REVIEW OF LITERATURE

Transcription

1 REVIEW OF LITERATURE The term mycorrhiza is defined as a symbiotic association of a fungus and the roots of a plant. Frank (1885) defined mycorrhiza as symbiotic associations between fungi and roots that are not pathogenic (i.e. intimate root fungus associations without disease symptoms). Mycorrhiza form mycelium and penetrate it in between cells and inside the cells. Based on their operation they are of three types, intraradical, periradical and extraradical. The intraradical phase is comprised of simple hyphae, with or without additional structures which occurs within the root epidermis and cortex, either between or within individual cells or just between them. The fungus is restricted to the cortex and epidermis. It does not cross the endodermis and enter the stele. The periradical phase is comprised of a layer of hyphae that surrounds the root like a sock or glove. The extraradical phase is comprised of hyphae which extend in typical mycelial fashion into the soil surrounding the root. Part of the extraradical phase may also consist of rhizomorphs, which are relatively tough aggregations of many hyphae. They often are visible with the naked eye. Mycorrhiza reproduces by asexual spores, called as chlamydospores which are produced within a fruiting body called as sporocarp. On the basis of anatomy, mycorrhizae are traditionally classified in two classes namely ectomycorrhiza and endomycorrhiza. However, over the years as more observations have been made and we come to know that this simple two part categorization cannot precisely describe the range of diversity in mycorrhizas. Both palaeobiological as well as molecular evidence of mycorrhiza indicate that AM fungi are an ancient group of symbiotic fungi that originated at least 460 million years ago (Simon et al., 1993). AM symbiosis is ubiquitous among land plants, which suggests that mycorrhiza were present in the early ancestors of extant land plants. This positive association with plants may have facilitated the development of land plants. The Rhynie chert of the lower Devonian period has showed the fossils of the earliest land plants in which AM fungi have been observed (Remy et al., 1994). The fossilized plants containing mycorrhizal fungi were preserved in silica. Plants of the Rhynie chert from the lower Devonian period i.e. 400 million years ago were found to contain structures resembling vesicles and spores of present Glomus species. Colonized fossil roots have 11

2 been observed in Aglaophyton major and Rhynia, which were ancient plants possessing characteristics of vascular plants with primitive protostelic rhizome (Remy et al., 1994). Intraradical mycelium was observed in roots and intracellular spaces and arbuscules were observed in the thin wall cells similar to palisade parenchyma. The fossil arbuscules appear very similar to those of existing AM Fungi. The cells containing arbuscules have thickened walls, which were also observed in extant colonized cells (Remy et al., 1994). The records of mycorrhiza from the Miocene period exhibit a vesicular morphology which closely resembles to that of present Glomerales. The need for further evolution may have been lost due to the readily available food source provided by the plant host (Kar et al., 2005). There was some fossil evidence which suggested that the parasitic fungi do not kill the host cells immediately after invasion, although a response to the invasion was observed in the host cells. This response may have evolved into the chemical signaling processes required for symbiosis (Remy et al., 1994). In both cases, the symbiotic plant-fungi interaction is thought to have evolved from a relationship in which the fungi was taking nutrients from the plant into a symbiotic relationship where the plant and fungi exchange nutrients. According to the fossil records the Glomeromycota seems to be an old group with an estimated origin of around 600 to 620 million years ago. Their evolutionary origin, therefore predates the split of the Ascomycota and Basidiomycota (Berbee and Taylor, 2000; Redecker et al., 2000). Spores and hyphae of glomalean fungi were discovered in 460 million year old rocks from the Ordovician period (Redecker et al., 2000), thus they were among the oldest recognized fungal fossils to date. The oldest arbuscules found thus far were in early land plants from the Devonian period, 400 million years ago; (Remy et al., 1994). The findings of Pirozynski showed that the Glomeromycota were present during the very earliest stages of the colonization of land by plants (Pirozynski and Malloch, 1975; Simon et al., 1993; Blackwell, 2000). Today AM fungi are found in most, major lineages of land plants. The arbuscular mycorrhizal fungi are placed in the taxonomic order Glomales which currently comprises six genera with species from the genus Glomus. Glomus like fungi are believed to have originated around million years ago based on fossil evidence (Taylor et al., 1995) and DNA sequence analysis of ribosomal genes (Simon et 12

3 al., 1993). Recognition of the presence of these symbioses occurred in the last century but subsequent investigation of their function only began in more recent times. The members of order Glomales are often called as VAM fungi because they often form so called vesicular arbuscular mycorrhizae, also sometimes called as endomycorrhizae. The members of Glomales form mycorrhizal relationships with most agronomically important angiosperms, almost all gymnosperms, as well as certain bryophytes, pteridophytes and even in few algae (Sieverding, 1991). An order Glomales comprises of three families, Glomaceae, Acaulosporaceae and Gigasporaceae (Morton and Benny, 1990). These three families include six genera; each family comprises of two genera. The family Glomaceae includes Glomus and Sclerocystis. The family Acaulosporaceae includes Acaulospora and Entrophospora. Whereas the family Gigasporaceae has two genera namely Gigaspora and Scutellospora. These fungi had formerly been placed in the order Endogonales of family Endogonaceae (Benjamin, 1979). The similarity in all the genera of AM fungi is as follows. 1. They are soil borne in nature. 2. They are obligate symbionts. 3. They produce vesicles as well as arbuscules. The difference between the genera of AM fungi is as under. 1. The genera like Acaulospora, Glomus and Sclerocystis forms sporocarp whereas Entrophospora, Gigaspora and Scutellospora do not form sporocarp. 2. Acaulospora, Entrophospora, Gigaspora, and Scutellospora forms azygospore, but it is not formed in Glomus and Sclerocystis. 3. The genera like Acaulospora, Glomus and Sclerocystis form chlamydospores which are not formed in Entrophospora, Gigaspora, and Scutellospora. 4. The genus Acaulospora forms both azygospores and chlamydospores. There are only six genera of fungi that contain species which are known to produce VA mycorrhizae with plants. However, not all of the species in each of these genera have been shown to form VA mycorrhizae (Morton and Benny, 1988). The genera are distinguished by their spore characteristics and the relationship of the spore to the 13

4 associated hyphal attachments. The existent genera were established to reflect the manner in which the spores were produced, except for the genus Scutellospora. Species in Scutellospora are distinguished from species in the closely allied genus Gigaspora by wall characteristics, method of germ tube formation and auxiliary cell characteristics etc. Many research workers, Schenck et al., (1984), Rothwell and Trappe (1979), Morton (1986), Trappe and Janos (1982), Schenck and Nicolson (1979), Trappe (1977), Ames and Schneider (1979), Spain et al., (1989), Schenck and Smith (1982), Walker and Rhodes (1981), Smith and Schenck (1985), Berch (1983), Thaxter (1922), Sieverding (1988), Tang and Zang (1984), Gerdemann and Trappe (1974), Mukerji et al., (1993), Trappe et al., (1984), Becker and Gerdemann (1977), Walker and Koske (1987), Skou and Jakobsen (1989), Walker (1982), Berch and Trappe (1985), Miller and Walker (1986), Hall (1977), Berch and Koske (1986), Tandy (1975), Bhattacharjee and Mukerji (1980), Rose and Trappe (1980), Koske and Halvorson (1989), Koske and Walker (1985,1986), Morton and Koske (1988), Ferrer and Herrera (1980) published illustrative literature on species of these genera. Many new species of AM fungi are reported from India. They are Sclerocystis sinuosa, (Gerdemann and Bakshi, 1976), Glomus reticulatum (Bhattacharjee and Mukerji, 1980) Gigaspora candida (Bhattacharjee et al., 1982), Glomus delhiense (Mukerji et al., 1993), Glomus sterilum, (Mehrotra and Baijal, 1992), Glomus bagyarajii (Mehrotra, 1997), Glomus hyderabadensis (Swarupa et al., 2004). Mycorrhizal fungi were classified by various workers in recent past. VAM fungi have two independent orders, (Morton and Benny, 1990) Endogonales and Glomales which were classified on the basis of their spore structure and mycorrhizal development. There are two systems of classification of mycorrhizal fungi i.e. old classification system proposed by Gerdemann and Trappe (1974) and the new or the present classification system proposed by Morton and Benny (1990). There are total 164 species of mycorrhizal fungi. Among known 164 species 111 species are reported from India (Mukerji, 1996; Bagyaraj and Padmavati, 1997; Mehrotra and Mehrotra, 1999; Manoharachary et al., 2002; Aggarwal et al., 2005). Before 1974 most arbuscular mycorrhizal fungi were included under the genus Endogone until Gerdemann and Trappe (1974) placed them in four different genera in the order Endogonales (Glomus, Sclerocystis, Gigaspora, Acaulospora). Morton and Benny 14

5 (1990) established a new order Glomales in the Zygomycota, comprising six genera. Since then evidence has accumulated supporting the view that arbuscular mycorrhizal fungi are distinct from Zygomycota. The classification proposed by Gerdemann and Trappe (1974) includes only one order, single family and eight genera. They included all mycorrhizal fungi in order Endogonales and family Endogonaceae. The genera included in family Endogonaceae were Endogone, Sclerogone, Glomus, Sclerocystis, Acaulospora, Entrophospora, Gigaspora, and Scutellospora. Morton and Benny (1990) classified the mycorrhizal fungi in one order, two suborders, three families and six genera. The classification system was as follows. Order: Glomales Sub-order: Gigasporineae Family: Gigasporaceae Genus: Gigaspora (Presence of inner membrane wall and giant spore in nature). Genus: Scutellospora (Endospore formation and germination shield on membrane wall). Sub-order: Glomineae Family: Glomaceae Genus: Glomus (Spore at the end of funnel shaped hyphae). Genus: Sclerocystis (Spores in asymmetrical fashion on a central sterile hyphal plexus). Family: Acaulosporaceae Genus: Acaulospora (Spores present laterally on swollen hyphae, sessile spores). Genus: Entrophospora (Form spores within the neck of hyphal terminus). 15

6 Schußler et al., (2001b) classified AM fungi based on their rdna phylogeny and proposed that AM fungi are the sister group of Ascomycota and Basidiomycota and not the monophyletic with any part of the Zygomycota. Therefore the Glomales was raised up to the rank of a phylum Glomeromycota (Schußler et al., 2001b). In the same study the grammatically incorrect order named Glomales was corrected to Glomerales and several new orders were established. It however must be emphasized that Glomales of (Morton and Benny, 1990) is synonymous with the Glomeromycota of Schußler et al., (2001b) and Glomerales of Schußler et al., (2001a) comprises a smaller subset of these taxa. In general molecular phylogenies have showed that glomeromycotan diversity at the phylum and genus level is much higher than expected through microscopic observation of spore morphology (Redecker et al., 2000; Schwarzott et al., 2001). Some of the morphological characters that were used previously to delimit genera and families probably have evolved multiple times independently. The classification of AM fungi proposed by Schußler et al., (2001b) is as follows. Phylum: Glomeromycota Order: Glomerales Family: Glomaceae Genera: Glomus, Pacispora, and Geosiphon. Order: Archaeosporales Family: Arcaheosporaceae Genus: Archaeospora Order: Paraglomerales Family: Paraglomaceae Genus: Paraglomus Order: Diversisporales Family Diversisporaceae Genus: Diversispora 16

7 Family: Gigasporaceae Genera: Gigaspora and Scutellospora Family: Acaulosporaceae Genera: Acaulospora and Entrophospora Above the genus level the family Glomaceae was erected by Pirozynski and Dalpe (1989). Gigasporaceae and Acaulosporaceae were established by Morton and Benny (1990). Morton and Redecker (2001) described new families Archaeosporaceae and Paraglomaceae to comprise deeply divergent lineages of AM fungi. Schußler et al., (2001b) divided the new phylum Glomeromycota into four orders Glomerales, Archaeosporales, Paraglomerales and Diversisporales. Walker and Schußler (2004) proposed new genus Diversispora, in family Diversisporaceae. The first molecular phylogeny of AM fungi was reported by Simon et al., (1993) using ribosomal small subunit sequences. These authors addressed the phylogenetic relationships among the three families known at that time and attempted to date their divergence by a molecular clock analysis. Morphological characters and fatty acid methyl ester profiles were evaluated phylogenetically by Bentivenga and Morton (1996). For instance phylogenies based on alpha and beta tubulin gene sequences were obscured by multiple paralogues (Corradi et al., 2004). A multigene phylogeny of fungi including basal lineages used nuclear ribosomal small, large subunit and 5.8 r subunit, tef, rpb1 and rpb2 sequences and placed the Glomeromycota as a sister group of Ascomycota and Basidiomycota (James et al., 2006). The Glomeromycota is supported consistently as a monophyletic group in phylogenetic analyses of ribosomal DNA and protein genes. Ribosomal RNA analyses place these species as a sister group of Ascomycota and Basidiomycota. Arbuscular Mycorrhizal fungi are key components of soil micro biota and obviously interact with other microorganisms in the rhizosphere which is the zone of influence of plant roots on microbial populations and other soil constituents. The microbial associates are more prone to general and annual fluctuations besides several abiotic factors such as environment and climatic factors which influence the whole 17

8 process of AM fungi and microbial interactions. These are ubiquitous in phosphorus deficient soil and form obligate symbiotic association with roots and other underground parts of most plants (Kunvar et al., 1999). The occurrence of vesicular-arbuscular mycorrhizae (VAM) has been reported from both terrestrial (Kendrick and Berch, 1985) and aquatic plants (Bagyaraj et al., 1979; Tanner and Clayton, 1985). During the development of infection, VAM fungi are known to produce two characteristic structures within the host roots viz., arbuscules and vesicles. Arbuscules are finely branched hyphae, which are now well known to be the major site of nutrient exchange between the host and the fungus (Smith and Smith, 1990). Members of Endogonaceae are widely distributed in agricultural and forest soils worldwide. Over 81 % of the shrub species in arid and semi arid areas of the world are endomycorrhizal (Venktesh et al., 2009). The mycorrhizal species are distributed almost in all kinds of soil. The black soils which hold more amount of water and remain in wet condition almost throughout the year show the association of mycorrhiza. The medium soil which has less water holding capacity show abundant amount of mycorrhiza as compared to black soil. The red soil is considered as good soils for cultivation of agricultural crops also possess many species of mycorrhiza. The west land in India is utilized for the cultivation of biodiesel plants like Jatropha curcas, Ricinus communis, etc. These plants show abundant mycorrhizal species associated with their roots. The coastal sand dunes exhibit favourable conditions for the association and development of arbuscular mycorrhizal fungi with plants since they are deficient in phosphorus (Ranwell, 1972; Koske and Halvorson, 1981). The importance of AM fungi for the growth and succession of plant species in coastal sand dunes was first recognized by Nicolson (1959). Aggregation of sand grains and colonization of AM fungi with dune plants significantly stabilize the sand dunes (Sutton and Sheppard, 1976; Koske and Polson, 1984). All kinds of soils like black soil, alluvial soil, clay loam soil, red soil, sandy soil etc. contain AM fungi. The chlamydospores of AM fungi are disseminated by various biotic and abiotic factors. The biotic factors include earthworm, ants and millipedes (Harinikumar and Bagyaraj, 1994), rodents may also be agents of dispersal as Endogone spores were shown to remain viable after passage through their alimentary tracts (Godfrey, 1957; David et al., 1995). The abiotic factors include air and water. Harinikumar and Bagyaraj (1994) 18

9 studied that earthworms, termites, ants, and millipedes contribute to the dissemination of vesicular arbuscular mycorrhizal (VAM) propagules. Interactions between mycorrhizal fungi and other organisms occur and may influence the function of the fungi. While grazing of mycorrhizal hyphae by fungivorous collembolan or springtails, may also disseminate mycorrhizal fungal propagules (Klironomos and Moutoglis, 1999). Other fungi and actinomycetes also play role in dispersal of AM spores (Fitter and Sanders, 1992). Some of the interactions among mycorrhizal fungi and other soil organisms have been summarized by Azcon and Barea (1992); Fitter and Sanders (1992). Arbuscular mycorrhizal fungi are commonly occurring soil microbes whose association with roots has numerous effects on growth of the host plant (Klironomos, 2003). There are many physical and chemical parameters which affect the colonization and sporulation. The factors like moisture content of soil, fertility of soil, temperature, ph, and soil type etc. matters most in AM spore colonization and sporulation. The autoecology of the arbuscular mycorrhizal fungi has been the subject of research for many years. The effect of soil ph, temperature and soil moisture on mycorrhization has been investigated by several workers (Lohman, 1927; Porter et al., 1987; Furlan and Fortin, 1973; Hayman, 1974; Reid and Bowen, 1979). Soil micro-organisms influence AM fungal development and symbiosis establishment. There were different responses recorded, some of them were positive (Meyer and Linderman, 1986; Bagyaraj and Menge, 1978; Gryndler et al., 1996), some of them were negative (Wyss et al., 1992; Mc. Allister et al., 1995) and other recorded were neutral (Edwards et al., 1998). Negative impacts upon the AM fungi include a reduction in spore germination and hyphal length in the extramatrical stage, decreased root colonization and a decline in the metabolic activity of the internal mycelium. Temperature and light greatly influence the development of vesicular-arbuscular mycorrhiza and growth of onion and other plants in a phosphate deficient soil, (Haymann, 1974). The size of arbuscules is more if the light is more and vice versa. Less light and low temperature do not stimulate growth of mycorrhiza although the soil is with low phosphate contents. Haymann (1974) recorded that there was more AM infection at 19

10 12 and 18 hours daylengths than 6 hour daylengths. The effect of infection increases in longer daylengths and higher light intensities. The effect of moisture on AM colonization and sporulation was positive as there was increase in moisture in soil (Lisa et al., 1988). If the moisture was too high the colonization and sporulation decreased and at least moisture or at the dried soils the spore count was relatively less. The percent colonization decreased as soil water availability decreased. The effect of ph and soil type greatly influenced the infection and spore count of AM fungi (Wang et al., 1993). The effects of greenhouse conditions are positive for the colonization and sporulation of mycorrhizal fungi. The plants grown in natural conditions if transplanted to the green house conditions along with the same soil where they had grown earlier showed more percentage root colonization and spore count than in natural conditions (Lansac et al., 1995). There was a positive relationship between root biomass and rootshoot ratio with mycorrhization percentage, and a positive response of water potential to that percentage also occurred. In the virgin soil i.e. the soil in its natural conditions do not show the AM spores but the agricultural practices increases the mycorrhizae qualitatively and quantitatively (Collins and Pfleger, 1992). The cropping sequence influences the population of AM fungi. The density of mycorrhizal spores decline when soils are kept uncultivated for long time (Black and Tinker, 1979; Yocom and Larsen, 1985; Thompson, 1987). The effect of heat on AM fungi was studied by Schreiner et al., (2001) and concluded that soil solarization i.e. the process of soil heating by covering fields with clear plastic, is a promising method to reduce populations of soil borne pests and weeds without the use of pesticides. However, the destruction of beneficial organisms such as arbuscular mycorrhizal fungi may also occur, thereby reducing positive effects of solarization. Solarization apparently reduces AM fungi in soil indirectly by reducing weed populations which maintain infective propagules over the winter. The fumigants like metam sodium and methyl bromide reduces the infectivity of AM fungi. Healthy, fertile and soils under cultivation are characterized by the presence of a diverse population of mycorrhizal fungi. Arbuscular mycorrhizal fungi are the most 20

11 common types of all mycorrhizae, which constitute a major group of soil microbial community (Linderman, 1992). AM fungal association is not restricted to the roots of plants only, but it is also found in all those organs of plants which are concerned with the absorption of substances from the soil. Srivastava et al., (1996) and Nasim (1990) reviewed the presence of AM fungi associated with the portions other than roots in 21 angiosperms and some non angiosperm species. All cultivated plants are associated with AM fungi. The application of pesticides like Afugan, Brominal, Gramoxone, Endosulphan etc. reduces the AM sporulation and root colonization in plants. The applications of fungicides like carbendazim, mancozeb, copper sulphate and aureofungin inhibits the AM colonization significantly, but the effect of fungicides reduces significantly with time. The effect of these fungicides has been studied in apple by Narender (2011). The effect of fungicide like Benlate has negative effects on the numbers of living internal hyphae, arbuscules, fungal-plant interface and living external hyphae in the soil. It markedly reduces the length of root in infected and uninfected onion plants (Sukarno et al., 1993). Mycorrhizal fungi are now days used as biofertilizers as it has been understood that they play vital role in plant life and in soil ecology. Biofertilizers are preparations which contain living microorganisms and increase microbial activity in the soil. Some of the important functions of soil microbes are as follows. 1. Convert ambient nitrogen into forms that the plants can use (Nitrate and Ammonia), 2. Increase porosity of soil. 3. Defend plants against pathogens by competing with pathogens for food. 4. Saprophytic fungi in the soil break leaf litter down into usable nutrients. The high soil porosity caused by microbes is important, because it aids water percolation. If pore spaces are too small, they cannot break the surface tension of a water droplet, and water will run off, instead of saturating the soil, where it can be taken up by plant roots. Chemical fertilizers are often over-applied, and end up polluting the water because they are not used up. The chemicals are less expensive in the short term, but 21

12 must be continuously reapplied, and are therefore more expensive over the long-term. A combination of chemical fertilizers and biofertilizers gives the plants a jump-start and maintains them until the microbes can get established. Mycorrhizal fungi form a bridge between the roots and the soil, gathering nutrients from the soil and giving them to the roots. Mycorrhizae also benefit plants indirectly by enhancing the structure of the soil. AM hyphae excrete gluey, sugar-based compounds called Glomalin, which helps to bind soil particles together, and make stable soil aggregates. This improves air and water percolation, as well as enhancing carbon and nutrient storage (Peters, 2002). Most natural, undisturbed soils have an adequate supply of mycorrhizae for plant benefits; however, the practices like erosion, grading, excavation, loss of original topsoil can reduce mycorrhizae populations to inadequate levels (Peters, 2002). Research on the potential value of arbuscular mycorrhizal fungi in agriculture and land reclamation followed from the discoveries in the 1950s, 1960s and 1970s that they could substantially increase P uptake and plant growth under certain circumstances. Arbuscular mycorrhizal fungi have an extraordinary importance as they increases nutrient acquisition by the plant as well as resistance to biotic and abiotic stresses (Barea and Jeffries, 1995; Barea et al., 2002). In fact, the symbiosis with arbuscular mycorrhizal fungi has been proposed as one of the mechanisms of heavy metal plant tolerance (Hildebrandt et al., 2007) and water stress avoidance (Ruiz-Lozano et al., 1995; Ruiz- Lozano and Azcon, 1996; Auge, 2004). The arid and contaminated soils are generally characterized by poor soil structure, low water holding capacity, lack of organic matter and nutrient deficient. Therefore, in order to carry out successful reafforestation, it is necessary to improve soil quality and the ability of the plant species to resist these harsh environments. In this respect, the application of organic amendments to the soil, prior to the inoculation of AM fungi, has been recommended (Medina et al., 2004 a, b). The beneficial effects of organic amendments include provision of plant nutrients, increased humus content and thereby increased water holding capacity, improved soil structure, and increased microbial activity (Caravaca et al., 2002). The nutrient most commonly associated with mycorrhizal benefit is phosphorus (P) which is highly immobile in most of the soils and limiting to plant growth and reproduction. The main areas where the benefits of introducing inoculant of AM fungi 22

13 into a plant growth system will increase the growth of plant where they are lacking indigenous inoculum of AM fungi, the effect is subsequently to increase early growth and nutrient uptake by phosphate. This uptake and transfer of mineral elements is done by the AM fungi once established in agro-systems (Dodd, 2000). It is well-documented that high inputs of chemical fertilizers especially phosphates and high nitrates along with certain fungicides like benomyl and soil sterilants have negative effects on AM fungi. Stressed and arid areas are the result of progressive degradation of vegetal cover i.e. species diversity and soil quality, both of which involve soil structure, nutrient availability and microbial activity (Barea and Jeffries, 1995). The establishment of a plant cover is most effective strategy for reclaiming degraded lands in semiarid mediterranean areas. Revegetation programmes based on planting drought tolerant, native shrub species would assist in the conservation of biodiversity and help to prevent the process of erosion and desertification (Caravaca et al., 2003). This is because of AM fungi which harbors the growth of plant and improve soil quality. The vegetal cover helps to avoid soil loss, together with improving physical properties of soil. However, in these semiarid areas the low productivity and fertility of the soil and the severe water deficits seriously limit plant growth. The establishment of a mycorrhizal symbiosis can improve the performance of the shrubs. Mycorrhiza represents ecological key factors governing the cycles of major plant nutrients and has significant influence on plant health and productivity (Jeffries et al., 2003; Requena et al., 2001). On the other hand, the quality and productivity of degraded soils can be improved by organic amendments (Roldan et al., 1996). Desertified areas are often facing the severe climate with little and irregular precipitation and frequent drought. Therefore, the main objective in restoration purposes is the improvement of plant drought tolerance. The osmolytes like proline and total sugars are considered as indexes of drought avoidance and antioxidative plant defense response (Ruiz-Lozano et al., 1995). The effect of AM fungi on the water stress tolerance was positive and it make plants tolerant in water stressed conditions by increasing proline content up to some extent. The proline content in the non mycorrhizal plants was more as compared to mycorrhizal plants. But still the growth and vigour index of mycorrhizal plants under stress was more than non mycorrhizal stressed plants. Thus it was evident that mycorrhizal fungi were helpful for the plants in the water stressed conditions. 23

14 Moreover, the benefits of AM fungi were also due to the improvement of the physical characteristics of the soil, which in turn favours the establishment of a stable plant cover (Alguacil et al., 2008). Soil structure has a lead and prime role in soil percolation and biogeochemical processes. Therefore, improved soil structure means increased water retention, nutrient uptake, drainage, aeration and root growth. Arbuscular mycorrhizal fungi increase phosphate (PO 4 ) uptake and plant growth under low phosphate conditions. The benefits of AM Fungi are greatest in systems where inputs are low. Heavy usage of phosphorus fertilizer can inhibit mycorrhizal colonization and growth. As the soil phosphorus level available to the plants increases, the amount of phosphorus also increases in the plant tissues and carbon drain on the plant by the AM fungi symbiosis become non-beneficial to the plant (Grant et al., 2005). A decrease in mycorrhizal colonization due to high soil phosphorus levels can lead to plant deficiencies in other micronutrients that have mycorrhiza mediated uptake such as copper (Timmer and Leyden, 1980). However mycorrhizae may only benefit plants during times of high phosphorus demand, i.e. during flowering or seed development (Fitter, 1991). AM fungi have showed to increase resistance to root infecting pathogenic fungi e.g. Pythium ultimatum, Phytophthora parasitica and root invading nematodes. This topic has been extensively reviewed by Linderman (1994) and Cordier et al., (1996). The increased surface of hyphae may also play an indirect role in influencing pathogen levels and in aiding nutrient acquisition and soil stabilization (Dodd, 2000). Tomato, brinjal and chili are solanaceous vegetables, often found affected by damping off disease during nursery stage. The disease is reported to be caused by species of Pythium, Fusarium and Phytophthora (Rahman and Bhattiprolu, 2005). AM fungi are found effective against damping off in these crops. The infection of arbuscular mycorrhizal fungi in plants has effect on foliarfeeding insects. In Plantago lanceolata L., Gange and West (1994), showed that the application of fungicide successfully reduced mycorrhizal infection, and this led to reductions in foliar biomass, caused by a lower leaf number. However the plants inoculated with mycorrhiza showed less incidence of insects as compared to plants treated with fungicides. Studies with foliar endophytes in grasses have showed that these 24

15 fungi can produce chemical toxins that reduce the growth of the phytophage, and hence the parasitoid (Barker and Addison, 1996). Furthermore, the community structure of parasitoids associated with foliar-feeding insects can be different on hosts with or without the fungus (Omacini et al., 2001). Pre inoculation of tomato plants with Glomus coronatum stimulated plant growth and reduced nematode Meloidogyne incognita infestation (Diedhiou et al., 2003). Combined application of the AM fungus and Fusarium sp. enhanced mycorrhization of tomato roots but did not increase overall nematode control or plant growth. Nutrient loss from ecosystems is among the top environmental threats to ecosystems worldwide, leading to reduced plant productivity in nutrient poor ecosystems and eutrophication of surface water near nutrient rich ecosystems. Hence, it is of pivotal importance to understand which factors influence nutrient loss. AM fungi are widespread soil fungi that form mutualistic relationships with the majority of land plants; reduce nutrient loss from grassland microcosms during rain induced leaching events (Heijden and Van der, 2010). Grassland microcosms with AM fungi lost 60% less phosphorus and 7.5% less ammonium compared to control microcosms without AM fungi. Similar results were obtained for microcosms planted with each of three different grass species. Communities of plants, biological soil crusts and arbuscular mycorrhizal fungi are known to influence soil stability individually (Chaudhary et al., 2009). Arbuscular mycorrhizal fungi and plants contribute the surface and subsurface stability of soil. There are various methods of mass multiplication of AM fungi namely trap culture, pot culture, funnel technique, etc. In the method of trap culture, soil or plants from the field are placed into sterile substrata in pots to increase the colonization of a susceptible host root systems e.g. maize or jowar. This process will lead to spore production by the fungus and allow identification of species. AM fungi need the symbiotic association with plants for proliferation. Therefore, inoculation of AM fungi with host plant is done to grow the inoculated plant and increase the number of spores. For the AM fungal inoculum, spores collected from soil can be used. However, spores in soil are not always active in colonizing plants. Therefore, trapping culture is often employed. Soil or sieving of soil is used as inoculum (Soil Trap Culture). To isolate AM fungi colonizing roots, mycorrhizal plants collected from field can be transplanted to 25

16 potting medium as plant trap culture (Murakoshi et al., 1998). The pot culture technique is generally followed to multiply the AM spores. The starter inoculum of selected AM fungus is generally raised by funnel technique (Menge and Timmer, 1982) using wheat as a host. A set up include a funnel on the bottle, the funnel is first filled with small amount of sterilized sand and soil mixture at the ratio of 1:1 up to neck portion. After 45 days, seedling roots are processed to detect colonization (Koske and Gemma, 1989). Soil sample (10 g) also is studied for spore quantification by using Gerdemann and Nicolson (1963) method. The complete system, including soil and seedlings were then transferred to bigger earthen pots containing sterilized sand soil for the multiplication of individual spores again using wheat as a host plant. The MPN assay was developed to estimate the density of organisms in a liquid culture (Cochran, 1950). Porter (1979) first used it to estimate the density of AM propagules in soil. It provides a relative measure of the density of propagules capable of colonizing roots. Four main assumptions of the MPN method are: (1) The propagules are randomly distributed in the soil; (2) The propagules are single and aggregates; (3) That dilution is proportional to the number of propagules; and (4) That if one organism is present it will be detected by the assay method (Tommerup, 1994). In Recent past, molecular methods have been developed which are primarily based on exploitation of genetic variation (De Souza et al., 2004) that allow identification of AMF in the host plant roots or directly in the soil without the necessity of spore formation. The nuclear encoded ribosomal DNA (rdna) has been well established as a marker for characterization of AMF, both in the lab as well as in natural assemblages (Clapp et al., 2002). Recently molecular phylogenetic analysis, based on SSU, rdna sequences, has resulted in profound changes in AM fungi classification, with proposal of a separate phylum Glomeromycota, containing new orders, families and genera (Schußler et al., 2001a). The molecular technologies are definitely making inroads into the problematic and unapproachable areas of AMF. The next one or two decades are going to witness tremendous activity in this area and one can visualize a clear and holistic picture of AMF. 26

17 The current researchers are likely to provide direction to future research. Following are some of the areas for future line of research. A new interesting target for PCR cloning is the enzymes involved in lipid biosynthesis. This assumes significance in view of the recent findings that AMF are unable to grow asymbiotically due to the lack of storage lipid biosynthesis (Bago et al., 1998). The construction of cdna libraries followed by random sequencing to obtain expressed sequence tags (ESTs) and screening cdna assays will also be useful tools for analysis of AMF gene expression. These libraries will form a starting material for more systematic work on gene expression of AMF. Selection of model isolate is necessary for this type of work. It is also recommended to consider at least one representative from each suborder for the systematic sequencing and expression analysis (Franken and Requena, 2001). As an alternative to rdna gene analysis, Franken and Requena (2001) suggested a method to measure functional biodiversity in ecosystems. This approach has several advantages like (1) A Glomalean- specific primer could be designed; (2) It detects not only the presence of isolates but their activity, and (3) It is also useful for formulation and control inocula for field inoculations. Hence, such types of approaches may have to be given top priority in future. Proteome analysis followed by application of reverse genetics to identify genes of interest in AMF is also underway (Samara et al., 1999; Benabdellah et al.,1998). Another emerging technology is laser capture micro dissection, which utilizes a laser to remove the contents of the cell of interest (Bonner, 1997). Transcripts from these preparations could be used to generate arbuscule cdna libraries. Bioimaging using confocal microscopy is another technology applied to mycorrhizal research (Barea, 1998) which, when coupled with developing AM transformation techniques (Forbes et al., 1998), may help to link gene expression studies with structural aspects of symbiosis. The association of AM fungi with the rhizosphere of tuberous plants has been studied by Nisha et al., (2010), In Ginger it has been studied by Kunwar et al., (1999); Muthukumar and Udaiyan (2000); Selvaraj et al., (2001); Panwar and Tarafdar, (2006); Khade and Rodrigues, (2007); Radhika and Rodrigues, (2010). Taber and Trappe, (1982) reported for the first time, the presence of AM fungi in the vascular system of rhizomatous tissue and the scale like leaves of ginger. Stasz and Sakai (1984) reported AMF structures occurring on scale-like leaves of six genera of Zingiberaceae. 27

18 In ginger the reports on chlorophyll, protein, DNA, RNA proline and carbohydrate contents are very scarce. The effect of salt stress on chlorophyll pigment and soluble protein content has been studied by Zeynep et al., (2010) in tomato cultivars. Dhanpackiam and Ilyas (2010) studied the effect of salt stress on chlorophyll and carbohydrate contents in seedlings of Sesbania grandiflora. The effect of salinity on chlorophyll, proline and antioxidant enzymes content in Capsicum annum has been studied by Sumalee (2011). So far various crop plants have been studied with respect to salt stress and biochemical contents but in ginger there are no such reports. Rosilda et al., (2010), studied the effect of AM fungi and phosphate fertilizer in micro propagated ginger with respect to survival, growth, development. They also studied the leaf number, leaf area, number of shoots per plants, shoot length as well as root colonization in ginger. The studies were also undertaken with respect to relative mycorrhizal dependency of ginger. The effect of AM fungi on chlorophyll fluorescence in Cassava plants under water stress has studied by Oyetunji et al., (2007). VAM fungi and their effect on morphology, growth, photosynthesis and metabolism in various hosts have studied by Huixing (2005). The amount of proline under the water stress in coriander has studied by Aliabadi et al., (2008). Rahman and Bhattiprolu (2005) studied the efficacy of fungicides and arbuscular mycorrhizal fungi for the control of damping off disease in tomato, chilli and brinjal. The plant inoculated with mycorrhiza enhances the water stress tolerance in Trifolium (Zeze et al., 2007). Medina and Azcon (2010), showed the effectiveness and application of AM fungi and organic amendments improve soil quality and plant performance under heavy metal and water stress conditions. The isolation and amplification of DNA from the rhizome of ginger and turmeric was carried out by Syamkumar et al., (2003) by PCR technique. The isolation of RNA from the rhizome of ginger has very few reports. But, there are no reports on effect of salt stress on the nucleic acid contents in ginger. The essential oil named 6 Gingerol from the rhizome of ginger has been quantified by Pawar et al., (2011). Arbuscular mycorrhizal fungi and its influence on the development of oleoresins in micro propagated 28

19 ginger have been studied by Maicon et al., (2008). The antibacterial effect of various essential oils isolated from ginger rhizome has revealed by Krittika et al., (2007). There are no reports on ginger with respect to water stress, salt stress and disease resistance. The changes in the physiological conditions e.g. water stress and salt stress changes the biochemical contents of the plants. The incidence of any disease in plants also affects the growth and yield of that plant. In current studies the emphasis has given to find out the effect of AM fungi on biochemical contents in ginger plants grown under salt and water stress. Moreover the studies are also carried out with respect to growth and yield in pot as well as in field conditions. The effect of AM fungi on Pythium ultimatum was also studied in pot cultures with respect to growth response and disease resistance. The various methods used in present investigation are described in next chapter of material and methods. 29