CLIAMS:1. A medium for culturing Arbuscular mycorrhizal fungi comprising a SGL medium, wherein the SGL medium is prepared by a combination of a GS2 medium and a SG2 medium.

2. The medium as claimed in claim 1, wherein the GS2 medium and the SG2 medium are in the ratio of 2.5:1 in liquid form.

3. An in vitro method for isolating Arbuscular mycorrhizal fungi comprising:
a) Cleaning at least one of a root bit of a crop;
b) Washing the root bit with water;
c) Screening the root bit for a mycorrhizal spore;
d) Surface sterilizing the root bit with the mycorrhizal spore;
e) Inoculating the root bit on a SGL medium;
f) Culturing an arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time;
g) Screening for the arbuscular mycorrhizal fungi;
h) Separating the arbuscular mycorrhizal fungi;
i) Inoculating the arbuscular mycorrhizal fungi in new petri plates in the SGL medium;
j) Growing a pure culture of the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time; and
k) Harvesting the pure culture of the arbuscular mycorrhizal fungi.

4. An in vitro method for mass production of arbuscular mycorrhizal fungi comprising:
a) Growing a pure culture of an arbuscular mycorrhizal fungi;
b) Transferring the pure culture of the arbuscular mycorrhizal fungi into a container with a SGL medium; and
c) Culturing the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time.

5. An in vitro method for preparing an inoculum of an arbuscular mycorrhizal fungi comprising:
a) Culturing an arbuscular mycorrhizal fungi in a SGL medium;
b) Mixing thoroughly the arbuscular mycorrhizal fungi with the SGL medium; and
c) Preparing at least one of a wet inoculum or a dry inoculum.

6. The method as claimed in claim 5, wherein the dry inoculum is prepared by drying the arbuscular mycorrhizal fungi in an oven at a predetermined temperature for a predetermined period of time followed by powdering the arbuscular mycorrhizal fungi. ,TagSPECI:TECHNICAL FIELD

[0001] The present disclosure generally relates to the field of media for fungal growth. More particularly, the present disclosure relates to a novel medium for the culture of arbuscular mycorrhizal fungi and its isolation and mass production technology that is free from root association.

BACKGROUND

[0002] Mycorrhiza (Greek: µ????, mykós, "fungus" and ???a, riza, "roots", pl. mycorrhizae or mycorrhizas) is a symbiotic (generally mutualistic, but occasionally weakly pathogenic) association between a fungus and the roots of a vascular plant. In a mycorrhizal association, the fungus colonizes the roots of host plants, either intracellularly as in arbuscular mycorrhizal fungi (AMF or AM), or extracellularly as in ectomycorrhizal fungi.

[0003] Mycorrhizal networks (also known as common mycorrhizal networks - CMN) are underground hyphal networks created by mycorrhizal fungi that connect individual plants together and transfer water, phosphorous, nitrogen and other minerals and nutrients benefitting the plant host. The formation of these networks is context dependent, and can be influenced by soil fertility, resource availability, host or myco-symbiont genotype, disturbance and seasonal variation. Several studies have also demonstrated that mycorrhizal networks can transport, phosphorus, nitrogen, water, defence compounds, and allelochemicals from plant to plant.

[0004] Types of mycorrhizal networks: There are two main types of mycorrhizal networks: arbuscular mycorrhizal (endomycorrhizal) networks and ectomycorrhizal networks. The two types are differentiated by the fact that the hyphae of ectomycorrhizal fungi do not penetrate individual cells within the root, while the hyphae of endomycorrhizal fungi penetrate the cell wall and invaginate the cell membrane.

[0005] Arbuscular mycorrhizal networks are formed between plants that associate with Glomeromycetes. Arbuscular mycorrhizal associations (also called endomycorrhizas) predominate among land plants, and are formed with 150-200 known fungal species, although true fungal diversity may be much higher. It has generally been assumed that this association has low host specificity. However, recent studies have demonstrated preferences of some host plants for some Glomeromycete species.

[0006] Ectomycorrhizal networks are formed between plants that associate with ectomycorrhizal fungi and proliferate by way of Ectomycorrhizal extrametrical mycelium. In contrast to Glomeromycetes, ectomycorrhizal fungal are a highly diverse and polyphyletic group consisting of 10,000 fungal species. These associations tend to be more specific, and predominate in temperate and boreal forests.

[0007] Endomycorrhizas are variable and have been further classified as arbuscular, ericoid, and orchid mycorrhiza, while arbutoid mycorrhizas can be classified as ectoendomycorrhizas. Monotropoid mycorrhizas form a special category. AM, formerly known as vesicular-arbuscular mycorrhizas, or VAM, are mycorrhizas whose hyphae enter into the plant cells, producing structures that are either balloon-like (vesicles) or dichotomously branching invaginations (arbuscules) or both. The fungal hyphae do not in fact penetrate the protoplast (i.e. the interior of the cell), but invaginate the cell membrane. The structure of the arbuscules greatly increases the contact surface area between the hypha and the cell cytoplasm to facilitate the transfer of nutrients between them. Fossil evidence and DNA sequence analysis suggest that this mutualism appeared 400-460 million years ago, when the first plants were colonizing land. Arbuscular mycorrhizas are found in 85% of all plant families, and occur in many crop species. The hyphae of arbuscular mycorrhizal fungi produce the glycoprotein glomalin, which may be one of the major stores of carbon in the soil. Arbuscular mycorrhizas are formed only by fungi in the Division Glomeromycota.

[0008] The Genus Glomus which belongs to Phylum Glomeromycota is a largest and diverse arbuscular mycorrhizal (AM) fungi and all species form symbiotic relationships (mycorrhizas) with plant roots. As with other AM fungi, all Glomus species are thought to be obligate symbionts, dependent on their mycorrhizal association with plant roots to complete their life cycle. They cannot be cultured in the laboratory in the absence of a plant host. Glomus species are found in nearly all terrestrial habitats, including arable land, deserts, grasslands, tropical forests. Several species of Glomus are cultured in vivo and in vitro root organ culture and sold as mycorrhizal inoculant for agricultural soils.

Benefits of mycorrhizal networks for plants:
[0009] Several positive effects of mycorrhizal networks on plants have been reported. These include increased establishment success, higher growth rate and survivorship of seedlings; transfer of water, carbon, nitrogen and other limiting resources to the plants there by increasing the probability for colonization even in less favourable conditions. These benefits are identified as the primary drivers of positive interactions and feedbacks between plants and mycorrhizal fungi that influence plant species abundance. Basic studies have demonstrated that AM association play an important role in plant nutrition. Plant colonised by AM fungi harbour greater amount of phosphorus and other trace elements specially when they are meanly soluble. The symbiosis has a sustainable net benefit to both partners. This benefit can be physiological, nutritional, ecological or any combination of these processes.

[0010] AM associated plants also exhibit improved resistance towards drought, environmental stress and some root pathogens. Exploiting and managing mycorrhizas has important and sustainable consequences for both agricultural and natural ecosystems. AM fungi have broad host range, hence may be used as inoculants to improve plant production in agriculture, horticulture and forestry. Commercially important benefits that can be derived from their use include increase in plant growth and yield, improved crop uniformity, reduction in phosphorus and trace metal fertilizers requirement, reduced losses due to environmental stress and root diseases, improved transplant establishment and reduced cropping times. All these benefits translate into increased profits for farmer and nurserymen. The most obvious effect of AM fungi has been attributed to amelioration of nutrient uptake (P and others), resulting in more vigorously growing plants which are able to ward off or tolerate root disease. The use of mycorrhizal biofertilizer also helps to improve higher branching of plant roots, and the mycorrhizal hyphae grow from the root to soil enabling the plant roots to contact with wider area of soil surface, hence, increasing the absorbing area for water and nutrients absorption of the plant root system. Therefore, plants with mycorrhizal association will have higher efficiency for nutrients absorption, such as nitrogen, phosphorus, potassium, calcium, magnesium, zinc, and copper; and also increase plant resistance to drought. In exchange for carbohydrates produced by the host through photosynthesis, the fungi help the plant take up water and immobile soil nutrients such as phosphorus (P), copper, and zinc. The fungus extends from the plant root and expands the volume of soil that the root system can explore by itself. Soil hyphae are likely to have an important role in nutrient cycling by helping to prevent losses from the system, especially at times when roots are inactive. Soil hyphae may have an important role in nutrient cycling by acquiring nutrients from saprophytic fungi. Mycorrhizal fungi contribute to carbon storage in soil by altering the quality and quantity of soil organic matter.

[0011] Most of the globally important important food crops are known to form symbiosis naturally with arbuscular mycorrhizal fungi which help the crops to efficiently obtain phosphate from the soils. The threat to global food security due to limited availability of phosphate fertilizers can be reduced significantly by promoting better phosphate acquisition through the arbuscular mycorrhizal fungal symbiosis. The benefits will be more in tropical soils which have low bioavailable phosphate content and high phosphate retention capacities.

Benefits that mycorrhiza secure from plants:
[0012] Mycorrhizal association provides AM fungi constant access to carbohydrates from the host.

Other Benefits of Mycorrhiza:
[0013] Glomalin discovered by Sara F Wright, is a glycoprotein produced abundantly on hyphae and spores of arbuscular mycorrhizal (AM) fungi in soil and in roots. Glomalin contain 30 to 40 percent carbon, and forms clumps of soil granules i.e aggregates which adds structure to soil and keep other stored soil carbon from escaping. As a glycoprotein, glomalin stores carbon in both its protein and carbohydrate (glucose or sugar) subunits. Glomalin-related soil proteins (GRSPs), along with humic acid, are a significant component of soil organic matter and act to bind mineral particles together, improving soil quality. Nichols reported that glomalin accounts for 27 percent of the carbon in soil and is a major component of soil organic matter. Glomalin has been investigated for its carbon and nitrogen storing properties, including as a potential method of carbon sequestration and takes 7–42. Glomalin improves soil aggregate, water stability and decrease soil erosion. Glomalin also contains from 1 to 9% tightly bound iron. Arbuscular mycorrhizal fungi appear to be the only producers of glomalin.

Impediments:
[0014] Despite the potential of AM fungi, inoculum production has been a major limitation due to many factors including lack of suitable and cost effective technologies. Production of pure culture of AM fungi at reasonable cost is still a big challenge for microbiologists. For the commercial development of inoculum, a number of strategies have been followed from time to time with their own merits and demerits. Currently two systems are available a soil and to soil less technologies. As far as soil technologies are concerned they are cost effective with low inputs and thousands of infected propagules can be extracted in a gm of soil. Main drawback associated with the soil system is the bulk amount, vulnerability of pest infestation and nutrient management. To rule out this problem soil less technologies i.e hydroponic, aeroponic, root organ culture were developed. Hydroponic system provides nutrients to the plants in the form of thin layers on the roots which results in grater proliferation of the roots, production of higher no of spores /cm of inoculated roots and it also reduces the chances of pest infestation and isolation of roots and spores easily. In aeroponic system, nutrients are provided in the form of evenly distributed fine mesh. This system reduces the infestation and gives higher sporulation but it is a costly affair. Many attempts have also been made to grow on defined and complex medium but they could support only the hyphal growth. Proper selection and efficiency of sterilization processes are key for the success of axenic or monoxenic AM fungal cultures. Isolated spores are often surface sterilized using a two-steps procedure. Then, vesicles are easily isolated by lacering heavily colonized roots. Basically, the surface sterilization involve baths in chloramines T (2%) solution with traces of a surfactant (Tween 20/80) and antibiotics such as streptomycin or gentamycin. Sterilized AM propagules must be stored at 4°C until use. The pre-symbiotic growth of AM is characterized by formation of running hyphae. Several factors such as nutrition, chemical treatments and genetical factors are critical for growth of the extraradical phase of AM fungi.

[0015] The monoxenic system is reported to be cheaper than greenhouse culture. The comparisons between in vivo and in vitro systems for production of spores are reported to be very much in favour for the axenic systems. However the effectiveness of these propagules under adverse conditions remains unclear. Some AM fungi lose their infectivity after several successive in vitro subcultures. Therefore it will be necessary to evaluate inoculum potential of different generations of AM propagules in continous monoxenic cultures. In order to preserve permanent fungal biodiversity, in vitro and in vivo collections must always be maintained. All types of AM propagules (isolated spores or vesicles, mycelia, sheared mycorrhizal roots) are virtually able to initiate AM symbiosis. Considering the inability of some AM fungi to produce spores, the use of intraradical forms of AM fungi seems to be a good opportunity to establish AM symbiosis. As intravesicles in mycorrhizal roots act as reserves and propagules, they have a higher inoculum potential than other AM propagules such as spores and hyphae. Several authors have exploited the intraradical forms of AM fungi to achieve mass production of Glomus species. The different in vivo and in vitro culture techniques of AM fungi are described below.

[0016] The large number of attempts at mycorrhization which have been carried out so far have consisted of using inoculums prepared from complete plants which are cultivated in pots or a greenhouse. Inoculation is nearly always carried out with specially gathered mycorrhizal roots, or sometimes with a suspension of spores, lyophilized roots or soil pellets mixed with infected roots.

[0017] The US Patent No. 5,554,530 discloses an invention wherein is described a method of producing mycorrhizal fungal propagules in vitro in a two-compartment container having a gellified medium, which comprises the steps of: a) cultivating aseptically transformed dicotyledon root organs, capable of autonomous growth in vitro, in a first compartment containing a mineral minimal medium with sugar, wherein the medium is suitable for root growth; b) inoculating the transformed root organs with endomycorrhizal spores; and c) cultivating the inoculated transformed root organs for a time sufficient for the mycorrhizal fungi to transfer to a second root-free and root exudate-free compartment containing the mineral minimal medium of step a) and for the mass production of fungal propagules to occur in the second compartment.

[0018] The US Patent No. 4,945,059 discloses an invention wherein is described a method of proliferating vesicular arbuscular mycorrhizal fungi (i.e., VAM fungi) is disclosed which comprises inoculating VAM fungi in a soil medium containing a potato and a porous amphoteric ion exchanger, an accelerator and, optionally, in the presence of a VAM formation accelerator. An advantage gained by the use of VAM fungi in cultivation of plants can be found in the fact that smaller amounts of fertilizers need be used when combined with VAM fungi, as opposed to the use of fertilizers alone.

In vitro culture of AM fungi
[0019] Currently AM fungi are cultivated using axenic or monoxenic techniques using different sources of inoculum.

Root Organ Cultures:
[0020] In this in vitro culture technique, the isolated root can be propagated continuously in different solid and liquid media with high reproducibility. Initiation of isolated roots requires pre-germination of seeds, previously surface sterilized with classical disinfectants (sodium hypochlorite, hydrogen peroxide), then thoroughly washed in sterile distilled water. Germination of seeds occurs after 48 h at 28°C in the dark on water agar or moistened filter papers. The tips (2cm) of emerged roots can be transferred to a rich medium such as modified White medium or Strullu and Romand medium. The pH of the medium is adjusted to 5.5 before autoclaving. Fast-growing roots are cloned by repeated subcultures.

Monoxenic Cultures:
[0021] Transformation of roots by the soil-borne microorganism Agrobacterium rhizogenes has provided a new way to obtain mass production of roots in a very short time. A 2-day old loopful of a bacterial suspension is used to inoculate sections of root organs. Genetically modified caroot (Daucus carrota L.) roots by A. rhizogenes show profuse roots two to four weeks later. Then, the tips are aseptically cultivated on rich medium. Several subcultures (3 to 4) are necessary in this medium enriched with antibiotics such as carbenicillin or ampicillin, to obtain free living roots without bacteria. A clonal culture derived from a single root is then established.

[0022] The entire vegetative development of AM fungi in monoxenic cultures is followed by several works using either transformed or non-transformed roots. A non-exhaustive list of AM fungi cultivated monoxenically has been given. The modified White medium (Minimal medium M) and the modified Strullu and Romand medium (MSR medium) are the most suitable medium to standardize the in vitro culture of AM fungi. The long term behaviour of G. margarita on Ri-TDNA transformed roots of carrot showed 80% of the fungal infections units were produced during the period of root aging.

[0023] The mass inoculum technology developed at TERI (Tata Energy Research Institute) exploited the genetically modified host roots using the bacterium Agrobacterium rhizogenes carrying Ri T-DNA plasmid. The TERI technology offers the mass production of viable, healthy, genetically pure and high quality fungal propagules without any pathogenic contamination under in vitro sterile environment. Encapsulation stabilize the biological properties of mycorrhizal roots and isolated vesicles or spores.

Methodology for recovering, for purifying, for cultivating tropical AM fungi:
[0024] AM fungi can be isolated from surface soil layers to soil depths greater than 35 m. They are multiplied in association with a mycotrophic plant under greenhouse conditions. Isolated spores or vesicles can be monoxenically cultivated in presence of isolated roots or on entire plant host. Intraradical forms of AM fungi are used as starter inoculum to have monoxenic cultures on M medium or on MSR medium and a bank of germplasms of AM monoxenically cultivated in association with isolated tomato or transformed carrot roots is established.

[0025] Although the facts and figures of potential role of mycorrhiza in enhanced nutritional needs of plants in laboratories were established, the major bottleneck for its widespread application to reach the end-users was its bulk production to cater the huge requirement. A known fact that culturing mycorrhiza in laboratory conditions like other microbes was not possible due to its strict biotrophic nature of proliferation in the presence of suitable host was the major reservation of its future contribution in agriculture. All the existing in vitro techniques are expensive requiring sophisticated equipment and expensive media.

[0026] In the light of the aforementioned discussion, there exists a need for developing simpler, low cost techniques for cultivating AM fungi. The present invention discloses a novel low cost media for culturing AM fungi and simpler and cost effective in vitro culture technique that can be used for the mass production of AM fungi.

BRIEF SUMMARY

[0027] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0028] Exemplary embodiments of the present disclosure are directed towards a medium for culturing Arbuscular mycorrhizal fungi comprising a SGL medium, wherein the SGL medium is prepared by a combination of a GS2 medium and a SG2 medium.

[0029] Another exemplary aspect of the present subject matter is directed towards an in vitro method for isolating Arbuscular mycorrhizal fungi comprising: Cleaning at least one of a root bit of a crop; Washing the root bit with water; Screening the root bit for a mycorrhizal spore; Surface sterilizing the root bit with the mycorrhizal spore; Inoculating the root bit on a SGL medium; Culturing an arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time; Screening for the arbuscular mycorrhizal fungi; Separating the arbuscular mycorrhizal fungi; Inoculating the arbuscular mycorrhizal fungi in new petri plates in the SGL medium; Growing a pure culture of the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time; and harvesting the pure culture of the arbuscular mycorrhizal fungi.

[0030] Another exemplary aspect of the present subject matter is directed towards an in vitro method for mass production of arbuscular mycorrhizal fungi comprising: Growing a pure culture of an arbuscular mycorrhizal fungi; Transferring the pure culture of the arbuscular mycorrhizal fungi into a container with a SGL medium; and culturing the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time.

[0031] Yet another exemplary aspect of the present subject matter is directed towards an in vitro method for preparing an inoculum of an arbuscular mycorrhizal fungi comprising: Culturing an arbuscular mycorrhizal fungi in a SGL medium; Mixing thoroughly the arbuscular mycorrhizal fungi with the SGL medium; and preparing at least one of a wet inoculum or a dry inoculum.

[0032] It is an object of the present invention to disclose a novel medium that is cost effective for culturing arbuscular mycorrhizal fungi

[0033] It is another object of the present invention to disclose an in vitro culture technique for growing arbuscular mycorrhizal fungi that is simpler and cost effective.

[0034] It is another object of the present invention to disclose an in vitro culture technique for mass production of arbuscular mycorrhizal fungi that does not rely on root organ culture for the mass production of the AM fungi spp. particularly those belonging to the genus Glomus and also an inexpensive technique for isolation of AM fungi.

BRIEF DESCRIPTION OF DRAWINGS

[0035] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

[0036] FIG.1 is an image showing AM fungi isolation from root bits.

[0037] FIG. 2 is an image showing extraradical hyphae of AM fungi.

[0038] FIG. 3 is an image showing arbuscules of AM fungi.

[0039] FIG. 4 is an image showing vesicles of AM fungi.

[0040] FIG. 5 is an image showing Glomus spp. spores inside root.

[0041] FIG. 6 is an image showing Glomus spp. spores inside root.

[0042] FIG. 7 is an image showing AM fungal spores inside root.

[0043] FIG. 8 is an image showing spores in SGL medium.

[0044] FIG. 9 is an image showing benefits from AM fungi inoculated in coriander.

[0045] FIG. 10 is an image showing benefits from AM fungi inoculated in wheat and sorghum.

[0046] FIG. 11 is an image showing benefits from AM fungi inoculated in cowpea.

[0047] FIG. 12 is an image showing benefits from AM fungi inoculated in cowpea.

DETAILED DESCRIPTION

[0048] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0049] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0050] According to a non limiting exemplary embodiment of the present disclosure, a medium for culturing Arbuscular mycorrhizal fungi comprising a SGL medium is disclosed, wherein the SGL medium is prepared by a combination of a GS2 medium and a SG2 medium.

[0051] In accordance with a non limiting exemplary embodiment of the present subject matter, an in vitro method for isolating Arbuscular mycorrhizal fungi is disclosed, wherein the method comprises of: Cleaning at least one of a root bit of a crop; Washing the root bit with water; Screening the root bit for a mycorrhizal spore; Surface sterilizing the root bit with the mycorrhizal spore; Inoculating the root bit on a SGL medium; Culturing an arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time; Screening for the arbuscular mycorrhizal fungi; Separating the arbuscular mycorrhizal fungi; Inoculating the arbuscular mycorrhizal fungi in new petri plates in the SGL medium; Growing a pure culture of the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time; and harvesting the pure culture of the arbuscular mycorrhizal fungi.

[0052] According to a non limiting exemplary embodiment of the present disclosure, an in vitro method for mass production of arbuscular mycorrhizal fungi is disclosed, wherein the method comprises of: Growing a pure culture of an arbuscular mycorrhizal fungi; Transferring the pure culture of the arbuscular mycorrhizal fungi into a container with a SGL medium; and culturing the arbuscular mycorrhizal fungi at a predetermined temperature for a predetermined period of time.

[0053] In accordance with a non limiting exemplary embodiment of the present disclosure, an in vitro method for preparing an inoculum of an arbuscular mycorrhizal fungi is disclosed, wherein the method comprises of: Culturing an arbuscular mycorrhizal fungi in a SGL medium; Mixing thoroughly the arbuscular mycorrhizal fungi with the SGL medium; and preparing at least one of a wet inoculum or a dry inoculum.

[0054] The present invention discloses a new medium –SGL which can support growth of AM fungi. Isolation of AM fungi from roots of wide varieties of plants with the new low cost SGL medium can be done. The AM fungi were isolated from the Onion, Hibiscus, Methi, Coriander, sesamum, paddy and some grasses etc. The present invention also discloses development of pure culture of AM fungi for mass multiplication. In this method, AM fungi is multiplied at room temperature at low cost without requirement of sophisticated equipment and costly medium and without root association. Rhizophagus / Glomus species could be grown non-symbiotically, with extraradical and intraradical hyphae and spores developing profusely in the medium. AM fungal spores germinate after 48 to 72 hours in SGL medium. The AM culture –dry or wet can be used as inoculum for growing plants. On inoculation with the culture, different plants formed mycorrhizal association and derive benefit recording better growth both in roots and shoots when compared with control plants.

Example 1:
Preparation of SGL media:
[0055] Several media such as GS2, SG2 and Potato dextrose agar were used alone and in combinations for examining the growth of the AM fungi without root association. AM fungi could be grown without root association in a special medium “SGL medium”. (SGL-Dr Korlapati Satyagopal IAS and Dr Girish Anantrao Gunjotikar Liquid medium), which is prepared by a combination of GS2 and SG2 (Patent application numbers: 1753/CHE/2014 published on 25/4/2014 and 5051/CHE/2014 published on 31.10.2014) in different proportions preferably 2.5: 1, ratio in liquid form. AM fungi could be cultured easily when compared with other media. The SGL media provides carbon &other nutrients that are required for growth of AM fungi. The SGL medium also provides the nutrients in ideal proportion to facilitate growth of AM fungi even in the absence of the roots (without symbiotic association). In nature it is observed that AM fungi establishes association with roots from germinating seeds ( blotter tests) to absorb the nutrients from roots of plants immediately after germination of seed(available in endosperm, and may be from seed coat) while benefitting the growing plant.( when compared with control blotter tests).. The association and growth of AM fungi right from germination stage of the plant implies that AM may grow even without symbiotic association if critical nutrients required for AM growth are provided. The current innovation incorporated above aspects while developing the media.

[0056] Referring to FIG.1, it is an image showing AM fungi isolation from root bits.

[0057] Referring to FIG. 2, it is an image showing extraradical hyphae of AM fungi.

[0058] Referring to FIG. 3, it is an image showing arbuscules of AM fungi.

[0059] Referring to FIG. 4, it is an image showing vesicles of AM fungi.

[0060] Referring to FIG. 5, it is an image showing Glomus spp. spores inside root.

[0061] Referring to FIG. 6, it is an image showing Glomus spp. spores inside root.

[0062] Referring to FIG. 7, it is an image showing AM fungal spores inside root.

[0063] Referring to FIG. 8, it is an image showing spores in SGL medium.

[0064] Referring to FIG. 9, it is an image showing benefits from AM fungi inoculated in coriander.

[0065] Referring to FIG. 10, it is an image showing benefits from AM fungi inoculated in wheat and sorghum.

[0066] Referring to FIG. 11, it is an image showing benefits from AM fungi inoculated in cowpea.

[0067] Referring to FIG. 12, it is an image showing benefits from AM fungi inoculated in cowpea.

Example 2:
Isolation of AM fungi:
[0068] The present innovation relied on isolating AM fungi from roots having mycorrhizal spores by following the procedure described below.

Cleaning the root bits:
[0069] Roots of several crops such as wheat, paddy, sorghum, cowpea, gongura, moth bean coriander and sesamum were collected for isolation of AM fungi.

Surface sterilisation:
[0070] All collected roots after washing with water were screened for mycorrhizal spores then roots with mycorrhizal spores are sterilised using 97 % ethanol in the ratio of 1 volume of ethanol: 9 volumes of water by soaking in the solution for approximately 5–10 minutes.

Inoculation in Media:
[0071] The surface sterilized roots are cut into bits and the root bits were aseptically inoculated on the media for isolation of AM fungi in the new SGL medium which facilitates quick and luxuriant growth.

Screening for AM fungi:
[0072] Fungi develop from the root bits after 48 to 72 hr at room temperature in the SGL medium. They are screened by preparing the slides and observing under compound microscope for isolating extraradical aseptate hyphae and the spores. The fungi developed on SGL medium includes Aspergillus, Trchoderma, Fusarium, Curvularia, Alternaria along with AM fungi. The AM fungi are separated and then inoculated in new petri plates in SGL medium for development of pure culture of AM fungi.

Pure culture of AM fungi:
[0073] After 48-72hr the pure AM fungi develop in SGL medium, which can be harvested for mass multiplication. The pure culture is once again screened under microscope prior to mass multiplication.

Example 3:
Mass multiplication of AM fungi and preparation of inoculum:
[0074] AM fungal pure culture developed on SGL medium after 72 hr is then transferred into containers with SGL medium for mass multiplication. The AM fungi are grown in SGL medium for 4- 7 days for luxuriant growth. +The AM fungi grown in the medium is mixed thoroughly since it is noted that extrarradical hyphae grow on surface of the medium and at the bottom and liquid portion of the medium, spores and germinating spores and growing hyphae are found in large numbers. After thorough mixing of the media with AM fungi, the culture can be used as inoculum source either as wet culture or dried culture (drying in oven at 60 oC for 72hr). The dried culture can be made in to powder in a mixer-grinder for use as dry powder inoculum for inoculation in pots /polybags/nurseries. Further studies were carried to verify the use of the AM inoculum on plants by inoculating seeds of different crops such as cereals and legumes and vegetable crops.

Confirmatory studies:
[0075] Seeds of different crops seeds such as wheat, paddy, sorghum, cowpea, hibiscus, moth bean coriander and sesamum were planted along with the dry and wet inoculum in pots/poly bags in several experiments as indicated below.

Example 4:
Experiments carried out to check establishment of cultured AM fungi with the roots
1. Planting the seeds along with Wet/Dry AM fungal inoculum
[0076] An appropriate media viz; red soil, coir pith and quartz sand were selected for inoculating wet and dry AM fungal culture along with above mentioned seeds. Coir pith and quartz sand, soil medium were sterilised by autoclaving method and after autoclaving, the media is cooled and used for the planting to avoid the chance of fungal contaminations. Nursery plastic bags and glass test tubes of 20 cm long filled with autoclaved quartz sand or soil or coir pith in 1/4th of the bottle and test tubes and 3/4th of the plastic bag. Then AM fungi wet/dry fresh culture is added to the growth media in bottles/test tubes or in plastic bags where the seed is to be planted. Seeds of different plant species such as corn, sorghum, sesamum, wheat, paddy, cow pea, mothbean, mungbean, mustered, ragi and onion were surface sterilised using a 97 % ethanol, in the ratio of 1 volume of ethanol: 9 volumes of water. The seeds were soaked in the solution for approximately 5–10 minutes. Surface sterilised seeds of different crops are planted in plastic bags and plastic bottles @ of 4 to 5 seeds in test tube. Then all bags /bottles /test tube are prepared in a laboratory in a well-lighted area to minimize contamination from microorganisms. Plants were watered once a day until water begins to drip from the bottom of the pot. Control bottles/test tubes/plastic bags were also maintained.

Observation of roots of plants inoculated with wet/dry inoculum:
[0077] After the development of the seedlings in the nursery plastic bags/test tubes/pots observation for the AM fungi were made besides testing the root and shoot length, leaf area. After germination of the seeds, observations were made on 3rd, 9th, 15th, 22nd and after one month. It was found that AM spores developed inside the roots after 9th to 15th days in the roots of all crops and complete matured spores were seen after 22to 30 days. The roots bits in which the spores were found, were again inoculated on SGL medium and after 5 days the colonies developed were microscopically checked and found that these are AM fungal colonies only.

[0078] Pure culture was used for the further study on spores of Glomus spp. Spores of Glomus and Rhizophzgus species are produced blastically at the end of a sporogenous hypha. Intercalary spore formation was also observed. The sporogenous hyphae develop from extraradical hyphae of mycorrhizal roots.Similar results have been described by Declerck et al. 2000. The details of arbuscules and spores in roots and intraradical vesicles of different crops studied are furnished below.

Extraradical hyphae:
[0079] The hyphae developed in the medium showed the colonisation in the seedling root observation.
Confirmation of the Glomus species with Molecular diagnosis:
[0080] The Glomus species grown on SGL medium sent for molecular diagnosis to Bioserve Biotechnologies (India) Pvt Ltd. The molecular analysis revealed that the culture belongs to Rhizophagus irregularies and sample is preserved under sample ID : CRO- 61.
Beneficial Impact of Mycorrhizal association:
[0081] Observations were also taken on the benefits of mycorrhizal association established when inoculated with wet/dry AM fungal culture produced in SGL medium. The observations have clearly revealed positive benefit both on the root and shoot growth when compared with control. Enhanced root growth in terms of length and more laterals and enhanced shoot growth in terms of length and width of leaves were observed in all the plants studied. The roots besides being longer are bushy. Details are furnished below:
Table.1.The impact of AM association on the root and shoot length and growth are furnished below.

Crop common name Shoot length (cm) Root length(cm) Shoot length(cm) Root length(cm)

Growth in Vermiculate after one month* Growth in Soil after one month*
Treated plants Untreated plants Treated plants Untreated plants Treated plants Untreated plants Treated plants Untreated plants
Cow pea 34 28 9 5 45 35 18 14
Moth Bean 38 29 10 6 40 34 16 14
Methi 22 16 9 7 24 20 14 9
Paddy 15 12 14 10 30 26 26 22
Sesamum 16 12 8 6 - - 12 9
Sorghum 12 8 14 12 15 12 20 14
Wheat 18 14 18 13 22 18 24 18

*Data represents average of two experiments [Three replications/per experiment (3 plants /replication )]
The benefit of the AM culture were tested for its impact on other parameters such as leaf length & width, no of lateral roots etc. The results of the study are furnished below.
Table.2.Bio-efficacy of Mycorrhiza for crop improvement in different crops (30 days)
S.No Growth parameters Paddy
(30 DAS) Wheat
(30 DAS) Groundnut
(30 DAS)
VAM Control VAM Control VAM Control
1 Germination (%) 75 75 100 100 75 75
2 Root length (cm) 18.4 17.1 7.3 6.1 27.0 23.0
3 Shoot length (cm) 19.6 14.1 32.7 33.5 11.5 9.0
4 No. of lateral roots 16.2 10.7 13.5 8.0 64.9 38.8
5 Root biomass (mg) 0.355 0.107 0.79 0.49 0.90 0.61
6 No. of leaves 7.5 5.2 21.3 18.7 9.9 7.7
7 Leaf length (cm) 13.4 9.3 24.1 23.5 4.2 3.4
8 Leaf breadth (cm) 0.5 0.5 7.7 7.0 1.6 1.5
9 Weight of the plant (g) 0.61 0.31 4.32 3.84 2.76 1.82

Table.3. Bio-efficacy of Mycorrhiza for crop improvement in different crops (35 – 38 days)
S.No Growth parameters Tomato
(35 DAS) Redgram
(35 DAS) Brinjal
(38 DAS)
VAM Control VAM Control VAM Control
1 Germination (%) 46 43 67 60 33.3 40
2 Root length (cm) 9.5 10.6 13.1 5.9 9.0 5.7
3 Shoot length (cm) 26.7 21.1 32.4 27.7 12.9 10.6
4 No. of lateral roots 26.7 21.1 22.1 11.5 42.7 28.0
5 Root biomass (mg) 0.33 0.15 0.21 0.26 0.11 0.17
6 No. of leaves 5.6 4.2 22.1 16.2 5.33 6.0
7 Leaf length (cm) 8.7 5.8 5.2 4.5 6.1 3.9
8 Leaf breadth (cm) 4.5 3.6 1.9 1.5 4.1 2.9
9 Weight of the plant(g) 6.3 3.3 2.01 1.63 2.7 2.7

Table.4. Bio-efficacy of Mycorrhiza for crop improvement in different crops (40 – 45 days)
1 Growth parameters Coriander
(40 DAS) Methi
(45 DAS)
2 VAM Control VAM Control
3 Germination (%) 70 30 90 90
4 Root length (cm) 8.7 3.9 34.4 26.6
5 Shoot length (cm) 12.2 4.2 27.7 24.9
6 No. of lateral roots 18.7 3.0 21.0 9.1
7 Root biomass (mg) 0.9 0.05 2.1 1.0
8 No. of leaves 12.7 4.7 42.7 32.7
9 Leaf length (cm) 10.5 4.2 2.2 2.9
10 Leaf breadth (cm) 3.0 2.0 2.2 2.4
11 Weight of the plant(g) 2.20 0.33 10.30 6.19

[0082] Although the present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[0083] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.