Rhizosphere microorganisms

The quantitative and qualitative nature of microflora around root has, received enormous amount of research attention since I904, when Prof Lorenz Hinder (Hinder I904) coined the term "rhizosphere". Population and activities of the rhizosphere micro flora (Reid 1990) and their influence on crop productivity (Whipps and Lynch 1986) have been thoroughly documented. Therefore it will suffice at this point to look at specific group of microorganisms which relate prominently to the health of tissue culture raised plant and which may be affected by environmental factor. Microbial populations, stimulated by rhizodepositions, are almost invariably higher in the rhizosphere than in root-free-soil. Four groups of organisms all directly or indirectly related to plant growth and health are the plant growth promoting rhizobacteria (PGPRs), root pathogens, biocontrol organisms and symbiotic organisms.

In general population of fungi are lower than those of bacteria and actinomycetes, though I 00,000 colony-forming units g-1 dry wt. of soil is not uncommon (Rouatt et al I960). The rhizosphere/nonrhizosphere (R/S) ratio derived from plate counts may range from 3: I to 100: I, but most frequently are 10: I to 20: I for crop plants. By the use of selective inhibitors and measurement of C02 production, the metabolically active biomass ratio of fungi/bacteria has been found to range from 1.5 to 9.0 in agricultural, grassland and forest soils, and from 0.13 to 1.5 in rhizospheres of crop plants and prairie grass (Anderson and Domsch I975, 1980~ Vancura and Kunc I977~ Nakas and Klein 1980). Thus whereas, fungi in bulk soil usually exceed bacteria in biomass, either group in the rhizosphere may have the larger biomass (Newman 1985). Saprophytic fungi as well as bacteria in the rhizosphere create a competitive deterrent to pathogen colonization of the rhizoplane. Some saprophytes, such as species of Trichoderma and Gliocladium, produce toxins or become directly parasitic on root pathogen. Other common fungi in the rhizosphere includes species of Penicillium, Aspergillus, Fusarium, Cladosporium, Cephalosporium, mucorales and nematode trapping hyphomycetes.

Ravira et a/ (1990) categorized root-infecting fungi as either specialized root­inhabiting fungi characterized by a declining or weakly competitive saprophytic phase after death of the host plant, or soil-inhabiting pathogens characterized by a strong competitive ability (expanding saprophytic phase in absence of the host plant). Most highly specialized are the symbiotic fungi represented by ectomycorrhizal species of basidiomycetes in the genera Pisolithus, Laccaria, Boletus, Thelephora, Suillus, Amanita, Lactarius, Corticium and Rhizopogon.

Endomycorrhizal species of the endogonaceae are found in the genera Gigaspora, Glomus, Scutellospora, Endogone, Acaulospora, and Sclerocystis. Specialized root pathogens are represented by such common species as Plasmodiophora brassica,

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Phymatotrichum omnivorum, Gaeumannomyces graminis, Verticillium dahliae and the vascular wilt Fusaria. Root-infecting fungi with high competitive saprophytic ability include Pythium sp., Rhizoctonia so/ani, Aphanomyces euteiches, Fusarium so/ani f sp. phaseoli and Cochliobolus sativus .

. These fungi collectively produce a variety of propagules (conidia, chlamydospores, sporangia, zoospores, sclerotia and rhizomorphs) which serve as inocula and/or survival structures. They provide opportunities for mycophagous invertebrate animals to influence pathogen behaviour or survival, either in the passive state of the fungus or during root colonization and infection. Schematic representation of influence of rhizosphere organism on the root symbiotic/pathogenic activity is shown in Fig. I.

Mycorrhizal fungi are heterogeneous group of about 6000 species belonging to the Zygomycotina, Ascomycotina and Basidiomycotina. They are grouped into a number of different types depending on their morphological relationship with the host plants, which comprise about 240,000 plant species. Mycorrhizal fungi occupy different niche during their life cycle, they reside in the rhizosphere as spores, hyphae and propagules, they occupy the rhizoplane during their interaction with the root, and finally develop inside the root tissues during the symbiotic phase (Bianciotto and Bonfante 1999). Despite the diversity of the taxa involved, mycorrhizal fungi share substantial features. They live in close association with the roots and accomplish their life cycle due to the establishment of symbiotic relationships. During the interaction, a bidirectional transfer of mineral nutrients and carbon occurs, ensuring a continuous flow of nutrients between the partners. There is extensive literature on the molecular, cellular and. physiological aspects of mycorrhizal fungi (Bonfante and Perotto 1995; Martinet a/1995; Harrison 1997). The positive effect of mycorrhizal fungi on plant nutrition, health and soil stability have valuable agro-biotechnological importance for low input agriculture. However, previous knowledge of biodiversity of mycorrhizal fungi in the rhizosphere is important. Potential exploitation of these fungi in agro-biotechnological systems for example, physiological traits and effectiveness of mycorrhizal fungi differ widely, depending on their taxonomic position and (at a lower rank) on the individual isolate. Polymerase chain reaction (PCR) base technique has been used to provide molecular tools for the identification of both endo- and ectomycorrhizal fungi when their morphological characters are ambiguous or missing. They have also been used to examine relations between closely related species and populations of a single species, a level of relation usually beyond the reach of morphological studies.

Most plants on earth have a symbiotic association in their roots with plant groWth promoting fungi and bacteria. There is wide spread agreement among plant and soil scientists that mycorrhizae are beneficial to the growth and health of plants. However, some arbuscular mycorrhizae (AM) and plant growth promoting fungal species and strains vary greatly in their association with plants ranging from mutualistic symbiosis to mild

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Fig. 1: Microbial activity in the rhizosphere

Schematic representation of rhizosphere influence on soil pathogens and symbionts, ultimately affecting root symbiotic/pathogenic activities

assoctattons gtvmg some benefits. Suffice to say that growth promoting fungi and its association is critical to the well being of plants, especially in natural ecosystems. Most plants have evolved with mycorrhizae and their very existence depends on the mycorrhizal association. In all ecosystems rhizosphere microorganism link plant and soil and that coupling influences most of the dynamics occur in mycorrhizosphere. In agro-ecosystem, current practices must have damaged or disturbed that coupling, and there is a need to develop an understanding and technology to re-couple plants and soil with mycorrhizae, emulating the balance that occur in un-disturbed ecosystem and returning our crop production system to a level of sustainability that allows for reduced inputs of pesticide and fertilizer.

Plant-microbe interactions

Indirect interaction occurs when the microbial population produces biologically active substances which affect plant growth. These may be positive when these substances promote plant growth, as seems to be the case when biotin and pantothenic acid are produced (Ravira 1959~ Brown 1972) or when iron uptake by the root is enhanced by agrobactin production by Agrobacterium tumefacienes. Some species of Psuedomonas produce cyanide, resulting in an indirect negative interaction with respect to the root (Schippers et a/ 1987). Reduction of pH by the root can result in both indirect positive and negative interactions within the microbial population. The most widely studied interaction between the root and microorganisms are direct. This is because of their economic importance. These are negative when paras!tism by bacteria or fungi causes disease, or positive when the result is beneficial, such as with legume Rhizobium association, mycorrhiza and other plant growth promoting fungal association.

The relationship between exudation and secretion from roots and vesicular arbuscular mycorrhizal infection has received considerable attention. Work by Harris eta/ (1985), Koch and Johnson (1984), Me cool and Munge (1983), Schwab eta/ (1984) have suggested that VA mycorrhizal infection is directly related to the amount of exudation of reducing sugar, and in some cases amino acids, that occur prior to infection. Thus condition which increase membrane permeability or increase exudation per se are likely to stimulate VA mycorrhizal infection. For instance, wheat plants with low exudation rates had lower levels of VA mycorrhizal infection than wheat plants with higher exudation rates (Azcon and Ocampo 1984) and plant species that did. not form association with VA mycorrhizas had low exudation rates than plants that did (Schwab et a/1984). However, in other studies, no relationship was found between the degree of VA mycorrhizal infection and total sugar content of root exudates from several plant species with differing degree of mycorrhizal susceptibility (Azcon and Ocampo 1984). In none of these experiments was a relationship found between the amount of reducing sugar and amino acid in the plant root and VA mycorrhizal infection however, a close relationship was found on the concentration of

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reducing sugar within the root of Trifolium subterranium and the percentage root length infected by Glomus fasCiculatum (Same et al 1983) and no clear explanation for these differences is apparent. In arbuscular mycorrhiza, hexokinase activity was found to be high in the intraradical hyphae of Gigaspora margarita, implying that glucose is used by the fungal hyphae (Saito 1995). NMR study also showed that Glomus etunicatum in leek roots was capable of utilizing glucose and synthesized trehalose and glycogen when glucose was applied exogenously to mycorrhizas (Shachar-Hill et a/1995). Thus glucose is one of the most likely substrates for the AM fungi in the symbiotic state. In the host plant, the expression of a sugar transporter gene in Medicago truncata roots was found to be localized in the areas colonized by Glomus versiforme (Harrison 1996), implying that carbohydrate supply to the mycobiont is regulated by the host plant. The first direct evidence of use of glucose by the intraradical hyphae of AM fungi in the symbiote state was given by radiorespirometry (Solaiman and Saito 1997).

When infection by G. fascicu/atum occurred in Sorghum vulgare growing under low P availability and in tomato growing under ozone stress, exudation from the root was decreased in comparison with non-mycorrhizal plants (Graham et al1981; Me Cool and Munge 1983) in the first instance it was suggested that P nutrition was improved following VA mycorrhizal infection and that the membranes of the roots had become less leaky; in the latter case it was proposed that the mycorrhizal fungus competed for the available carbohydrate so less leaked out.

Arbuscular mycorrhizas improve plant acquisition of soil nutrients, especially the less mobile elements such as phosphate. Because roots effectively acquire P only within a few millimeters from the root surface over a period of several days. AM hyphae can enhance procurement of P by extending the effective absorptive zone around the root surface (Reid 1990; Jakobsen et a/ 1994). Also hyphae can acquire nutrients several centimeters from the root surface (Li et a/ 1991; Johansen et a/ 1993; Pearson and Jakobsen 1993a,b). Similar to roots (Hutchings and De Kroon 1994) mycorrhizal hyphae can also exhibit foraging behaviour in that they often proliferate when encountering organic matter rich microsites in the soil (loner and Jakobsen 1995).

Nitrate (NOJ) and Phosphorus are the major plant nutrients whose diffusivities in the soil differ by at least four orders of magnitude. In calcareous soils, mobility of soil P is further reduced. By effectively extending the range of roots for absorption and foraging activity, mycorrhizal fungi can contribute to the acquisition of P also when nutrients are heterogenously distributed on the scale of centimeters (Cui and Caldwell 1996). By contrast a significant contribution of hyphal uptake to N03 acquisition is not expected since roots already effectively acquire this relatively more abundant and mobile nutrient.

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Roots exhibit mechanisms apart from mycorrhizal associators that can facilitate acquisition of nutrients from enriched soil microbes. Roots proliferation (Jackson and Caldwell 1989~ Caldwell 1994) and enhanced physiological uptake capacity of roots in fertile soil patches (Caldwell 1994) have been shown for Agropyron desertorum. If background level of P in soil are low, acquisition of P can be enhanced if added P is concentrated in small soil volumes rather than diluted over a large volume (Kovar and Barber 1989). This occurs even without root proliferation in the P enriched microsites because soil solution P increases disproportionately more than do mother forms of P as P is added to the soil. This can greatly augment root uptake of solution P (Caldwell 1994). Since several root and soil factors already contribute to exploitation of enriched patch nutrients, root colonization by mycorrhizal fungi confers an additional advantage in nutrient acquisition from patches.

The expectation was significant interaction between nutrient distribution of soil and mycorrhizal infection for P acquisition, i.e., mycorrhizas would contribute more toP uptake in a uniform than in patchy nutrient treatment, because soil P would be low in the uniform­nutrient treatment. No facilitation or interaction with distribution pattern were expected for NOJ.

The main effect of AM fungi on plant supply with P and N occurs via the uptake of these nutrients by the extraradical hyphae. Mycorrhizal colonization also changes some plant characteristics important to nutrient uptake and this may in tum have an effect on nutrient accumulation by mycorrhized plants. These changes may be related to better P nutrition of mycorrhizal plants or they may be independent of plant P status. The most often observed effect is a change in root/shoot ratio (George et al1994a, b) and in specific root length (root fitness) after mycorrhizal colonization (George et a! 1995). Effect on root branching may be related to effects of mycorrhizal colonization on root tip activity (Fieschi et a/1992).

Not only root morphology but root metabolism may be affected by fungal colonization. Mycorrhizal colonization increased the proportion of amino acid in shoot and root of Phleum pratense (Ciapperton and Reid 1992) and increased the concentration of some amino acids in Plantago /anciolata (Gange and West 1994), but the physiological interpretation of such effects is often difficult when differences in shoot dry weight between mycorrhizal and non-mycorrhizal plants are large. In another study, carbohydrate and amino acid compositions were not distinctly different between mycorrhizal and non­mycorrhizal white clover or onion (Tawaraya and Saito 1994). Change brought about by AM fungi obviously are not casually related to increased nutrients supply in mycorrhizal plants, but can affects plant growth and thus nutrient uptake under stress conditions. Mycorrhizal maize for example, showed less leaf growth inhibition in response to non­hydrolic signals from roots after partial soil drying (Auge ·~I a/1994). In Sorghum this effect

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was related to slower soil water uptake by mycorrhizal roots and possibly to differences between mycorrhizal and non-mycorrhizal plant in abscisic acid concentration in the xylem sap (Ebel et a/ 1994). There are also several reports of modified disease and pest susceptibility in mycorrhizal plant. For example increased glycoside contents in leaves of mycorrhizal P. lanceolata may increase resistance against leaf chewing insects (Gange and West 1994). Using cultures ofRi-T-DNA transformed roots, it was shown that an increased production of phenolic compounds in mycorrhizal carrots roots may be responsible for increased resistance of mycorrhizal roots against Fusarium infections (Benhmou et a/1994).

Jakobsen et a/ (1994) has discussed in detail on P uptake by AM hyphae. Introduction of hyphae-penetrable nets in the rhizosphere to restrict root growth, hyphae extend several centimeters in the soil (Li et a/1991; Jakobsen et a/1992a), 2mm per day. In compartmentised experimental systems, more than 70% of the plants P contents was due to P uptake by hyphae (George et a/ 1994b). Following P uptake, accumulation of polyphosphates in the hyphae (Smith and Gianinazzi-Pearson 1988) allows a large transport rate of P to the plants. Hyphae have a capacity to almost completely meet the P demand of the plant by supply of P from soil to the cortical cell of roots. AM fungal isolates differ in their capacity to trasnsport P (Jakobsen et a/I992b), so that the mycorrhizal efficiency also differs among fungal isolates (Pearson and Jakobsen 1993a). The host plant species also influences the P transport capacity of the associated hyphae. Mycorrhizal plants often have higher shoot P concentration and shoot dry weight than non-mycorrhizal plants or plants with reduced colonization levels. In contrast, in most cases reported, N concentration in the shoot of mycorrhizal plants was not affected or reduced when compared with non­mycorrhizal plants. Mycorrhizal plants typically have higher PIN (Cuenca and Azcon 1994; Tobar et a/1994a) and C/N (Gange and West 1994) ratios than non-mycorrhizal plants.

Ames eta/ (1983) first studied the uptake ofN by AM hyphae in boxes divided into root and hypha! growing zones. AM hyphae can absorb and translocate considerable amounts of N when provided as "NI-4 + and N03. Fery and Schuepp (1993) estimated for maize grown in compartmented boxes that approximately 30 per cent of the total plant N uptake was due toN uptake by AM hyphae. Uptake and translocation ofN by AM hyphae is also regulated by N demand of the host plant. The conventional view was that in AM associations, N transfer is of secondary importance only (Read et a/ 1989). Not all AM fungi are equally efficient transporters of N (Frey and Schuepp 1993).

The nitrogen metabolism in mycelium of ectomycorrhizal fungi is rather well understood (Martin eta/ 1994). Quantitative evidence on ammonium assimilation or nitrate reduction in AM hyphae is scared because AM fungi can not be grown adequately without a host plant (Ho and Trappe 1975; Smith et a/ 1985). Nitrogen metabolism in AM fungi obtained from analysis of mycorrhizal and non-mycorrhizal roots (Azcon et a/ i 992) are questionable because NIP ratios were different in mycorrhizal and non-mycorrhizal plants

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and nitrogen metabolism in plant tissues itself may therefore also have differed. Quantification of enzyme involved in the nitrogen assimilation by AM mycelium may be possible in the near feature in hyphae that can be isolated from hyphal soil compartments (Frey et a/1994).

The form in which N and P is transferred in the intraradical mycelium from the fungus to the plant is not known, but Smith et a! (1994) suggested that H2P04 (for P) and amino acids and amides (for N) are the main forms involved. P transporting ion channels in the fungal plasmamembrane could transfer large amounts of P from fungus to plant in a short time (Tester et a/1992).

The P transfer across the symbiotic interface is believed to follow a pattern similar to the oppositely directed carbon transfer. Passive transport from the fungus into the interface followed by active uptake by the plant cell. Calculated rates of P flux across the periarbuscular membrane (Cox and Tinker 1976) are similar to rates of P uptake by other fungi and algae (Beever and Burns 1980). While carbon loss is common from plant root cells, membrane process for P transport absorption over loss in both plant and fungi and most P lost by effiux is reabsorbed (Beever and Burns 1980~ Clarkson 1985). Special features are therefore required to explain the abnormally high P loss from the arbuscule. Two principally different mechanisms have been proposed (Tester et a/ 1992~ Smith et a/ 1994). The first is that a high arbuscular P concentration will reduce hyphal reabsorption of lost P in accordance with the general control of P uptake by the internal P concentration. Secondly, P effiux may be promoted by altered operation of trans-membrane carriers and opening of ion channels. The transport of P in the direction, fungus to plant will be supported by a low Pi concentration in the plant cytosol as compared to the fungal cytosol and by W ATPase on the periarbuscular membrane (Gianinazzi-Pearson et a/ 1991 ). P loss from the fungus into the periarbuscular space appears to be the most unusual step in the hyphal P transport from soil to plant and could be an important determinant for the P transport efficiency of VA mycorrhizal fungi (Smith and Smith 1990). The active interface is another probable determinant of efficiency (Smith and Dickson 1991). Although it is questionable whether variation in efficiency of P transport by different fungi would be caused by a single factor, biochemical marker would be useful to evaluate the functional state of VA mycorrhizas. Alkaline phosphatse in the intraradical structure is a possible marker of symbiotic efficiency in terms of P transport as its activity increases markedly during early stage of root colonization (Gianinazzi-Pearson and Gianinazzi 1978). They were also present in the vacuoles of mature hyphae and arbuscules, corresponding to the distribution of polyphosphate granules (Gianinazzi and Gianinazzi-Pearson 1979). eDNA that encodes a transmembrane phosphate transporter (GvPT) (Harrison and Buuren 1995) have been identified and sequenced from Glomus versiforme. The functioning of the protein encoded by GvPT was confirmed by complementation of a yeast phosphate transport mutant. Expression of GvPT was localized to the external hyphae of G. versiforme during

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mycorrhizal association, these being the initial site of phosphate uptake from the soil. An increased enzyme activity of alkaline phosphatase was succeeded by mycorrhizal growth response (Tesserant et a! 1993). eDNA clones, MtPT1 and MtPT2 encoding phosphate transporters from Medicago tnmcata colonized root by Glomus versiforme have been identified (Liu et a/1998). The eDNA represent M truncata genes and the encoded protein share identity with high affinity phosphate transporters from Arabidopsis, potato, yeast, Neurospora crassa and an AM fungus G. versiforme.

Experiments with N 15 -labeled nitrate indicated that this anion is taken up by Glomus mosseae hyphae (George et a! 1992) and is transferred from the fungus to the plant under water stress conditions (Tobar et al1994b ). Earlier investigators (Carling eta! 1978; Oliver eta! 1983) demonstrated increased total activity of nitrate reductase (NR) in both roots and shoots of AM plants and concluded that this enhancement is brought about by a relief of phosphate stress. Nitrate might be the preferential nitrogen source for AM fungi (Azcon et a/1996). First demonstration of differential formation of transcripts of a gene coding for the same function in both the symbiotic partners (Kaldorf et a! 1998) indicates that nitrate formation catalyzed by NR was mainly NADPH-dependent in roots of AM colonized plants but not in those of controls, which is consistent with the fact that NRs of fungi preferentially utilize NADPH as reductant. The fungal NR and mRNA was detected in arbuscule but not in vesicle by in situ RNA hybridization experiments.

Protein synthesis is enhanced in relation to fungal colonization development. Electrophoretic analysis of soluble extracts from mycorrhizal roots has shown that the host plant produces a number of new proteins, which have been called endomycorrhizins, in response to colonization by the fungal symbiont, but their function has not yet been determined (Dumas et a! 1990; Wyss et a! 1990; Schllenbanum et a! 1991 ). Detailed investigation using bidimensional PAGE (Tahiri-Alaoui 1992) or in vitro translation of total RNA (Garcia-Garido et a! 1993) has shown that not only a new polypeptides synthesized during AM colonization but quantitative changes also occur, and reduction in the expression of certain host genes leads to the disappearance of some polypeptides. Reduction in gene expression is frequently overlooked or considered unimportant in studies of plant-microbe interactions, it may represent an underestimated component of the morphological and physiological processes involved in the establishment of compatible endomycorrhizal associations (Gianinazzi-Pearson et a/1995).

Cell wall degrading enzymes

Fungi synthesize a variety of cell wall polysaccharide and specific combination of major wall polymers have been used in classification of systematic analysis (Bartnicki­Garcia 1968, 1970, 1987). With the exception of the Entomopthorales, zygomycetes are

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characterized as having a chitosan-chitin wall (Bartnicki-Garcia 1987 ~Lewis 1991). The presence of chitin oligomers in the spore and hyphal wall of some Glomus and Gigaspora species have been shown by both affinity, cytochemistry and biochemical analysis (Bonfante-Fasolo et al 1986~ Grandmaison et al 1988~ Bonfante-Fasolo et al 1990). However, the possibility of additional glucan component have been suggested by Bonfante­Fasolo et al (1990). Fluorescence, electronmicroscopic and cytochemical studies (Varma 1999a) indicated that the blocking ofthe fungal chitin-synthase activity induces alteration in hyphal morphology, a reduction in fungal wall thickness, and several other changes in the hyphal wall structural organizations. Examination of the ultrastructure and chitin WGA gold specific labelling on Gi. margarita hyphae revealed that Nikkomycin Z induced a noticeable reduction in the wall thickness in the hyphae that was proportional to antibiotic concentration. In contrast, the cell wall of G. intraradices showed a very thick cell wall consisting of an electron-transport outer wall and multilayered inner one, with alternating electron-dense and electron-transparent layers (Bago et al1996a, b).

Plants have the ability to perceive and respond to signals resulting from pathogenic attack. This recognition suggests the induction of a large arsenal of defence measures that plant employ to protect themselves from the invading pathogen. The defense responses may be induced specifically, as in a gene for gene type of interaction (Flor 1971~ Keen 1990~ Martin et a/1993 ~ Staskawick et a/1995) or non-specifically by a range of biotic and abiotic elicitors (Darvill and Albersheim 1984~ Darvill et al 1992~ Rohwer et al 1987~ Templeton and Lamb 1988). Many fungal and bacterial pathogens secrete a large arsenal of hydrolytic enzyme that digest the plant cell wall allowing the pathogen to have access to nutrients (Salmond 1994; Walton 1994). Such enzymes can release plant cell wall derived elicitors that activate plant defense responses (Davis et al 1984~ Hahn et al 1981~ Nathnagel et al 1983). How the molecules interact with the plant cells to induce the defense mechanism is not clear, but a number of studies have identified putative elicitors-binding receptors in the plasma membrane from different plant cells (Cossio et al 1992~ Ebel et al 1993, 1995~ Farmer et a/1991).

Most of the inducible plant defense mechanisms include both local and systemic transcriptional activation of defense genes. Local defense responses are thought to have the direct effect on the pathogens and they involve chains in ion fluxes across the plasma membrane, generation of active oxygen species (Atkinson 1993~ Medhy 1994), the hypersensitive response (Klement 1982), the synthesis of several proteins, biosynthesis of phytoalexins and pathogenesis-related (PR) proteins. PR proteins accumulate as a result of infection by pathogens or after treatments with elicitors (Linthorst 1991; Stintzi et al 1993;

' Van Loon 1985; White and Antoniw 1991). Some PR proteins appear to have protective properties against the pathogens (Alexander et al 1993; Broglie et al 1991; Mauch et al 1988), but in many cases no direct function has been assigned (Neuhaus et al 1992). Depending on the pathogen a local infection can also lead to the activation of defense

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response in the uninfected parts of the plants. An example of this is the systemic acquired resistance (SAR), which is characterized by a general, induced disease resistance in uninfected parts of the plant and is effective against a broad range of pathogens (Mal amy and Klessig I 992; Ryals et a/ I 994). SAR is associated with the systematic coordinated synthesis of a large number of PR proteins, especially the acidic (extracellular) isoforms (Uknes et a/1992; Word et a/1991). The correlation between PR protein synthesis and the acquired resistance suggest a direct involvement of these proteins in plant defense against pathogens (Bol et a/ I 990). The penetration of root by AM fungi involves cell-wall degrading hydrolytic enzymes (Bonfante et a! 1990; Bonfante and Perotto 1995; Bonfante 1997). These enzymes may lead to an organized colonization of the root (Blilou et a! 1996). There is agreement that most hydrolytic enzymes from higher plants differ from those of microbial origin in optimum pH: the optimum pH for plant hydrolytic enzymes is in the acid range (Rexova-Benk:ova and Murkovic 1973). Biotrophic fungi are usually thought to penetrate host tissue mechanically through formation of a penetration peg "appressorium". Some wall components such as melanin are considered to play an important role in the increase of hydrostatic pressure, since they act to trap solutes within appresorium causing water to be absorbed because of the increasing osmotic gradient. In contrast, in many pathogenic interactions, however, the infection process is under the control of cell wall degrading enzymes, since the plant cell wall, the first barrier to be overcome, may be partially degraded by enzymes of microbial origin; among these polygalacturonases are considered to be important determinants of pathogenicity (Walton I 994). In biotrophic fungi, the infection process is carried out by low and regulated production of key cell wall degrading enzymes by the fungus. Investigations have demonstrated the production of Pectinase, Cellulase, X ylanase and Chitinase ( Garcia-Romera et a/ 1990, I 99 I a, b; Varma and Bonfante 1994; Perotto et al1995a, b, I 997) from the hyphae and mycorrhizal roots. It seems that mycorrhizal fungi colonize the root tissue of their host plant by combination of mechanical and enzymatic mechanisms (Bonfante and Perotto 1995; Gianinazzi-Pearson et a/ 1996). Very weak and localized production of enzymes might ensure that viability of the host is maintained, defense responses are not triggered and a high degree of compatibility is reached (Varma 1999a).

The composition of dicotyledonous cell wall is typically 25-40% cellulose, I 5-25% hemicellulose, 15-40% pectic materials , 5-10% protein (hydroxyproline-rich glycoproteins and enzymes) and a very small proportion of phenolic compounds (Bartnicki-Garcia 1987; Bonfante 1988). The cell wall comprises a crystalline microfibrillar array of cellulose embedded in an amorphous mass of pectic and hemicellulose materials. The AM fungi hydrolyze these cellular complexes in a very organized manner to make their entry into the root cortical cells: They induce and/or activate a combination of enzymes to stimulate the targets (Saito 1996).

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Endoglucanase (E C 3.2.1.4.) was purified from roots of onion colonized with Glomus mossea. The endoglucanase have a relative molecular weight of about 27 kDa (Garcia-Romera eta! 1996). Application of extracellular cellulase from Aspergillus niger increased the growth of mycorrhizal inoculated plants of the family Crucifereae and Chenopodiaceae (Blilou et a/1996), cellulases have been shown to increase the germination and hyphal growth of AM spores (Persad-Chinnery et a! 1992). Xyloglucan-specific endoglucanase activity was reported from G. mosseae during the process of penetration and colonization of the host Allium cepa (Rejon-Palomares et a!I996b ).

The importance of hydrolytic enzymes in the degradation of the plant cell wall by many microorganisms is well documented. The production of hydrolytic enzymes has been observed not only in parasites but also in mutualistic organism such as Rhizobium (Mateos et a/1992) and arbuscular mycorrhiza (Garcia-Romera et a/1990; Rejon-Palomares 1996a). There is evidence that hydrolytic enzymes including hemicellulases are involved in the colonization of roots by the fungi ( Garcia-Romera et a! 1990, 1991 a; Garcia-Garido et a! 1992a, b, c).

Pectin is an important component of plant cell wall, but degradation of this polysaccharide has been reported only for a sterile ericoid mycelium isolated from Calluna vulgaris by Cairney and Burke (1996). Polygalacturonase seems to be an important component of the enzymatic arsenal secreted by ericoid fungi during saprophytic life. In addition, they could also play a role during root colonization, since penetration of the plant cell wall is prerequisite for the establishment of endomycorrhizal symbiosis. Information on pectin degradation is a missing link in AM fungi, except for a brief report by Garcia­Romera eta/ (1991b, 1996), who claimed to detect a complex of pectinase and cellulase enzyme from spores and external mycelium of G. mosseae colonizing the roots of Lactuca sativa.

Saprophytic fungi promoting t4e plant health

Very little work has been done on the plant growth promoting activity of rhisphere fungi. So far only a -few fungi like Trichoderma harzianum, T. koningii, non-pathogenic strains of Rhizoctonia so/ani, Phytopthora parasitica and sterile dark fungus (Jariwala and Rai 1998; Narita and Suzui 1991) have been reported as plant growth promoting fungi (PGPF). Preliminary studies revealed that several fungi including species of Phoma, Trichoderma and_Penicil/ium and also non- sporulating fungi isolated from zoysiagrass (Zoysia tenuifolia (wild.) Thiele.) rhizospheres promoted plant growth and suppressed soil­born fungal disease of a number of crop plants (Hyakumachi I 994; Shivanna et al 1994; Shivanna et a/1995a, b). Interestingly, all these PGPF were also found to be endowed with

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the ability of disease suppression of 'take-all' and common root rot of wheat (Shivanna et a/1996).

Piriformospora indica a new mycorrhiza like-fungus

A root endophyte was found (gold strike by chance) in the rhizosphere of spineless cacti and Cenchrus sp (desert grass), in sand desert of interior of Rajasthan, which was called for its characteristic spore morphology, Piriformospora indica (Verma et a/1998). A similar kind of fungus has been detected from the rhizosphere soil of Kallar grass and rice rhizosphere from Pakistan and Phillipines, respectively. However, none of the European soils tested were found to contain this fungus. Recently, fungus identical toP. indica was detected from the rhizosphere soil of Eucalyptus in the foothills of Himalayas- a semi temperate climate (personal communication with Prof B. Johri). In another communication Dr. A B Khan, New Southwales, Australia has described (personal communication) a fungus similar in description to P. indica. Although they have considered their isolate as one of the species of Glomus, however, to us it looks a prototype of P. indica. Geographical occurrence of P indica is shown in Plate I. The culture of Piriformospora indica was patented. Culture is deposited at; DSMZDEUTSCHE SAMMLUNE VDN, MIKROORGNISMEN UND ZELLKULTUREN GmbH, MascheroderWeg lb, 0-38124, Braunschweig, Date: 1997-10-24. The patent number is B 2822 PCT.

The fungus grows on a wide range of synthetic and complex media, e.g., on minimal medium normally used for in vitro germination of AM fungi with 1.0% sugar or glucose as carbon source, on two different media for Aspergillus sp (CM1 and MM2) and on Moser-B medium Plate II, Fig. a. Mass cultivation of the fungus can be easily achieved on simplified broth culture Plate II, Fig. b. Recently the fungus was grown on a typical pathogen (Gaeumannomyces graminis) medium containing inorganic nutrients and yeast extract with sugar as the carbon source. Optimum pH was 5.8 (4.8- 6.8) and temperature around 30 °C (25- 35 °C).

Significant quantitative and morphological changes have been detected, when the fungus was challenged to grow on different media. The incubation in the shaking state retarded the growth in MM liquid cultures, whereas no such negative effect was ever observed during cultivation in any other substrate. In fact the fungal biomass was considerably enhanced on shaking cultures with aspergillus (CM) medium. On Moser-B medium, the colonies appeared compact, wrinkled with furrows and constricted. The mycelium produced defined zonation and high amount of white aerial hyphae.

The hyphae were highly interwoven, often adhered together and gave the appearance of simple cords Plate III. Young mycelia were white and almost hyaline (Plate IV, Fig. b), but inconspicuous zonations were recorded in older cultures. Hyphae were thin walled and

14

Plate 1: Eco-geographical occurrence of Piriformospora indica

Figure shows the geographical distribution of P. indica in different continents. The new. fungus was found in India, Pakistan and Philippines. In vitro PCR hybridization analysis showed the absence of the fungus from Europe, Marburg (Germany). Unpublished data

y ~~<·Jlll •m.l

Marburg •1<(!1'!/M' ¢.10to:l!lt!'*Mf

tdt~oJtf

•

Plate 1

Lvzon

San Fernando MA~-Ov•z.o;o

Batqa,.

MllldOtU Samar

Plate ll : Axenic culture of P. indica

Fig. a, growth pattern of mycelia formed on aspergillus synthetic medium in 90 mm petriplate, after incubation for twenty days at 25 ± 2 °C. Fig. b, fast growth of fungal biomass in Erlenmeyer flasks on a gyratory shaker (125 rpm) maintained at 25 ± 2 °C

Plate II

Plate ID: Spore germination

Plate shows the cluster of spore germination and intermingling of hyphae

Plate Ill

Plate IV: Morphological characterization of Piriformospora indica

Fig. a, sporulation of P. indica on minimal agar media; the inserted sector shows the characteristic pear shaped spores with multilayered structure. Fig.b, highly anastomised and intertwined hyphae. Fig. c, typical dolipore septum with continuous parenthosomes a characteristic feature of the members of basidiomycetes

Plate IV

of different diameter ranging from 0.7-3 .5 11m. The septate hyphae showed sometimes anastomosis. New branches emerged irregularly arid the hypha! wall showed some external deposits at regular intervals, perhaps polysaccharide and /or some hydrophobic proteins, deeply stained with toluidine blue. Since septation was irregular, the single compartment could contain more than one nucleus.

Clamydospores were very sticky (almost glued) and it required drastic and vigorous physical treatments to segregate them. The submerge mycelium produced on MM1, CM, and MM2 also produced chlamydospores. The Chlamydospores were scanty and often in loose clusters. Schematic diagram of the pear shaped chlamydospores are shown in Fig. 2.

Chlamydospores were formed from thin-walled vesicles at the tips of the hyphae Plate IV, Fig. a. They appeared singly, or in clusters. These chlamydospores were distinctive due to their pear shaped structure measuring 16-25 11m in length and 10-17 11m in width. Sometimes they were produced in chains within the hyphae. Very young spores have one­layered thin, hyaline walls. At maturity, they appeared thick (1 .5 11m), double walled, smooth and pale yellow. The cytoplasm was densely packed with granular materials and usually contained 8-25 nuclei (Plate V, Fig. a). Neither clamp connections nor sexual structures could be observed. Spores germinate at 36 °C (30-40) within 6-10 hours, producing one single main germiling, followed by several short side branches.

The walls were very thin and showed multilayered structures. The septal wall consisted of dolipores (Plate IV, Fig. c), surrounded on two sides by the continuous parenthosomes (see arrow), which proofs the systematic position within the Hymenomycetes. The dolipores were very prominent with a multilayered cross wall and a median swelling mainly consisting of electron transport material. The parenthosomes were always straight and had the same diameter as the corresponding doli pore. Any kind of pores could not be detected, this means that they are flat disc without any perforation. The parenthosomes consisted of an electron dense outer layer and a less dense inner layer, which showed an inconspicuous dark line in the median region.

Most of the plant tested were colonized by P. indica . The hyphae first colonize the surface, later enter the cells (Plate VI, Fig. a), traverse through the cells and produce structures similar to appressoria, arbuscules and vesicles, normally observed for AM fungi. At maturity external and internal spores were produced (Plate V, Fig. b and Plate VI, Fig. b) .

15

Fig 2: Piriformospora indica- spores

Schematic representation of the multi-layered (2-4 ), multi-nucleate (8-25) and pear shaped spores of Piriformospora indica, attached with hyphae

Plate V: Sporulation inside the root

Fig. a, colonized root stained with acridine orange to observe the number of nuclei into the spore. Each spore contains 8-25 nuclei . Fig. b, transmission fluorescent microscopy of colonized root. Spores were seen inside the host tissue and their nuclei fluoresced greenish­yellow

Plate V

Plate VI: Root coloniz~tHHi

Fig. a, Intracellular branclHh.g ahd coilihg of hyphae (see arrows) . Fig. b, colonized root showing spore formatidh ih chain dn the root surface

Plate VI

Molecular studies

In order to get a more precise idea about the closest relatives of P. indica, part of 18S rRNA was amplified, sequenced and compared with all coresponding data of different Basidiomycetes available in the gene bank sequence library, as well as of some Ascomycota and Basidiomycota. Based on these results a second analysis was carried out using only two out groups (one Asomycete and one Zygomycete) and only those Basidiomycota which contribute to the understanding of the evolutionary relationships of the new fungus . The dendrogram (Plate VII) mirrored the classification within Basidiomycota into different orders with the exception of the Aphyllophorales and Agaricales which were grouped together. The lowest evolutionary distance of the 18S rRNA sequence of the new fungus appeared to be the members of rhizoctonia group (Ceratobasidiales), namely Rhizoctonia so/ani and Thanatephoros praticola. The significance for a common branch shared by those fungi and P. indica in this reconstructed phylogeny had a bootstrap value of 61%. When the same analysis was carried out without the rhizoctonia group, the new fungus does not match up with any other species, but occupied an own branch. The further analysis neighbour­joining phenogram resulting from analysis of SSU rRNA gene sequences of fungi was carried out with 927 bootstrap. The taxonomic status of the new fungus did not alter.

The new fungus only produces chlamydospores at the apex of undifferentiated hyphae. Different kinds of substrate were tested to induce sexual development, like young and mature hemp leaves, young leaves of Cyanodon dactylon and pollen grains, oat meal, potato carrot, potato dextrose or tomato dextrose agar. Since all these efforts did not lead to desirable results, there were only a few features to characterize the fungus morphologically and to group it according to the classical species concept.

There is no existing genus which covers all the characters of the new fungus . Other groups ofBasidiomycota which may be related according to the molecular or ultrastructural data are not known to be able to produce chlamydospores or have a different mode of life. Therefore a new genus was erected and the fungus was called Piriformospora indica (Verma et al 1998). PCR analysis for taxon specific detection of P. indica in roots and shoots of rice with 18s rRNA-targated primers Prf and Pirrev, Plate VIII demonstrates the absence of P. indica in shoot of rice.

P. indica improves the growth and over all biomass production of different grasses, trees and herbaceous species (Varma et a/1998b; Sahay et a/1998). More recently we have identified a model plant which belongs to mono.cot, Settaria italica, the smallest genome next to rice for future studies. The roots were colonized and the plants highly promoted by interacting with P. indica. The new fungus also promotes several tropical legumes tested. Interestingly, like AMF the new fungus did not invade the root of Brassica sp (member of

16

Plate VU: Dendrogram for Piriformojpora indica

Neighbor joining phenogram resulting from analysis of SSU rRNA gene sequences (927 position) of fungi. Chytrids and zygomycetes were used as an outgroup. Bootstrap confidence value greater than 50 per cent are indicated at internodes. Topologies that are supported by more than 95 per cent are depicted in bold

83

99

,,._ __ Agaricus bisporus Cyatus striatus

Pluteus petasatus Albatrellus syringue

~111:801-- Phlebia radiata Gloeophyllum sepiarium Rhizoctonia solani

saccatum too Pseudocolus fusiformis

L--;;iC=~D~acrymyces chrysospermus 99 Heterotextus a/pinus

LiOO~==n Filobasidiella neoformans 100 Tremela sp.

Hyphomycetes

L---iOOC======~C~ro~nanium ribicola 100 Leucosporidium scottii

r _ _!lOO~==~Candida albicans Saccharomyces cerevisiae

elata Li;~====-;: Colletotrichum gloeosporioides

100 Leucostoma personii ..--------Glomus intraradices

._-t Acaulospora spinosa .._ __ Gigaspora margarita

._ ________ Chytrium confervae

..----------- Blastocladiella emersonii '~·----------------- Mucor racemosus

9.02

Plate VII

Basidiomycetes

Ascomycetes

Chytrids +

Zygomycetes

Plate Vlll: Taxon-specific PCR demonstrates absence of P. indica in shoots of rice

PCR "'nalysis revealed the specific detection of P.indica in roots (lanes 1-2) and shoots (3-4) of rice with 18S rDNA-targated primers Pirf and Pirrev (A). Plants from the P32

experiment were analyzed. Control PCR with generall8S rONA-targeted primers NSl and NS2 (B). Lanes 1 and 3, uninoculted plants, lanes 2 and 4, rice inoculated, lane 5, P. indica; lane 6, Acremonium alternatum, lane 7, water; lane 8, marker

---636 bp

Plate VIII

Brassicaceae) and the myc- mutants of Glycine max, (seeds obtained from Professor Peter Gresshoff, Knoxwille, USA).

Immunofluorescence and ELISA tests were carried after making the polyclonal antibodies from P. indica. Abs was raised from the crushed mycelium and spores. Immunofluorescence studies indicated the recognition of the P. indica and an affinity with the spores of Glomus intraradices. The antigenic similarities was shown with most of the members of Zygomycetes tested, with lesser cross-reactivity with members of Ectomycorrhiza (basidiomycetes) and the least with other fungi belonging to Ascomycotina and Deuteromycetes (Varma et a/1998c).

Stimulatory factors

Freshly harvested P. indica hyphae were washed with water and aliquots (lg fresh weight) were washed twice in 5 ml 80% aqueous methanol. The supernatant was used for analytical HPLC analysis. The analysis showed seven peaks in the hyphae and one main peak in the culture filtrate. Preparative HPLC analysis of hyphae and culture filtrate were done. The major compound detected was benzoic acid . Function of this compound is not clear. Benzoic acid was also the main component in the culture filtrate. At this stage of our knowledge we do not know the stimulating factor which promotes the plant growth. This is under investigation.

Freshly harvested whole roots of different plants were washed with water and aliquots were treated twice in 5 ml of 80% aqueous methanol. The supernatant was used for HPLC analysis. The cyclohexomone derivative blumenin accumulated in roots of cereals colonized by arbuscular mycorrhizal fungi was also present here. The exact function of accumulated cyclohexomones is still not known. It is speculated that these secondary compounds might be involved in the regulation of mycorrhizal colonization. HPLC analysis of methanolic extracts from six week old infected and not infected plants showed quantitative but not qualitative changes in the root samples as a result of interaction of P. indica with barley, maize, and fox tails (Setaria italica) roots. No changes were recorded for the host rice and wheat. The UV spectra, obtained from HPLC photodiode array detector showed a cluster of peaks between 7. 5 and 12.5 min of the HPLC chromatograph on interaction of maize, barley, rice and fox tail with P. indica, indicating the presence of indole-derivatives, e.g., tryptophane, tyrosine and their derivatives.

Carbon and phosphorus are the two essential nutrients which mycorrhizal fungi are considered to transport and assimilate. In new fungus, the ~hosphate translocation to carrot root organ cultures and intact rice plant was tested. When P 2 was incorporated, root became radioactive, similar to carrot roots incorporated with P32 was also accomplished by hyphae of P. indica. With rice plants the radioactivity appeared in shoot within 48 hours. Addition

l7

of sucrose to the fungal compartment drastically increased P- transport to the plant indicating some plant-dependent factors play an important role in hypha! P-transport. P transport is an active process, dependent on sucrose (Sudha 1998). These results suggest that more fungi than previously thought may assist in acquisition of P to plants.

Biological control of disease

Roots extracts of maize showed a significant presence of cyclic hydroxamic like D IMBOA (2, 4-dihydroxy-7 -methoxy-1, 4-benzoxazin-3-one) or D IBOA (2, 4-dihydroxy-1, 4-benzoxazin-3-one). Very low amounts were present in wheat and none in rice, barley and Setaria. As a result of the maize root colonization by P. indica, we obtained an enhancement of different benzoxazinone levels. HPLC analysis of methanol extracts of infected maize roots showed eight different peaks with photodiode array detector with typical UV spectra from benzoxazinone derivatives. Four of these peaks were strong, increased 2-3 folds in the colonized roots compared to the uninfected roots. It is a kind of chemical plant defense against fungal virulence. None of these peaks were identical to DIMBOA or DIBOA. The identification of chemical structure of these compounds is in progress.

P. indica was challenged with a virulent root and seed pathogen Gaeumannomyces graminis. They did not inhibit the growth of each other, a distinct demarcation zone was observed (Plate IX). In another experiment, when P. indica was allowed to grow before and the pathogen was placed later in the middle, the pathogen growth was completely blocked. The cultured filtrate also completely stopped the growth of the pathogen. These experiments indicate that the new fungus has potential to act as biological agent for the control of root disease, however, the chemical nature ofthe inhibitory factor is still not known.

P. indica was colonized with tissue culture raised plantlets of Bacopa monniera (medicinal plant), tobacco and brinjal. In general, the rate of plantlets survival was much higher than the untreated controls (unpublished work by author). Higher biomass was also recorded in the treated plants. A similar positive effect was seen on treatment with the low amounts of culture filtrate.

Comparative study with arbuscular mycorrhizae

A comparative analysis of the present fungus P. indica was made with established arbuscular mycorrhizal fungi. It seems the new fungus is close to AMF in morphology, functions, and serological features. But the sequence analysis of 18s rRNA is different, warranting to place the new fungus in Basidiomycetes instead of in Zygomycetes. Its similarity with AMF is represented in Table 1.

18

Pta•e \X~ Biological control

Piriformospora indica showing inhibition zone against A.\pergillus ~ydowi (Cultures were grown by Miss Archana Singh and photographed by Prof A. K. Sarbhoy, IARI, New­Delhi)

Plate IX

Table 1: Comparison of Piriformospora indica with AMF

Geographical distribution

Axenic culture

Morphology hyphal strands hyphae (diameter) spore (shape) spore (colour) no. of nuclei/spore doli pore parenthosome sexual stage

P. indica

India, Pakistan, Philippines

yes

undulating 0.7-3.5j.tm pear shaped faint yellow to golden

8-25 present present absent

AMF

ubiquitious

no

straight 10-20j.tm globose golden yellow > 1000

absent absent absent

Systematics Zygomycetes (based on ELISA) Zygomycetes Basidiomycetes (based on 18s rRNA sequencing)

Root colonization aerial portion colonization extramatrical hyphae appressorium sporulation (in root)

Colonization in crucifers

Enzymes chitinase mannase glucanase ferulase laccase tyrosinase

yes absent present yes yes

no

? ?

detected detected detected detected

yes absent present

yes yes

no

detected detected

? ? ? ?

amylase proteinase peroxidase catalase polyphenol oxidase polymethylgalacturonase

Phytohormone

Biological control agent for plant disease

Plant defense compounds

Mycelial HPLC benzoic acid hydroxamic acid

detected no no no no no

yes

yes

hydroxy amino­acidDIMBO, DIBOA

analyzed yes yes

Myc- mutants G~vcine max Pea

root colonization absent root colonization absent

Table I. contd.

? yes yes yes yes yes

yes

yes

callose, phenolic compounds, PR­protein, silicon

not analyzed not known not known

root colonization absent root colonization absent

In viro application of AMF in micropropagation

Tissue culture has been an essential tool in the field of plant pathology for many decades. White and Braun ( 1941) for the first time used the tissue culture technique for the induction of tumor by Agrobacterium tumejaciens in sunflower, that was devoid of viable bacteria. Carlson (1973) was first to demonstrate that tissue culture could be used to select for pathogen resistance in plants. Using an structural analogue of the fire blight toxin, he selected resistant cells, developed callus and eventually regenerated whole plants that were resistant to fire blight. A coat protein of tobacco mosaic virus was incorporated into tobacco cells, which endowed the plants with resistance to subsequent viral infections and significantly delayed disease development (Abel et al 1986). Recently, protoplast fusion technique have facilitated the transfer of resistance between plant species (Hampp et al 1998).

In addition to providing novel methods to develop disease resistant plants, tissue culture has become an important tool in the study of molecular aspects of plant/pathogen interactions. Plant cells react with environmental and pathogen-related stress with a variety of responses (i.e., changes in ion fluxes across the plasmamembrane, increased synthesis or activation of specific enzyme systems and increased production of defense- related products) the use of plant cell suspension cultures or individual protoplasts has enabled easier and more accurate monitoring of these cellular processes at biochemical level. Many of these biochemical processes can not be monitored in whole plants. Unlike intact plants, plant suspension cultures provide relatively homogenous population of cells that are immediately accessible to treatment with pathogenesis or pathogen-related products. Due to unculturable nature of arbuscular mycorrhizal fungi, we do not know the responses of these symbiotic fungi in tissue culture condition. To fill this gap, a new AMF like organism, which belongs to Basidiomycota, Piriformospora indica (Verma et al 1998) has been discovered. Culturable nature ofthis fungus and plant promoting effect (Varma et al 1998 a,b,c~ Sahay et a/1998) will be worth to study in vitro plant symbiosis.

Micropropagation is inherently a cleaner system for producing plants compared with traditional production methods, since the plants are grown in culture, disease are not transmitted from the field into the green house and on subsequent generations. A single disease free mother plant can theoretically produce unlimited disease free daughter plants · without the possibility of re-infection.

Research done at various universities indicates that it is possible to grow plants much more quickly to size than is traditionally seen in nurseries today, regardless of how the plants are propagated. Such rapid growth requires optimization of all growing conditions, including nutrients, light and temperature. However, it is amazing to note that the results can be achieved with even modest adjustments of growing practices. Several

19

field growers are now producing well formed, small branched trees of Prunus "K wanzan", Morus alba 'chaparral' (mulberry), and others in one year instead of two. Blue berry plant production can be dramatically speeded up. Use of micropropagated cherry under stock yields increased the vigor and earlier fiuitset (and early pay back) for the orchardist (Vestberg and Estaun 1994).

Plant regeneration in vitro provides an alternative to root cutting for the propagation of woody plants. However, rooting of in vitro produced shoots is often the limiting step during propagation. Two patterns of adventitious root formation on cutting have been recognized in both herbaceous and woody plants. One consists of direct development of adventitious root primordia from cell associated with or in close proximity to the vascular system. The other is an indirect process in which adventitious root formation is proceeded by proliferation of un-differentiated cells, which usually starts in the parenchyma of epidermal cells, certain cells within the undifferentiated tissue then become organized and initiate an adventitious root primordium. In general, the direct pattern is found in herbaceous species and easy to root woody species, and the indirect pattern is difficult to root species. A diversity of factors control morphogenesis in vitro and no theory clearly explains all the responses observed.

The acclimatization phase raises problems concerning survival and development of plantlets. While cultural practices play an important role in the ultimate performance of any block of plant material, the two key attributes are much greater uniformity and low survival (50-600/o) rates. Low survival rate of micropropagated plant is a million dollar question. In recent years, the role of mycorrhizae in the out planting performance of plantlets/seedlings raised through tissue culture has been exploited (Bojan et a/1995). Varma and Schuepp (I 994, 1995) reported that the endomycorrhizal root colonization is affected by the host fungus combination in micropropagated strawberry, raspberry and hortensia. The effects ranged from mutualistic through neutral to negative. Mycorrhized micropropagated plantlets surviving acclimatization was 100% (Varma and Schuepp 1995). Out planting performance of V AM inocutated tissue culture raised sugarcane was 80% with Glomus aggregatum (Bojan et a/1995). A comprehensive review of the literature on the outplanting performance of inoculated seedlings by Castellano ( 1996) gives a ready references to the literature to reforestation workers and scientists studying the effects of mycorrhizal inoculation on seedling performance after outplanting in the field. Most of the micropropagated plants inoculated in ex vitro experiments are highly mycorrhiza dependent high value plants like grape vine (Ravolarina et a/1989, Schubert eta!, 1990, Schllenbanum et a/1991), oil palm (Bial et a/1990), apple (Branzati et a/1992; Vestberg and Estaun 1994 }, plum (Fortuna et a! 1992}, pineapple (Guillemin eta! 1992; Lovato et a/1992) and avocado (Azcon-Aguilar et a! 1992). Therefore, there is a high potential for introducing AMF into the micropropagation system of these plants. Micropropagation represents a potential application niche for arbuscular mycorrhizal (AM) fungi for the following reasons:

20

>- Micropropagated plants often show symptoms of vitrification (syn. hyperhydrocity), i.e., they have poorly developed cuticles and non-or partial functional stomata which results in drought stress at acclimatization in vivo.

>- Micropropagated plants, as a consequence of vitrification, can have poorly developed constitutive disease resistance, with hypolignification and reduced phytoalexine content.

>- Aseptic micropropagated plants represents "bilological vacuum" available for promiscuous facultative pathogens.

A prior inoculation with symbiotic fungi (AM) in vitro would appear to offer solutions to both the above quality issues in micropropagation and to problems in AM application, e.g., uncertainity regarding the influence of environmental microorganisms on the inoculation stage (Lovata et a/1996).

AM fungi have been reported to confer protection against biotic and abiotic stresses (Azcon-Aguilar and Bago 1994 ). Conventionally the exploitation in bio-protection has been by applications to seedlings and vegetative propagules. Inoculation is based on the incorporation of AM propagules in the region of plant roots. As the AM inoculum is usually produced on plant roots in the environment, it is non-sterile. Indeed, some of the contaminating microorganisms have been postulated to have a 'helper' role in mycorrhizae (Azcon-Aguilar et a/1986). 71/ _ 7 .3 7 g

The application of bio-protectant microorganisms, both biocontrol and growth promoting rhizosphere bacteria, mycorrhizal fungi and other soil born fungi to plants is the subject of extensive current research. Wang et a! (1993), for example showed beneficial effects in the weaning of micro plants of three ornamental species, Gerbera, Nephrolepis and Syngonium following inoculation with Glomus intraradices and G. vesiculiferum.

Salamanca et a! ( 1992) showed that inoculation with Glomus Jasciculatum to micropropagated shrub legumes Anthyllis cytisoides and Spartium juncium shortened their acclimatization process by eight weeks. These shrubs are used in revegetation programs for Mediterranean areas, and their shorter propagation cycle is of high value. Micropropgation is of increasing importance as an effective tool to multiply all kinds of agricultural and horticultural plants, or even plants used in regeneration and revegetation programs. Although micropropagation is a well established technique, each plant is a new challenge and specific protocol have to be designed to match its characteristics. Some of these plants have problems during the micropropagation process. That could be over come, atleast to some extent by introducing symbiotic organisms, (bacteria/fungi). Some woody plants difficult to root have been shown to improv~ their survival when inoculated with AMF.

21

Another problem that could be resolved by mycorrhizal symbiosis is the dormancy that some micropropagated plants (certain Pnmus and Malus root stocks) present once they have been acclimatized. Symbiotic fungi/bacteria have potential to improve the out planting performance, if they are colonized by these organisms under in vitro conditions, since in nature they will have to face not only physical stresses but also biological stresses.

The survival rates and initial '1ransplant shock" on transfer of the plantlets to field is very high. Often stunted growth leads to non-recovery of the plants and are attacked by soil fungi. Today about 50% flori-horticulture plants are produced by micropropagation techniques, but at weaning about I 0-40 % of plantlets either die or do not attain market standard, causing significant loss at the commercial level.

The technology used for micropropagating plants does not take into consideration the existence of mutualistic symbiosis of mycorrhizae and other associative plant growth promoting rhizobacteria (PGPRs) and symbiotic saprophytic fungi. The medium used were devoid of mycorrhizal propagules and therefore the plants obtained from these systems are non-mycorrhizal. Such non-mycorrhizal plants obtained from the micropropagation process eventually become mycorrhizal when they are planted in field soils. However, an early inoculation of these plants with selected mycorrhizal and associative bacterial/fungal inocula promises to improve plant survival, performance and allow lower chemical inputs. Steps in tissue culture where microorganism can be applied potentially to give beneficial/fruitful results are schematically shown in Fig 3. A glimpse of culture room and mist chamber used during the experimental period is shown in Plate X, Fig. a, b.

22

Fig. 3: Strategy to use microbes in tissue culture

I STAGE 11

Initiation of sterile culture from explant

? Pathogen 1 > <==:::11 Symbiont

(Phytopthora/tobacco)

jSTAGE2j

fSTAGE3j

lsTAGE4j

Multiplication of shoot/propagules

<.________, Symbionts (AMF/ __ _.I P. indica)

Rooting of shoots to produce individual plantlets

Symbionts (AMF/ <===:=J P. indica/ PGPRs)

Acclimatization of plantlets in green house (physical/

chemical/biological)

Schematic representation of potential use of symbiotic organisms in various steps of micropropagation to study their effect in tissue culture and plant-symbiont interaction

Plate X: A view of the tissue culture room and mist chamber

Fig. a, shows the culture racks equipped with fluorescent light. PR lamps were also provided in each rack. Figure shows a glimpse of the growing cultures. Fig. b, shows the growing plantlets in pots in the mist chamber

Plate X