Indian Journal of Biotechnology Vol 2. January 2003. pp 65-75

Biotechnological Importance of Piriformospora indica Verma et al-A Novel Symbiotic -like : An Overview

2 3 Anjana Singh I, Archana Singh , Meera Kumari', Mahendra K Rai 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 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 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. 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 (). 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 (v) Ericoid mycorrhizae and (vi) The Australian lily Most terrestrial plants on earth have a symbiotic Thysanotus (Malia et aI, 2002). Here current association in their roots with soil fungi, known as hypotheses of phylogenetic relationships within mycorrhizae, which are beneficial to the growth and heterobasidiomycetous- Hymenomycetes wi th pal1icu lar health of plants and soil (Cruz et aI, 2002; Hodge et reference to Auriculariales is presented. The family aI, 2001; Jeffries & Barea, 2001; Rausch et aI, 2001). Sebacinaceae contained two genera, namely The following six different types of associations of Piriformospora and Sebacina (Weiss et aI, 2002). plants with mycorrhizae have been recognized: (i) AM fungi are the most widespread and probably Yesicular-arbuscular mycorrhizae (YAM or AM) most ancient symbionts in the world, found inmost (Smith, 1995; Walker, 1995), (ii) Ectomycorrhizae biomes and with most plant species. The co-evolution (ECM), (iii) Ectendo-, arbutoid-and monotropoid of the symbionts in this intimate relationship since mycorrhizal associations, (iv) Orchid mycorrhizae, 350 million years has involved a multitude of ecological, physiological and molecular interactions *Author for correspondence: enabling the formation of a partnership of mutual Tel: 26704511. 26107676 Ext-45 I I; Fax: 26187338, 26198234 benefit (Franken et aI, 2000; Kaldorf et aI, 1998). The E-mail: [email protected]@mail.jnu.ac.in partners in this association are members of Abbreviations: AM: arbuscular mycorrhiza; cDNA: complementary Basidiomycetes, Ascomycetes, Zygomycetes and these deoxyribonucleic acid; ECM: ectomycorrhizae; gmPGPRs: colonize most vascular plants belonging to gene'ically modified plant growth promoting rhizobacteria; Cryptogams, Gymnosperms and Angiosperms (Read. PGPRs: plant growth promoting rhizobacteria; Pitefl: plant 1999; Smith & Read, 1997). Mycorrhizal associations translation elongation factor; rRNA: rhibosomal ribonucleic acid; Ri T'-DNA: root inducing transfer DNA; YAM: vascular involve 3-way interactions between host plants. arbuscular mycorrhiza. mutualistic fungi and soil factors (Declerck et aI, 2000; Franken & Requena, 2001; Morton & Bruns, transformed as well as non-transformed roots, leading 2000; Morton & Redecker, 2001; Schuessler & to complete control of the life cycle of a few species Kluge, 2001). of AM fungi. There are also some reports of the The characteristic features of mycorrhizal enhancement of growth by in vitro culturable associations are summarized in Table 1. It is (Addy et ai, 2000; Dix & Webster, 1995; postulated that about 1.5 million fungi exist in nature, Froehlich et ai, 2000; Schulthess & Faeth, 1998). In however, only 0.7 million have been described to a nature, individual species infect plant species taxonomical status. Among them about 6000 \ belonging to different genera, families, orders and mycorrhizal species have been reported (Sutton, 1996; classes (Schuessler & Kluge, 2001). However. they Lilleskov et ai, 2002). do not establish symbiotic relationships with the AM fungi are ubiquitous, important for terrestrial species of some plant families, such as Brassicaceae, ecosystems and are obligate biotrophs (Harrison, Chenopodiaceae, Cyperaceae, ]unceaceae, Proteaceae 1999) exhibiting little host specificity (Bonfante, or with Lupinus spp (Gianinazzi-Pearson et ai, 1996; 2001). The colonization of plant roots by AM fungi Gollotte et ai, 1996). Non-mycorrhizal species and can greatly affect the pla:1t uptake of mineral genera have also been reported. in mycorrhizal nutrients. It may also protect plants from harmful families (Hirrel et ai, 1978; Trappe, 1987). Tester et elements in soil (Rufyikiri et ai, 2000). The potential al (1987) have given the details of the occurrence of of AM fungi for growth promotion of plants has been mycorrhizae in non-mycorrhizal families. well established (Azcon-Aguilar et ai, 1994; Bagyaraj Inoculum production of AM fungi presents a very & Varma, 1995; Morte et ai, 1996; Varma, 1995, difficult problem. These fungi do not grow like any 1998, 1999a; Varma & Schuepp, 1995). Mosse & other fungi, apart from with their hosts. Obligate Hepper (1975) were the first to produce a simplified symbiotic mode of growth, non-availability of pure in vitro system for the study of AM development culture and expensive means of production and their using excised roots in place of whole plants. Mugnier unreliability for the beneficial effects have greatly & Mosse (1987) modified the technique by using Ri jeopardized/undermined the mycorrhizal science. T-DNA transformed roots (hairy roots) as host tissue. Non-availability of authentic pure cultures on Becard & Piche (1992) presented an in-depth commercial scale is the greatest bottleneck in the evaluation of the root organ culture method and application of AM fungi in plant biotechnology. improved the procedures so that typical vesicular- However, mass production of several thousand viable arbuscular mycorrhiza can now be obtained on 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 & 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-scale In order to get a more precise idea about the closer application of AM fungi (Declerck et aI, 1996a,b, relati ves of P. indica, a part of 18S rRN A was 1998, 2000). amplified, sequenced and compared with corresponding data on a number of different Piriformospora indica-AM-like fungus Basidiomycota from GenBank. Sclerotinia sclerotia Verma et al (1998) have discovered a new plant (Ascomycota) and Glomus mosseae (Zygomycota) growth promoting fungus, Piriformospora indica were used as outgroups. Based on the results. a from the desert soils of North-west India. The fungus dendogram of the molecular phylogeny was grows on a wide range of synthetic and complex constructed (Fig. 3), which indicated the lowest media, e.g., minimal media, MM1, MM2, Moser B evolutionary distance of the 18S rRNA sequence of and Aspergillus (Kaefer, 1977) with 2% sucrose or the new fungus to members of the Rhizoctonia group glucose as a carbon and energy source. Young (Ceratobasidiales), namely Rhizoctonia solani Kuhn 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 0.7-3.5 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-) 16-25 (-33) flm in length and (9-) 10-17 (-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 electron- transparent 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 Fig. l--{a) Piriformospora indica growth on aspergillus agar conesponding dolipore. Parenthosomes are flat discs medium (Kaefer. 1977). Black arrow shows the origin of the without any detectable perforation, and consist of an inoculum and the white arrows indicate the rhythmic zonation. (b) electron dense outer layer and a less dense inner layer typical pear-shaped chlamydospores stained with trypan blue as seen under light microscope (x 320). Inset shows the magnified. (Verma et aI, 1998). view of a spore with granulated cytoplasm. 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 Fig. 2-Dolipore and parenthosomes of P. indica. Sections of y' hyphae were observed by TEM. Arrows indicate the dolipore (1) aI, 1999) including members of terrestrial orchids and the continuous parenthosomes (2). This septal pore type is (Sahay & Varma, 1999,2000; Singh & Varma 2000: typical for Hymenomycetes. Singh et aI, 2000; Singh et aI, 2001).

Agaricus bisporus L36658 Cyathus striatus AF026617 P/uteus petasatus AF026634 A/batrel/us syringae AF026632 65 Spongipellis unic%r M59760 98 Ph/ebia radiata AF026606 53 G/oeophyllum sepiarium AF026608 100 Rhizoctonia so/ani E17097 Rhizoctonia so/ani 085630 87 Rhizoctonia so/ani 085636 Rhizoctonia so/ani 085641 100 Piriformospora indica •••••_---- Basidiomycota Rhizoctonia zea e 085647 Geastrum saccatum AF026620 96 Pseudoco/us fusiform is AF026623 Dacrymyces chrysospermus L22257 Heterotextus a/pinus L22259 Fi/obasidiella neoformans 012804 Tremel/a moriformis T00977 100 Trichosporon /aibachii AB001760 Leucosporidium scottii X53499 91 Cronartium ribico/a M94338 Colletotrichum g/oeosporioides M55640 Leucostoma persoonii M83259 Eremascus a/bus M83258 Ta/aromyces flavus M83262 Ascomycota Saccharomyces cerevisiae T10409 98 Candida a/bicans M60302 G /om us intraradices X58725 Gigaspora margarita X58726 I Zygomycota 0.05 Acau/ospora rugosa Z14005 su 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 other representatives of the Basidiomycetes. Branches with support values below 55% were collapsed. Puzzeling support indices are shown at each branch. 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 Adhatoda zeylanica Medic. syn. Beta vulgaris Linn. efficient phosphate solubilizer and transporter (Sudha A vasica Nees ef ai, 1999; Varma et ai, 2001). Like AM fungi, P. AneUl"apinguis (Linn.) Dumort. Brassica oleracea Linn. val'. indica does not colonize the members of Crucifereae botrytis Linn. Arabidopsis thaliana (Linn.) Brassica napus Linn. or the myc' mutants of soybean, Glycine max and pea, Heynh. PiSU111safivul11 (Singh, 2001). More than 90% of the Artemisia annua Linn. Dianthus carvophvlllls Linn. micropropagated plantlets of tested hosts treated with (carnation) the fungus survive transfer from laboratory to open Azadirachta indica A. Juss. Eruca sativa Mill. (Rocket environmental conditions (Sahay & Varma, 1999, (neem) salad) Bacopa lIIonnieri (Linn.) myc' Glycine lIIax cv. Frisson 2000). It also protects them from potent root Wettst. (two strains) pathogens (Varma ef aI, 2001). The similar host range Chlorophytum borivil/ianl/ln myc' Pisllll/ sariVll1IILinn. of P. indica and AM fungi suggests that this Baker (musli) phenomenon may be connected with some identical C. IlIberosum Baker Nastllrtilllll officinale R. Br.. functional aspects as indicated by the serological data Cicer arietinllln Linn. (chick Spinacea oleracea Linn. (ELISA, western blotting, immunofluorescence and pea) (spinach) Dact)'lorhi~afuchsi (Druce) immuno-gold labelling). One striking difference is Soo' that unlike AM fungi, the host range of P. indica also D. incamara (Linn.) Soo'. includes terrestrial orchids, Dactylorhiza purpurella D. lIIaculara (Linn.) Verno (Steph's) Soo, D. incamata (Linn.) Soo, D. majalis D majalis (Rchb. f.) Hunt & (Rchb.f.) Hunt & Summerh and D. fuchsii (Druce) Summerll Soo (Blechert ef ai, 1999; Singh & Varma, 2000; D. purpurel/a ( Steph's) Soo' Singh ef aI, 2001; Varma et ai, 2001). It would be Dalbergia sissoo Roxb. useful to assess the non-hosts of AM fungi with FlInaria hygrOllletrica Hedw. respect to their interaction with P. indica for its Glycine max (Linn.) Merrill further functional characterization. The host and non- (soybean) host spectrum is given in Table 2. The mechanisms Nicotiana tabaccllm Linn. which determine the non-host nature of plant species, (tobacco) N. attenllata Linn. preventing the establishment of a functional AM symbiosis, are not known at the genetic level. Oryza sativa Linn. Petroselinllm crispllln (Mill.) Comparison of salient features of P. indica with AM Airy-Shaw fungi is given in Table 3. Present knowledge of the PiSll1llsativlllII Linn. (pea) sequence of fungal development leading to Popllius tremllia Linn. establishment of functional AM symbiosis suggests Prosopis chilensis Stuntz syn. P that non-host nature of plants lies in their inability to jlllijlora DC. trigger expression of fungal genes involved in hyphal Quercus sp Linn. (oak) commitment to the symbiotic status. In order to obtain Setaria italica Linn. a tool for molecular studies on P. indica, Pitefl Solanlllllllleiongena Linn. encoding the translation elongation factor EF-la. in P. SorghulII vulgare Linn. indica has been cloned and analyzed. Comparison of Spilanthes calva DC. the genomic and cDNA sequence reveals the presence Tectona grandis Linn. f. of seven entrons in the coding part of the gene and at Terminalia Gljllna Linn. least one in the 5' untranslated region of Pitefl is only Withania somnifera (Linn.) present as one copy in the genome, as determined by Dunal Southern blot analysis. Interaction with roots of Zea Zea ma),s val' White (maize) mays in a time course experiment was analyzed in Zi~iphlls IIII/II/nlliaria Burm. f. relation to hyphal development and RNA accumulation showing high expression of the gene Data is based on the root colonization analysis in vivo and in (Buetehorn ef ai, 2000). The Pitefl promotor should vitro (c.f. Varma et ai, 2001). be a good tool to construct vectors for the 2001; Ghazi, 2001; Kranner, 1998; Varma. 1999b: development of a transformation system for P. indica. Varma et ai, 2001). The properties of P. indica have The gene Pitefl might in addition be useful for been patented (Varma & Franken P, 1997, European estimating the amount of active mycelium during in Patent Office, Muenchen, Germany. Patent No. planta development and for the calibration of RNA 97121440.8-2105, Nov. 1998). The culture has been accumulation analysis of differentially expressed deposited at Braunsweich, Germany (OMS No.1 1827) genes. Like AM fungi, P. indica functions as and 18S rDNA fragment deposited with GenBank. bioregulator, biofertilizer and bioprotectant against Bethesda, USA, AF 014929. root pathogens, overcomes the water stress (dehydration), delays the wilting of the leaves, Growth pattern of P. indica prolongs ageing and tissue lysis (Abdalla & Gamal, Circular agar disc (about 4 mm in diameter) 2000; AI-Karaki 2000a, b, c; Calvet, 2001; Elsen, infested with spores and actively growing hyphae of P. indica was placed onto petri-dishes (90 mm. Table 3-Comparison of salient features of P. indica with AM fungi disposable, Tarson) containing solidified Aspergillus medium (Kaefer, 1977). Inoculated petri-dishes were P. indica AM fungi incubated in an inverted position for 7d at 30±2°C in dark. Within seven days, the mycelia completely Geographical India, Pakistan, ubiquitous disiriblliion Philippines, cover the surface of the agar medium with several Australia rhythmic zonations (Fig. 1a). The rhythm indicates A.renic cllllllre yes no the production of the spores and their re-germination. Hyphal strains often undulating straight The physiological and metabolic regulation leading to Hyphal diameter 0.7-3.5 )..UTI 10-20 ).UTI rhythmic growth is not clear. Spores are produced Spore shape pear shaped globose No. of nuclei/spores 8-25 >1000 singly and/or in chains (Fig. 1b). Dolipore present absent Parenthosomes present absent Influence of P. indica on Plant Growth Extramatrical hyphae present present In vitro cultivation of young seedlings of Withania Appressorium present present somnifera (Linn.) Dunal, Zea mays var. White and Vesicle yes yes Arbuscule arbuscule like yes Spilanthus calva DC on interaction with fungus structures resulted in profused root proliferation (Fig. 4a.d). Acid phosphatase detected detected After the biological hardening of micro propagated Alkaline phosphatase detected detected plantlets of W. somnifera and S. calva in a mist Nitrate reductase detected detected chamber, the plants were transfened for a large-scale Chitinase detected detected Cellulase detected detected field trial. A significant increase in growth and yield Catalase no detected of both plant species was recorded relative to Monooxygenase detected not known untreated controls (Fig. 5, Table 4). Glucanase detected noi known The differences in growth observed between Ferulase detected not known inoculated and control plants may have been caused Laccase detected not known by greater absorption of water and mineral nutrients Tyrosinase detected not known Amylase detected not known due to extensive colonization of root by P. indica. The Proteinase detected detected ability of P. indica to continue improving growth of S. Polyphenoloxidase no detected calva and W. somnifera even during the hot March- Polymethy Igalacturonase no detected June summer season (day temperature above 40°C) PhI/II prol1lolional effeci yes yes Biocontrol agel}t for suggests that the fungus may improve drought plant disease (s) yes yes tolerance. Positive influence of this fungus on plant Crucifers coloni~alion no no growth clearly indicates the commercial potential for Glycine l1Iax Myc' absent absent large-scale cultivation of medicinal plants in gener ••1 PiSlI1Il salil'/Iln Myc absent absent Biological hardening and S. calva and W. somnifera in particular (Rai et al. agent for micropropaga- 2001; Rai & Varma, 2002). ted plants positive positive Like the medicinal plants, the tissue culture raised Orchid mycorrhiza yes no Adhatoda zeylanica Linn. and Nicotiana tabacUiIl Linn. c.f. Varma el al. 2001, 2002. were also allowed biological hardening under strict 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, 1999. 2000). 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, 2001. (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).

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 Fig. 4--Surface sterile seeds of Withania sOll1nifera and Zea mays AM fungi, Glomus mosseae and Scute//ospora var White were pre-germinated on water agar. About 3 cm long gilmorei and P. indica, and grown on un-autoclaved young seedlings were placed on MS agar slants. One agar disc 4 and sterile autoclaved potted soils for twelve months. mm in diam infested with spores and hyphae were placed by the Plants inoculated with P. indica were found to have a side of the radicle (a, d). Test tubes were incubated in a tissue better growth as compared to those inoculated with G. culture laboratory maintained at 25±2°C, 1000 lux and 70% humidity. Roots proliferations were photographed after 6 days. mosseae and S. gilmorei (Table 6). Black arrows show the extensive root proliferation as a result of interaction with fungus. Control plants did not receive any Culture filtrate of P. indica initially showed a fungus (b, c). significant increase in neem and maize plant growth 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±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 0.02 Wilhania Fungus 152.53±0.76 1O.70±0.26 63.03±0.15 4.63±0.15 0.23 671.90 sOll1/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 Ground Parts: UGP. Under Ground Parts; ED, Dependency. c.f. Rai el al. 2001; Rai & Varma 2002.

9.00±0.25 15.06±0.35 18.2±OAO

18.56±OAI 23.10±OAI 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 2002.

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±0.35 0.80±0.28 P. indica 28.14 1.17±0.29 46.25 2.55±0.05 , 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 portion Mycobionts 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 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 Further, these fungi confer increased protection impact was steadily slowed down over the period of against nematodes and suppressing nematode one year. Nevertheless, the plant height was reproduction and infection. However, the invariably higher than those received AM fungi or mechanism(s) by which these fungi induce resistance none (Kumari, 2002). in their hosts and the environmental conditions required for enhancing disease resistance need critical Conclusion evaluation and examination. AM fungi and P. indica act as bioprotective agents against root pathogens (bacteria, fungi and The concepts used In the of nematodes). These playa key role by increasing the Glomeromycota are based on the spore morphology tolerance of the host roots to soil-borne pathogens. with the main criteria used for species delimitation being spore size, shape, colour, basal structure, References ornamentation and wall structure. The use of wall Abdalla M E & Gamal M A-F, 2000. Influence of the structures is likely to be of increasing value for endomycorrhizal fungus Glomus mosseae on the development of peanut pod rot disease in Egypt. Mveorrhi::.a. separating taxa at the supra-specific level, since they 10,29-35. are useful ontogenetic studies, which should help in Addy H D, Hambelton S H & Currah R S, 2000. Distribution and determining the different nature of spores. The molecular characterization of the root endophyte introduction of molecular characters has been useful. Phialoeephalafortinii along an environmental gradient in the The study of bio-diversity requires tools, which boreal forest of Alberta. Mycol Res, 104, 1213-1221. provide criteria for defining and resolving biological AI-Karaki R, 2000a. Growth, water use efficiency and mineral acquisition by tomato cultivars grown under salt stress. J groups at different taxonomic levels, and different Plant Nutr, 23, 1-8. techniques can be applied to analyse genetic diversity AI-Karaki R, 2000b. Growth and mineral acquisition by depending on the level to be considered. In the case of mycorrhizal tomato grown under salt tolerance. Mvcorrhi::.a. anamorphic, mitotically reproducing fungi that do not 10,51-54. undergo sexual reproduction, like AM fungi and AI-Karaki R, 2000c. Germination of tomato cultivars as P. indica, molecular characters offer extremely influenced by salinity. Crop Res. 19.225-229. interesting possibilities for problems of diversity, Azcon-Aguilar C, Encina C L, Azcon R & Barea J M. 1994. complexity and phylogeneticity. There is clear Effect of arbuscular mycorrhizae on growth and developmelll of Annona eherimola micropropagated plants. Agric Sci evidence that progress with polymerase chain reaction Finland, 3, 281-288. is being made, both inter- and intra-specific taxa Bagyaraj D J & Varma A, 1995. Interaction between arbuscular apparently been identifiable through DNA mycorrhizal fungi and plants and their importance in polymorphisms, detected through the use of short sustainable agriculture in arid and semi-arid tropics. ill arbitrary primers. The species definition will not Advances in Microbial Ecology. Plenum Press, England. U, 119-142. usually allow generalization of biological behaviour Becard G & Piche Y, 1992. Establishment of vesicular arbuscular to be made (though some instances might), but it does mycorrhiza in root organ cultures: Review and proposed allow for the comparative species and the consequent methodology. in Methods in Microbiology. edited by J R gradual accumulation of knowledge that might later Norris, D J Read & A K Varma. Academic Press. London. be used in a phylogenetic classification. Pp 89-108. Blechert 0, Kost G, Hassel A, Rexer K-H & Varma A. 1999. First Studies of mycorrhiza are also beginning to take remarks on the symbiotic interaction between into account multi-trophic interactions. This, coupled Pirifonnospora indica and terrestrial orchids. ill Mycorrhiza: with a trend to consider these issues in the context of Structure, Function, Molecular Biology and Biotechnology. edited by A Varma & B Hock, 2nd edn. Springer-Verlag. natural environment is a healthy development. Berlin. Pp 683-688. Mycorrhizae are the structures of biological interest in Bonfante P, 2001. At the interface between mycorrhizal fungi and their own right. But the wider importance lies in their plants: The structural organization of cell wall. plasma contribution to ecosystem as components of plant and membrane and cytoskeleton. ill Mycota IX Series. edited by microbial communities. The first century of B Hock. Springer-Verlag, Berlin. Pp 45-61. mycorrhizal research has enabled us to appreciate the Buetehorn B, Rhody D & Franken P, 2000. Isolation and characterisation of Pitefl encoding the translation elongation main structural characteristics of mycorrhiza and their factor EF-Ia of the root endophyte Pirifonllospor{/ illdim. distribution in nature. The new millennium holds the Plant Bioi, 2,687-692. promise that we shall be able to identify the fuller Calvet C, Pinochet J, Hernandez-Dorrego A. Estaun V & impacts of the symbiosis on fitness of both partners Camprubi A, 2001. Field microplot performance of the and to understand the place of symbiosis in the peach-almond hybrid GF-677 after inoculation with context of ecosystem function. Discovery of a arbuscular mycorrhizal fungi in a replant soil infested with root-knot nematodes. Mycorrhiza, 10, 295-300. symbiotic and cultivable fungus, P. indica is an Cruz A F, Ishii T, Matsumoto I & Kadoya K, 2002. Network unique landmark of the millennium, which promises establishment of vesicular-arbuscular mycorrhizal hyphae in immense biotechnological applications. the rhizospheres between trifoliate orange and some plants. J JPN Soc Hortie Sci, 71, 19-25. Declerck S, Strullu D G & Plenchette C, 1996a. In vilm ma,s- Acknowledgement production of the arbuscular mycon'hizal fungus. Glo/llus versifonJze, associated with Ri T-DNA transformed carrot The authors are thankful to UGC, DBT, CSIR, roots. Mycol Res, 100, 1237-1242. DST, Government of India and Indo-German Bilateral Declerck S, Strullu D G, Plenchette C & Guillemette T. 1996b. Collaboration for partial financial support. Entrapment of in vitro produced spores of Glo/llus Joersifonl/e in alginate beads: in vitro and in vivo inoculum potentials. J cells. in Mycorrhiza Manual. edited by A Vanna. Springer- Biotechnol. 48. 51-57. Verlag, Berlin. Pp 227-242. Declerck S. Strullu D G & Plenchette C. 1998. Monoxenic culture Kumari M, 2002. Mycorrhizae for better establishemnt of neem of the intraradical forms of Glomus sp isolated from a plantlets (biological hardening) and enhancement of tropical ecosystem: A proposed methodology for germplasm therapeutic properties. Thesis submitted to B R Ambedkar collection. Mycologia. 90,579-585. Bihar University, Muzaffarpur for the award of the degree of Declerck S, Cranenbrouck. Dalpe Y, Seguin S, Grandmougin- Doctor of Philosophy in Botany. Feljani A. Fontaine J & Sancholle M, 2000. Glomus Lilleskov E A, Fahay T J. Horton T R & Lovett G M. 2002. proiliferulll sp nov: A description based on morphological, Below ground ectomycorrhizal fungal community change biochemical. molecular and monoxenic cultivation data. over a nitrogen deposition gradient in Alaska. £cologr. Mvcologia. 92. 1178-1187. 83,104-115. Dix N J & Webster J. 1995. Fungal Ecology. Chapman Hill, New Malia R. Singh An, Md. Z. Yadav V, Suniti. Verma A. Rai M & York. Varma A, 2002. Piriformospora indica and plant gro~th Elsen A. Declerck S & De Waele D, 2001. Effects of Glolllus promoting Rhizobacteria. in Frontiers of Fungal Diversity in inlraradices on the reproduction of the burrowing nematode India, edited by G P Rao, D J Bhat. T N Lakhanpal & C (Radopholus silllilis) in dixenic culture. Mycorrhiza, 11, 49- Manoharachari. International Book Distributing Co .. 51. Lucknow. Pp 1-19. Franken P. Requena N, Buetehorn B, Krajinski F, Kuhn G, Morte A. Grisela D & Honrubia M, 1996. Effect of arbuscular Lapopin L, Mann P, Rhody D & Stommel M, 2000. mycorrhizal inoculation on micropropagated Telraclinis Molecular analysis of the arbuscular mycorrhiza symbiosis. articulata growth and survival. Agronolllie, 16,633-637. A rch Acker P.f7an~enbau Bodenkd, 45, 271-286. Morton J B & Bruns T D. 2000. Ancestral lineages of arbuscular Franken P & Requena N. 2001. Analysis of gene expression in mycorrhizal fungi (Glomales). Mol Phvlogenel £\'01. 14. arbuscular mycorrhiza: New approaches and challenges, New 276-284. Phrlol. 150.431-439. Froehlich J. Hyde K D & Petrini 0, 2000. Endophytic fungi Morton J B & Redecker D, 2001. Two new families of Glomales. associated with palms. Mycol Res. 104, 1202-1212. Archaeosporaceae and Paraglomaceae with two new genera Ghazi N. AI-Karaki R, Hammad M & Rusan, 2001. Response of Archaeospora and Paraglolllus based on concordant two tomato cultivars differing in salt tolerance to inoculation molecular and morphological characters. Mvcologio. 93. with mycorrhizal fungi under salt stress. Mycorrhiza 11, 43- 181-195. 47. Mosse B & Hepper C M, 1975. Vesicular arbuscular mycorrhizal Gianinazzi-Pearson V. Dumas-Gaudot E, Gollote A. Tahiri- infection in root organ cultures. Phvsiol Plan I Phl'topmhol. Alaoui A & Gianinazzi S, 1996. Cellular and molecular 5,215-223. defense-related root responses to invasion by arbuscular Mugnier J & Mosse B. 1987. VAM infection in transformed root mycorrhizal fungi. New Phylol, 133.45-57. inducing T-DNA root grown axenically. Phytopathology. 77. Gollotte A. Lemoine M C & Gianinazzi-Pearson V, 1996. 1045- 1050. Morphofunctional integration and cellular compatibility Rai M & Varma A. 2002. Field performance of Wilhanio between endomycorrhizal symbionts. in Concepts in sOlllnifera Dunal after inoculation with three species of Mycorrhizal Research, edited by K G Mukerji. Handbook of Glomus. J Basic Appl Mycol. 1,74-80. Vegetation Science Series. Kluwer Academic/Plenum Rai M, Acharya D. Singh A & Varma A. 2001. Positive growth Publishers. New York. Pp 91-111. responses of the medicinal plants Spilanlhes calm and Harrison M, 1999. Molecular and cellular aspects of the Wirhania sOlllnifera to inoculation by Pirifonnospora indica arbuscular mycorrhizal symbiosis. Annu Rev Plant Physiol in a field trial. Mycorrhiza. 11, 123-128. Planl Mol Bioi. 50. 361-389. Rausch C, Daram p. Brunner S. Jansa J. Lalol M. Legge\\ ele G. Hirrel M C. Mehravaran H & Gerdemann J W, 1978. Vesicular Amrhein N & Bucher M. 2001. A phosphate transporter arbuscular mycorrhizae in the Chenopodiaceae and expressed in arbuscule-containing cells in potato. Nalllre Cruciferae: Do they occur? Can J BOI, 56, 2813-2817. (Lond). 414,462-466. Hodge A. Campbell C D & Fitter A H, 2001. An arbuscular Read D J, 1999. The state of art. in Mycorrhiza: Structure. mycorrhizal fungus accelerates decomposition and acquires Function, Molecular Biology and Biotechnology. edited by A nitrogen directly from organic material. Nalure (Lond), 413, Varma & B Hock. Springer-Verlag, Berlin. Pp 1-34. 297-299. Rufyikiri G. Declerck S. Dufey J E & Delvaux B. 2000. Jeffries P & Barea J M, 2001. Arbuscular mycon'hiza--A key Arbuscular mycorrhizal fungi might alleviate aluilliniulll component of sustainable plant soil ecosystems. in Mycota toxicity in banana plants. New Phyla I. 148,343-352. IX Series. edited by B Hock. Springer-Verlag, Berlin. Pp 95- 113. Sahay N S & Varma A. 1999. Piriforlllospora indica: A new Kaefer E. 1977. Meiotic and mitotic recombination in Aspergillus biological hardening tool for micropropagated plants. F£MS and its chromosomal aberrations. Adv Genel. 19. 33-131. Microbiol Leu. 181. 297-302. Kaldorf M. Schmelzer E & Bothe H, 1998. Expression of maize Sahay N S & Varma A, 2000. Biological appJOach towards and fungal nitrate reductase genes in arbuscular mycorrhiza. increasing the survival rates of micropropagated plants. CIIIT Mol Plam Microbe Inleraclioll, 11,439-448. Sci. 78,126-129. Kranner l. 1998. Determination of glutathione. glutathione Schulthess F M & Faeth S H. 1998. Distribution. abundances. and disulphide and two related enzymes-glutathione reductase associations of the endophyte fungal community of Ari:ono and glucose-6-phosphate dehydrogenase, in fungal and plant fescue (FeSluw ari~onica). Mycologia, 90.569-578. Schuessler A & Kluge M, 2001. Geosiphon pyriforll1e, an Varma A, 1995. Ecophysiology and application of Arbuscular Endocytosymbiosis between fungus and cyanobacteria, and Mycorrhizal Fungi in arid soils. in Mycorrhiza, editcd by A its meaning as a model system for Arbuscular Mycorrhizal Varma and B Hock. Springer-Verlag. Berlin. Pp 561-591. Research. in Mycota IX Series, edited by B Hock. Springer- Varma A, 1998. Mycorrhizae: Their role in sustaining wasteland. Verlag. Berlin. Pp 151-161. ill Problems of Wasteland Development and Role of Schuessler A. Schwarzott D & Walker C, 2001. A new fungal Microbes, edited by A Mishra. AMIFEM Publications. phylum, the Gloll1eroll1ycota: phylogeny and evolution. Bhubaneswar, Orissa. Pp 161-181. Mycol Reo, 105, 1413-1421. Varma A, 1999a. Functions and applications of arbuscular Singh AI' & Varma A, 2000. Orchidaceous Mycorrhizal fungi. in mycorrhizal fungi in arid and semi arid soils. in Mycorrhiza. Mycorrhizal Biology, edited by KG Mukerji, B P Chamola edited by A Varma & B Hock. Springer-Verlag. Berlin. Pp & Jagjit Singh. Kluwer Academic/Plenum Publishers, New 521-556. York. Pp 265-288. Varma A, 1999b. Hydrolytic enzymes from arbuscular Singh AI', Sharma J, Rexer K-H & Varma A, 2000. Plant mycorrhizae: The current status. in Mycorrhiza. edited by A productivity determinants beyond minerals, water and light. Varma & B Hock. Springer-Verlag, Berlin. Pp 373-389. Piriforll1ospora indica: A revolutionary plant promoting Varma A & Schuepp H. 1995. Mycorrhizae. their applications in fungus. CurrSci, 79, 101-106. micropropagated plantlets. Crit Rev Biotechnol. 15. 313-328. Singh AI', 2001. Intergenic hybridization between a yeast strain and a plant growth-promoting endophytic fungus by Varma A, Verma S, Sudha, Sahay N, Buetehorn B & Franken P. isolation, fusion and regeneration of somatic protoplasts. 1999. Piriforll1ospora illdica, a cultivable plant growth- Ph D Theiss, Jawaharlal Nehru University, New Delhi. promoting root endophyte. Appl Environ Microhiol. 65. 2741-2744. Singh An. Singh AI' and Varma A, 2001. Root endosymbiont: Piriforlllospora indica----A boon for orchids. J Orchid Soc Varma A, Singh A. Sudha, Sahay N. Sharma J. Roy A. Kumari India. 15. 89-102. M, Rana D, Thakran S. Deka D. Bharati K. Franken P. Hurd Smith S E. 1995. Discoveries, discussions and directions in T, Blechert 0, Rexer K-H. Kost G, Hahn A, Hock B. Maier W, Walter M, Strack D & Kranner 1,2001. Pirijrmnospol'l/ mycorrhizal research. ill Mycorrhizae, edited by A Varma and B Hock. Springer-Verlag, Berlin. Pp 3-24. indica: A cultivable mycorrhiza-like endosymbiotic fungus. in Mycota IX Series, Chapter 8, edited by B Hock. Springer- Smith S E & Read D J, 1997. Mycorrhizal Symbiosis. Academic Verlag, Berlin. Pp 123-150. Press. London. Pp 605. Strimmer K & Haeseler A, 1996. Quartet Puzzling: A quartet Varma A, Sudha, Sahay N S. Singh A. Kumari M. Bharti K. maximum likelihood method for reconstructing tree Sarbhoy A K. Maier W, Walter M. Strack D. Franken P. topologies. Mol Bioi Evol, 13,964-969. Singh An & Malia R, 2002. Piri/orll1oopora indica: A plant stimulator and pathogen inhibitor Arbuscular Mycorrhiza- Sudha, Kaur H, Narula A, Kumar S, Srivastava P S & Varma A, like fungus, edited by D K Makandey & N R Markandcy. 1999. Mycorrhizal aided biological hardening of ill vitro Capital Book Company LId.. New Delhi. Pp. 71-89. raised plantlets. ill Advances in Biotechnology, edited by J P Tiwari. T N Lakhanpal, J Singh, V P Chamola & R Gupta, Verma S, Varma A, Rexer K-H, Hassel A. Kost G. Sarbhoy A. APS Publishing Corporation, New Delhi. Pp 469-485. Bisen P, Buetehorn B & Franken P. 1998. Pirifonnospol'l/ Sutton C, 1996. A Century of Mycology, British Mycological indica gen. nov., a new root-colonizing fungus. Mycologio. Society, Centenary Symposi um. Cambridge Universi ty Press, 90,895-909. New York. Walker C, 1995. AM or VAM: What is in word? in Mycorrhiza. Swofford D L 1998. PAUP*' Phylogenic analysis using edited by A Varma and B Hock. Springer-Verlag. Berlin. Pp parsimony (*and other methods), Version 4. Sinauer 25-28. Associates, Sunderland. MA. Weiss M & Oberwinkler F. 2001. Phylogenic relationships in Tester M. Smith S E & Smith F A, 1987. The phenomenon of Auriculariales and related groups-hypotheses derived from "nonmycorrhizal" plants. Can J Bot, 65,419-431. nuclear ribosomal DNA sequences. Mycol Res. lOS, -l03- Trappe J M. 1987. Phylogenetic and ecological aspects of 415. mycotrophy in the Angiosperms from an evolutionary Weiss M, Rexer K-H & Oberwinkler F, 2002. Auriculorioles ancl standpoint. in Ecophysiology of VA Mycorrhizal Plants, related taxa. 7th Int Mycol Congr, 11-16 August. Stockhol m. edited by G R Safir. CRC Press, Boca Raton. Pp 5-25. Sweden. I' 7.