizospheres between trifoliate orange and some plants. J JPN Soc Hortie Sci, 71, Declerck S, Strullu D G & Plenchette C, 1996a. In vilm ma,sproduction of the arbuscular mycon'hizal fungus. Glo/llus versifonjze, associated with Ri T-DNA transformed carrot roots. Mycol Res, 100, Declerck S, Strullu D G, Plenchette C & Guillemette T. 1996b. Entrapment of in vitro produced spores of Glo/llus Joersifonl/e

10 in alginate beads: in vitro and in vivo inoculum potentials. J Biotechnol Declerck S. Strullu D G & Plenchette C Monoxenic culture of the intraradical forms of Glomus sp isolated from a tropical ecosystem: A proposed methodology for germplasm collection. Mycologia. 90, Declerck S, Cranenbrouck. Dalpe Y, Seguin S, Grandmougin- Feljani A. Fontaine J & Sancholle M, Glomus proiliferulll sp nov: A description based on morphological, biochemical. molecular and monoxenic cultivation data. Mvcologia Dix N J & Webster J Fungal Ecology. Chapman Hill, New York. Elsen A. Declerck S & De Waele D, Effects of Glolllus inlraradices on the reproduction of the burrowing nematode (Radopholus silllilis) in dixenic culture. Mycorrhiza, 11, Franken P. Requena N, Buetehorn B, Krajinski F, Kuhn G, Lapopin L, Mann P, Rhody D & Stommel M, Molecular analysis of the arbuscular mycorrhiza symbiosis. A rch Acker P.f7an~enbau Bodenkd, 45, Franken P & Requena N Analysis of gene expression in arbuscular mycorrhiza: New approaches and challenges, New Phrlol Froehlich J. Hyde K D & Petrini 0, Endophytic fungi associated with palms. Mycol Res. 104, Ghazi N. AI-Karaki R, Hammad M & Rusan, Response of two tomato cultivars differing in salt tolerance to inoculation with mycorrhizal fungi under salt stress. Mycorrhiza 11, Gianinazzi-Pearson V. Dumas-Gaudot E, Gollote A. Tahiri- Alaoui A & Gianinazzi S, Cellular and molecular defense-related root responses to invasion by arbuscular mycorrhizal fungi. New Phylol, Gollotte A. Lemoine M C & Gianinazzi-Pearson V, Morphofunctional integration and cellular compatibility between endomycorrhizal symbionts. in Concepts in Mycorrhizal Research, edited by K G Mukerji. Handbook of Vegetation Science Series. Kluwer Academic/Plenum Publishers. New York. Pp Harrison M, Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annu Rev Plant Physiol Planl Mol Bioi Hirrel M C. Mehravaran H & Gerdemann J W, Vesicular arbuscular mycorrhizae in the Chenopodiaceae and Cruciferae: Do they occur? Can J BOI, 56, Hodge A. Campbell C D & Fitter A H, An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nalure (Lond), 413, Jeffries P & Barea J M, Arbuscular mycon'hiza--a key component of sustainable plant soil ecosystems. in Mycota IX Series. edited by B Hock. Springer-Verlag, Berlin. Pp Kaefer E Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genel Kaldorf M. Schmelzer E & Bothe H, Expression of maize and fungal nitrate reductase genes in arbuscular mycorrhiza. Mol Plam Microbe Inleraclioll, 11, Kranner l Determination of glutathione. glutathione disulphide and two related enzymes-glutathione reductase and glucose-6-phosphate dehydrogenase, in fungal and plant cells. in Mycorrhiza Manual. edited by A Vanna. Springer- Verlag, Berlin. Pp Kumari M, Mycorrhizae for better establishemnt of neem plantlets (biological hardening) and enhancement of therapeutic properties. Thesis submitted to B R Ambedkar Bihar University, Muzaffarpur for the award of the degree of Doctor of Philosophy in Botany. Lilleskov E A, Fahay T J. Horton T R & Lovett G M Below ground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. cologr. 83, Malia R. Singh An, Md. Z. Yadav V, Suniti. Verma A. Rai M & Varma A, Piriformospora indica and plant gro~th promoting Rhizobacteria. in Frontiers of Fungal Diversity in India, edited by G P Rao, D J Bhat. T N Lakhanpal & C Manoharachari. International Book Distributing Co.. Lucknow. Pp Morte A. Grisela D & Honrubia M, Effect of arbuscular mycorrhizal inoculation on micropropagated Telraclinis articulata growth and survival. Agronolllie, 16, Morton J B & Bruns T D Ancestral lineages of arbuscular mycorrhizal fungi (Glomales). Mol Phvlogenel \' Morton J B & Redecker D, Two new families of Glomales. Archaeosporaceae and Paraglomaceae with two new genera Archaeospora and Paraglolllus based on concordant molecular and morphological characters. Mvcologio Mosse B & Hepper C M, Vesicular arbuscular mycorrhizal infection in root organ cultures. Phvsiol Plan I Phl'topmhol. 5, Mugnier J & Mosse B VAM infection in transformed root inducing T-DNA root grown axenically. Phytopathology Rai M & Varma A Field performance of Wilhanio solllnifera Dunal after inoculation with three species of Glomus. J Basic Appl Mycol. 1, Rai M, Acharya D. Singh A & Varma A Positive growth responses of the medicinal plants Spilanlhes calm and Wirhania solllnifera to inoculation by Pirifonnospora indica in a field trial. Mycorrhiza. 11, Rausch C, Daram p. Brunner S. Jansa J. Lalol M. Legge\\ ele G. Amrhein N & Bucher M A phosphate transporter expressed in arbuscule-containing cells in potato. 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11 Schuessler A & Kluge M, Geosiphon pyriforll1e, an Endocytosymbiosis between fungus and cyanobacteria, and its meaning as a model system for Arbuscular Mycorrhizal Research. in Mycota IX Series, edited by B Hock. Springer- Verlag. Berlin. Pp Schuessler A. Schwarzott D & Walker C, A new fungal phylum, the Gloll1eroll1ycota: phylogeny and evolution. Mycol Reo, 105, Singh AI' & Varma A, Orchidaceous Mycorrhizal fungi. in Mycorrhizal Biology, edited by KG Mukerji, B P Chamola & Jagjit Singh. Kluwer Academic/Plenum Publishers, New York. Pp Singh AI', Sharma J, Rexer K-H & Varma A, Plant productivity determinants beyond minerals, water and light. Piriforll1ospora indica: A revolutionary plant promoting fungus. CurrSci, 79, Singh AI', Intergenic hybridization between a yeast strain and a plant growth-promoting endophytic fungus by isolation, fusion and regeneration of somatic protoplasts. Ph D Theiss, Jawaharlal Nehru University, New Delhi. Singh An. Singh AI' and Varma A, Root endosymbiont: Piriforlllospora indica----a boon for orchids. J Orchid Soc India Smith S E Discoveries, discussions and directions in mycorrhizal research. ill Mycorrhizae, edited by A Varma and B Hock. Springer-Verlag, Berlin. Pp Smith S E & Read D J, Mycorrhizal Symbiosis. Academic Press. London. Pp 605. Strimmer K & Haeseler A, Quartet Puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol Bioi Evol, 13, Sudha, Kaur H, Narula A, Kumar S, Srivastava P S & Varma A, Mycorrhizal aided biological hardening of ill vitro raised plantlets. ill Advances in Biotechnology, edited by J P Tiwari. T N Lakhanpal, J Singh, V P Chamola & R Gupta, APS Publishing Corporation, New Delhi. Pp Sutton C, A Century of Mycology, British Mycological Society, Centenary Symposi um. Cambridge Universi ty Press, New York. Swofford D L PAUP*' Phylogenic analysis using parsimony (*and other methods), Version 4. Sinauer Associates, Sunderland. MA. Tester M. Smith S E & Smith F A, The phenomenon of "nonmycorrhizal" plants. Can J Bot, 65, Trappe J M Phylogenetic and ecological aspects of mycotrophy in the Angiosperms from an evolutionary standpoint. in Ecophysiology of VA Mycorrhizal Plants, edited by G R Safir. CRC Press, Boca Raton. Pp Varma A, Ecophysiology and application of Arbuscular Mycorrhizal Fungi in arid soils. in Mycorrhiza, editcd by A Varma and B Hock. Springer-Verlag. Berlin. Pp Varma A, Mycorrhizae: Their role in sustaining wasteland. ill Problems of Wasteland Development and Role of Microbes, edited by A Mishra. AMIFEM Publications. Bhubaneswar, Orissa. Pp Varma A, 1999a. Functions and applications of arbuscular mycorrhizal fungi in arid and semi arid soils. in Mycorrhiza. edited by A Varma & B Hock. Springer-Verlag. Berlin. Pp Varma A, 1999b. Hydrolytic enzymes from arbuscular mycorrhizae: The current status. in Mycorrhiza. edited by A Varma & B Hock. Springer-Verlag, Berlin. Pp Varma A & Schuepp H Mycorrhizae. their applications in micropropagated plantlets. Crit Rev Biotechnol Varma A, Verma S, Sudha, Sahay N, Buetehorn B & Franken P Piriforll1ospora illdica, a cultivable plant growthpromoting root endophyte. Appl Environ Microhiol Varma A, Singh A. Sudha, Sahay N. Sharma J. Roy A. Kumari M, Rana D, Thakran S. Deka D. Bharati K. Franken P. Hurd T, Blechert 0, Rexer K-H. Kost G, Hahn A, Hock B. Maier W, Walter M, Strack D & Kranner 1,2001. Pirijrmnospol'l/ indica: A cultivable mycorrhiza-like endosymbiotic fungus. in Mycota IX Series, Chapter 8, edited by B Hock. Springer- Verlag, Berlin. Pp Varma A, Sudha, Sahay N S. Singh A. Kumari M. Bharti K. Sarbhoy A K. Maier W, Walter M. Strack D. Franken P. Singh An & Malia R, Piri/orll1oopora indica: A plant stimulator and pathogen inhibitor Arbuscular Mycorrhizalike fungus, edited by D K Makandey & N R Markandcy. Capital Book Company LId.. New Delhi. Pp Verma S, Varma A, Rexer K-H, Hassel A. Kost G. Sarbhoy A. Bisen P, Buetehorn B & Franken P Pirifonnospol'l/ indica gen. nov., a new root-colonizing fungus. Mycologio. 90, Walker C, AM or VAM: What is in word? in Mycorrhiza. edited by A Varma and B Hock. Springer-Verlag. Berlin. Pp Weiss M & Oberwinkler F Phylogenic relationships in Auriculariales and related groups-hypotheses derived from nuclear ribosomal DNA sequences. Mycol Res. los, -l Weiss M, Rexer K-H & Oberwinkler F, Auriculorioles ancl related taxa. 7 th Int Mycol Congr, August. Stockhol m. Sweden. I' 7.