UNCULTIVABLE ENDOPHYTES"

TECHNICAL FIELD OF THE INVENTION:

The present invention relates to a process for the isolation of uncultivable endophytes from plant sources to obtain novel endophytes having plant growth and yield promoting attributes,biocontrol activity against insect pests and phyto- pathogens and to make host plants tolerant against abiotic stress conditions such as salinity, drought, temperature, pH etc. some unknown functions which may prove beneficial to plants.

Further, the present invention relates to bio-fertilizer compositions comprising uncultivable plant endophytes isolated by the present process.

BACKGROUND AND PRIOR ART OF THE INVENTION:

Endophytes are bacteria or fungi that, during part of their life, can survive inside a plant tissue without eliciting symptoms of disease. These endophytic microorganisms can reside internal to plant tissues while invading living tissues of the host plant deprived of producing any harm, with sometimes causing unapparent and asymptomatic infections. Myriad plant growth promoting bacterial endophytes (PGPBEs) have been identified which facilitate plant growth via three interrelated mechanisms: phyto-stimulation, bio-fertilization and bio-control.

The ability of diverse microbial endophytes to promote plant-growth occurs as a result of direct or indirect mechanisms. Direct promotion of plant growth occurs when either bacteria or fungi facilitate acquisition of essential nutrients or modulate levels of hormones within a plant. Nutrient acquisition facilitated by PGPBE typically includes nitrogen, phosphorus and iron. Modulation of hormone levels may entail PGPB synthesizing one or more phyto-hormones, i.e. auxin, cytokinin and gibberellin (Santoyo et al, Microbiol Res. 2016 Feb; 183:92-9). It has been estimated that 1 g of soil comprises 4000 to 5000 different bacterial "genomic units" based on DNA-DNA re-association, however, only 5000 bacterial species have been described and only 1% of soil microbes have been cultivable by standard laboratory practices. The general limitations in studying microbial diversity include:

(i) Limitations in methodology and lack of taxonomical knowledge;

(ii) Molecular techniques based on PCR raise issues such as cell lysis efficiency, hybridization efficiency, primer specificity, etc.

DNA-dependent meta-genomic techniques have revealed that over 99% endophytes remain to be isolated and identified. The conventional endophyte isolation on known synthetic media is not sufficient to obtain major portion of endophytes residing in various plant locations. Additionally, synthetic media are unable to allow growth of novel endophytes, which thus remains uncultivable. However, major part of endophytic microbial community holds great promise with respect to discovery of novel endophytes which would be exploited to achieve higher crop yield. These endophytes could possess enhanced bio-potential such as nutrient solubilisation, phytohormone production as well as harbour unknown beneficial attributes for host plants. Therefore, it becomes important to assess and evaluate effects of endophytes on host plant owing to plant/crop health.

The procedures for isolation and identification of endophytic bacteria from plant sources suffer from the difficulty or impossibility of absolute surface sterilization of external plant tissues. The impact of these problems can be reduced by using standardized protocols.

Using regular cultivation methods, 99% of bacterial species are not able to be cultured as they do not grow in conditions made in a laboratory, called the "Great Plate Count Anomaly". Presently, microbiological cultures are grown in solid agar- based growth media or in a liquid nutrient medium. After introduction of the starter bacteria into culture they are grown according to their nutritional requirements, temperature requirements, CO2/O2 optimal concentration, etc. However, many bacteria are fastidious, with very complex nutritional/ environmental requirements and culturing such bacteria is challenging. The idea of using synthetic media is still the foundation of microbial recovery and propagation. Moreover, microbiological techniques have not changed over the years compared to the rapid progress seen in genetics and molecular biology.

An isolation chip (iChip) was developed by Epstein et al. to grow "uncultivable" microorganisms and access novel bioactive molecules that might be developed as therapeutic antibiotics. The iChip applies an in situ cultivation model to isolate such inaccessible microorganisms. The iChip utilizes a system of hundreds of miniature diffusion chambers, each loaded with a single cell. The diffusion system allows the cells on the iChip to interact with naturally occurring nutrients and environmental factors. The development of an isolation chip, the 'iChip' (Epstein et al, Appl Environ Microbiol. 2010 Apr; 76(8): 2445-2450), has resulted in the culture of up to 50% microbes present in soil.

The iChip's are made of a central hydrophobic plastic poloxymethylene (POM) plate with an array of holes 1mm in diameter. It has been suggested by Kate Le Cocq in Molecular Plant Pathology (2017) 18(3), 469-473 that the isolation chip technology could be used to facilitate the culture of plant-associated microbiome. However, there have been no studies providing for isolation of plant endophytes by employing isolation chip.

US Patent No.7,011,957 relates to an isolation and cultivation of micro-organisms from natural environments and drug discovery based thereon. The growth chamber system used therein is sealed with a semi-permeable membrane which is permeable to components from the natural environment from which the sample is acquired, but is not permeable to cells of the microorganism. However, the environment to be tested for the presence of uncultivable microbes includes fresh water, seawater, sediments and soils. A large pool of uncultivable microbes from a habitat comprising a diverse microflora was under consideration in US'957, therefore resulting in isolation of non-target microbes.

However, there have been no attempts to isolate uncultivable endophytes from plant sources. Isolation and cultivation of uncultivable plant endophytes through in-situ incubation in the plant extract as a growth medium has not been attempted.

OBJECT OF THE INVENTION:

It is an object of the present invention to provide a process to isolate and cultivate uncultivable endophytes from different commercially important crop plants/wild- crop/medicinal plants/all type of plant material for use in enhancing crop yield, providing abiotic and biotic tolerance to crop/plants and other unknown functions which may be beneficial to plants/crops in agriculture.

SUMMARY OF THE INVENTION:

In an aspect, the present invention provides a process for the isolation and cultivation of uncultivable endophytes from plant extracts, the said process comprising;

(a) preparing a gel based nutrient medium comprising about 1% to about 10% of plant fresh weight (w/v) recovered as plant extract/juice after surface sterilization of roots, stems, leaves, flowers, fruits or seeds;

(b) contacting the central component of an isolation chip with the medium of step (a) to capture microbial cells embedded in the medium into the through holes;

(c) sealing the central plate with semi-permeable membranes and assembling the top plate and bottom plate symmetrically onto the central plate;

(d) incubating the assembled plates in a suspension of the plant extract to provide conditions suitable for growth of uncultivable endophytes for a duration ranging from 3 weeks to 4 weeks at a temperature ranging from about 25°C to about 30°C;

(e) examining the medium for presence of microbial colonies and sub-culturing cells onto a nutrient medium. Further, the microbial colonies isolated from the central plate are subjected to Random Amplification of Polymorphic DNA (RAPD) and subsequently sequencing of the 16S rRNA/ITS gene to identify if the endophyte isolates belong to the already cultivable range of species or the not yet cultivated strains of endophytes.

In another aspect, the present invention provides a kit for the detection and isolation of uncultivable endophytes from natural sources, the said kit comprising;

(a) an isolation chip comprising three components, i.e. a top plate, a central plate and a bottom plate,

(b) each plate having a pore size ranging from about 0.5 mm to about 5mm, and

(c) a minimum essential nutrient medium comprising carbon, nitrogen and other micronutrients.

DETAILED DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts the isolation chip being used in the present invention.

Figure2 depicts a schematic representation of the Isolation chip (Ichip) assembly for isolation of uncultivable endophytes from leaves/stem/roots/flower/seeds and fruits, (a) Central plate of the Ichip system placed in an agar based microbial suspension, (b) Ichip plate with microbial colony entrapped in a solid agar plug, (c) the central plate is sandwiched between four polycarbonate membranes and two side plates with coinciding matching holes of same dimensions. Finally all the components are assembled by tightening the screws; (d) the assembled Ichip is placed in its original environment (respective plant part juice/extract) for in situ incubation.

Figure 3 depicts Random Amplification of Polymorphic DNA (RAPD) profile of plant material sourced from Red gram seed

Figure 4 depicts RAPD profile of plant material sourced from Red gram fruit (pod) Figure 5 depicts RAPD profile of plant material sourced from Red gram stem Figure 6 depicts RAPD profile of plant material source from Red gram root Figure 7 depicts RAPD profile of plant material sourced from sugarcane leaf; wherein isolation chip is subjected to in-situ incubation for 4 weeks;

Figure 8 depicts RAPD profile of plant material sourced from sugarcane leaf; wherein isolation chip is subjected to in-situ incubation for 8 weeks;

Figure 9 (A) depicts RAPD profile of plant material sourced from Hibiscus leaf, wherein, microbial inoculum is subjected to in-situ incubation without Ichip;

Figure 9 (B) depicts RAPD profile of plant material sourced from Hibiscus leaf; wherein microbial inoculum is subjected to in-situ incubation with Ichip;

Figure 9(C) depicts RAPD profile of plant material sourced from Hibiscus flower

Figure 10 depicts Endo A, D and E showing antagonistic activity against

Pyricularia oryzae. Endophyte E corresponds to uncultivable endophyte from sugarcane leaf.

DETAILED DESCRIPTION OF THE INVENTION:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Source of biological material: Geographical location of plant materials being used during research work Cajanuscajan, Saccharum officinarum, Hibiscus rosasinensis - Wargal, District - Siddipet, State - Telangana, India Coordinates - 17°46'34"N 78°36'55"E

The term 'Uncultivable endophyte' referred to in the present invention are endophytic microorganisms which are unculturable and need culture-independent techniques to detect and identify the same.

The term 'uncultivable endophyte' in the present invention is also referred to as 'unculturable endophyte' for the purpose of the present invention. In a preferred embodiment, the present invention provides a process for the isolation and cultivation of uncultivable endophytes from plant extracts, the said process comprising;

(a) preparing a gel based nutrient medium comprising about 1% to aboutl0% of plant fresh weight recovered as plant juice/extract after surface sterilization of roots, stems leaves, flower, fruits or seeds;

(b) contacting the central component of an isolation chip with the medium of step (a) to capture microbial cells embedded in the medium into the through holes;

(c) sealing the central plate with semi-permeable membranes and assembling the top plate and bottom plate symmetrically onto the central plate;

(d) incubating the assembled plates in a suspension of the plant extract to provide conditions suitable for growth of uncultivable endophytes for a duration ranging from 3 weeks to 4 weeks at a temperature ranging from about 25°C to about 30°C;

(e) examining the medium for presence of microbial colonies and sub-culturing cells onto a nutrient medium.

The gel based nutrient medium comprises plant fresh weight recovered as a plant extract. The plant extract is selected from the group comprising a suspension or slurry of crushed leaves, fine powdered seed material or germinated seed, stem, flower sap and fruit pulp. About 60% to 80 % of the plant fresh weight was recovered as juice which was further filtered through 0.2 μπι membrane to ensure complete sterility in media for in-situ incubation of the isolation chip. Accordingly, the nutrient medium employed in step (a) of the present process comprises about 1% to aboutl0%of the filtered plant extract.

Alternatively, the in-situ incubation medium used in step (d) is artificially prepared in accordance with defined medium constituents.

In accordance with step (d) of the preferred embodiment, the suspension of plant extract for in-situ incubation was prepared from surface sterilized plant material (root/stem/leaves/seeds/flower and fruits) which was employed in the gel based nutrient medium of step (a) of the present process by blending with equal volume of sterile water (w/v) to maintain sterile conditions.

For the purposes of the present invention, the description of the isolation chip is provided. The isolation chip (also termed Ichip) is an assembly of a plurality of flat plates containing multiple registered through-holes (Fig 1). The plates are manufactured from blocks of hydrophobic plastic Acrylonitrile butadiene styrene (ABS). Alternatively, the plates constituting the isolation chip were prepared using autoclavable plastic material Polypropylene (PP).

More specifically, the isolation chip comprises 3 plates such that the through holes contained in each plate are placed symmetrically over each other.

The dimensions of each of the plates are as follows:

(a) central plate of 99 by 52 by 1 mm, and

(b) two symmetrical top and bottom plates of 99 by 52 by 7 mm (Fig 1).

The latter plates, i.e. top and bottom plates have ridges providing rigidity and have multiple through-holes having diameter ranging from 0.5 to 5 mm (corresponding to central plate), arranged in two arrays with 95 through-holes per array. After symmetric assembly of each of these three components, all holes in each plate are in sync. The size of the array is such that it can be completely covered by standard membranes having diameter ranging from about 25mm to about 50mm. The pore size of the polycarbonate membranes is ranging from about 0.02 μπι to about 0.05 μπι. More specifically, the assembly comprises a combination of 190 miniature diffusion chambers, containing on an average (theoretically) one cell per through- hole.

Prior to isolation and cultivation of the microbial cells, the components of the isolation chip and the plant part to be used are surface sterilized. Subsequently, the central plate of the isolation chip is immersed into a suspension comprising surface sterilized plant extract in a liquid agar-based medium for 5 min (Fig. 2 a). Each through-hole of the central plate captures a volume of suspension containing a definite number of cells (Fig. 2 b). Microbial cells were immobilized inside small agar plugs which are formed once the agar solidifies. The central plate of the isolation chip inserted with the microbial cells was removed from the agar based medium. The central plate with microbial cells trapped in its pores was carefully assembled by first placing O.C^m-pore-size, 47-mm polycarbonate membranes onto each array of through-holes from both sides of the central plate to prevent cell migration from in and out of the agar plugs. Finally, the top and bottom plates were applied and aligned and screws were tightened to provide pressure. Subsequently, the assembled Ichip was transferred to a liquid plant material extract suspension in order to provide in situ incubation. It provides the immobilized cells with their naturally occurring nutrients and growth factors. (Fig. 2d). The entire assembly of the isolation chip was incubated for 3-4 weeks at a temperature of about 25-30 °C. After incubation, Ichips were washed vigorously in particle-free DNA-grade water and disassembled.

The sap/juice inoculum was also incubated without Ichip, wherein the sap was inoculated in sterilejuice/sap for similar incubation time as that of Ichip. Herein the enrichment of uncultivable endophytes was achieved without using Ichip. After incubation, 100 μΐ of endophyte suspension from enrichment was plated onto nutrient agar plates to obtain enriched endophytes. The endophytes so obtained were compared by RAPD profiles and 16S rRNA/ITS gene sequence, with ones being isolated from Ichip and the endophytes obtained without enrichment protocol.

The present invention further provides isolation of endophytes from plant extracts of surface sterilized leaves, stems roots, flower, seeds and fruits, therefore resulting in the isolation of plant growth promoting (Nutrient solubilisation and phyto hormone production) endophytes/abiotic /biotic control endophytes. In general plants were selected from regions being reported for higher yield or microbial infestation (biotic stress) or abiotic stress like drought/salinity. The leaf, stem, root, flower, seed and fruit surfaces used for isolation of endophytes are selected from the group comprising but not restricted to chilli, rice, maize, sugarcane, tea, cardamom, wild plants. Further, hybrid and regular seeds of cotton, chilli and other plant sources were used for the purposes of the invention.

In the isolation step (the inoculums for Ichip) the nutrient medium employed for the cultivation of endophytes is a minimal essential nutrient medium comprising a carbon source, nitrogen source and micronutrients. For enrichment of uncultivable endophytes, respective plant material extract/sap is used for incubation. However, plant material extract (SAP) of one plant can also be used as in situ incubation media for isolation of endophyte from other plants. Accordingly, the medium used for in-situ incubation is a sterile suspension/slurry of crushed leaves, fine powdered seed material or powdered germinated seeds, extract of stems/stem/flower sap and fruit pulp respectively.

In an embodiment, the present invention provides an isolation chip comprising three components, i.e. a top plate, a central plate and a bottom plate, wherein each plate comprises a co-inciding wells having diameter ranging from about 0.5mm to about 5mm. The present assembled Isolation chip allows micro-encapsulation of microbes and at the same time inhibits environmental contamination in course of in-situ incubation.

In another preferred embodiment, the present invention provides a kit for the detection, isolation and cultivation of uncultivable endophytes from natural sources, the said kit comprising;

(a) an isolation chip comprising three components, i.e. a top plate, a central plate and a bottom plate, each plate having a pore size ranging from about 0.5 mm to about 5mm;

(b) a minimum essential nutrient medium comprising carbon, nitrogen and other micronutrients.

In another embodiment, the present invention provides the central plate of the isolation chip assembly comprising a plurality of wells/through holes having diameter ranging from about 0.5mm to about 5mm, more preferably having diameter ranging from about 1mm to about 3mm and most preferably having diameter of about 2mm.

Further, the microbes isolated from central plate are subjected to Random Amplification of Polymorphic DNA (RAPD) and subsequently sequencing of the 16S rRNA/ITS gene to identify if the endophytes isolates belong to the already cultivable range of species or the not yet cultivated strains of endophytes. In case when endophytes isolated from Ichip are not differing from cultivable pool of endophytes in 3-4 weeks period or very few are differing, it suggests uncultivable endophytes from the used inoculum did not get enough in situ incubation. In order to increase/enrich the number of uncultivable endophytes, in-situ incubation period for same inoculum via Ichip was increased from 1 month to 3 months. Multiple Ichips were inoculated with same inoculum and every month one Ichip was harvested for isolation of endophytes. After each harvest, incubation media was also changed so as to avoid limitation of nutrients in course of incubation. This strategy proved to be helpful in yielding higher number of uncultivable endophytes from same inoculum. It proves the hypothesis that with increase in in-situ incubation more number of uncultivable endophytes came to cultivability due to adaptation.

The present invention using Ichip is useful in isolation of both cultivable and uncultivable endophytes followed by their identification via 16S rRNA/ITS gene sequencing. On the contrary, metagenomics approach (WGS/amplicon) could help only in identification of endophytes but not the cultivation/isolation. However, metagenomics approach could be used to profile complete endophyte diversity from plant tissue which would aid and support data on uncultivable endophytes obtained using Ichip technology.

In yet another preferred embodiment, the present invention provides isolation of microbial strains selected from the group consisting of Azoarcus sp, Azospirillum lipoferum, Burkholderia, Enterobacter, Gluconacetobacter, Pseudomonas, Stenotrophomonas, Bacillus, Serratia and Microbacterium but not restricted to these Genera/endophytes.

In another embodiment, the present invention provides endophytes having at least one of the following functions;

(a) efficient nutrient uptake via nitrogen fixation, solubilization of phosphorus, potassium, zinc and silica and other nutrients;

(b) producing phyto-hormones selected from Indole acetic acid (IAA), Gibberellic acid (GA) and the like;

(c) Biocontrol potential such as fungicidal and insecticidal activities;

(d) providing tolerance against biotic stress involving insects and microbial pathogenesis;

(e) providing tolerance to abiotic stress such as drought, temperature and salinity for crops; and

(f) enhancing plant immunity against biotic/abiotic stress.

Accordingly, endophytes possessing nitrogenase enzyme cluster fix inert nitrogen gas from atmosphere into H3. Subsequently, NH3 is converted into NO2 & NO3 for further assimilation into plant system.

As regards solubilization of minerals, the isolated endophytes secrete different organic acids, viz., glucanic acid, oxalic acid, citric acid, formic acid, tartaric acid, malic acid, lactic acid, succinic acid, acetic acid, etc. The organic acids released decrease pH of the medium and release minerals from their respective insoluble ores. Furthermore, endophytes also release enzymes such as phytase/phosphatase which help in solubilizing insoluble phosphate source i.e. phytic acid and thus help in assimilation of soluble phosphate to plant system. Endophytes also help in iron acquisition for host plants by releasing high affinity siderophores. Apart from above mentioned nutrients, endophytes can also help plants in acquiring other micronutrients such as Ca, Mg, S, Mn, Cu, B, Mo, CI and Co. Endophytes can produce phytohormones such as Indole acetic acid (IAA), cytokinin and Gibberllic acid (GA) which are growth promoters for plants. These hormones help in germination of seeds followed by overall increased vegetative growth.

As regards biocontrol potential, endophytes employ multiple mechanisms to remediate biotic stress to plants. Several antibiotic, antimycotic, antiviral, antioxidant, nematicide, insecticide and immunosuppressive compounds have been reported from endophytes for bacterial phyto-pathogens endophytes produce secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), HCN etc. which act as antibiotics. Moreover, siderophore being released by endophytes helps against phytopathogen by quenching available iron from media, thus iron becomes limiting factor for pathogen growth. Similarly, metabolites such as surfactin, fengycin, subtilin etc. can be released by endophyte in response to fungal phytopathogens attack. Endophytes also release hydrolyzing enzymes such as chitin, lysozyme etc. in order to save plant against fungal pathogens. Additionally, metabolites such as cytochalasines, ambuic acid, oocydin, jesterone, cryptocandin, lolitrem B, and 3-hydroxypropionic acid and taxol, etc. are other useful weapons conferred by endophytes to plants against phytopathogens. Moreover, endophytes can also produce some toxic alkaloids and protect their hosts from herbivores. For plant insects/pests, volatile secondary metabolites such as naphthalene released by endophytes act as deterrent/repellent for the insects. Moreover, toxins such as flavotoxin are also produced by endophytes which if ingested by insect, kill the pest by septicemia. In respect of providing tolerance to abiotic stress such as drought, temperature and salinity for crops; endophytes produce an enzyme ACC deaminase which halts after effects of abiotic stress to plants. When a plant is exposed to biotic stress such as drought/high salinity,the phytohormone ethylene is released which mediates all negative effects of stress involving leave fall, shrinking etc. ACC deaminse cleaves substrate i.e. ACC for ethylene production and thus limits the concentration of this hormone in plant. In order to help plant to resist high salinity conditions, osmolytes such as glycine, salicylic acid etc. counteract against osmolyte imbalance brought about by high salinity conditions. Enhancing plant immunity against biotic/abiotic stress wherein endophytes release small molecular weight phytohormones such as Jasmonic acid (JA), salicylic acid (SA) which enhances overall plant immunity of plants against different biotic and abiotic stress conditions. Phytohormones act at gene expression level and thus regulate enzymes such as hydroxylase, oxygenase etc. being involved remediating stress conditions. JA exerts direct control over production of chemical defence compounds that confer resistance to a remarkable spectrum of plant-associated organisms, ranging from microbial pathogens to vertebrate herbivores.

Owing to novelty of uncultivable endophytes, novel and yet to be characterized attributes, will be explored which might be playing important role in overall plant yield/health. For instance, endophytes secrete an array of biochemical, among which very few are characterised for their role in plant growth promotion eg. IAA is a phytohormone for growth. Similarly, novel endophytes would possess solubilisation potential of some more unknown minerals or on the contrary novel mechanism of known mineral solubilisation. Some unknown bio-controlling mechanisms can also be explored against yet to be explored phyto-pathogens. Similarly, unknown mechanisms against different abiotic stress conditions can be explored from uncultivable endophytes. Thus, uncultivable endophytes hold great possibilities to possess different and novel biomolecules with unknown biopotentials leading to enhanced growth.

Endophytes imparting enhanced phenotypic traits/characters such as plant height, fruit weight, leaf width etc. These enhanced traits can be acquired by plants owing to biochemical signals/elicitors being released or some other unknown mechanisms carried out by endophytes.

The endophytes isolated by the present process are tested for the aforementioned activities. Accordingly, the nitrogen fixation potential was evaluated by estimating the fixed nitrogen by endophytes in nitrogen free Ashby's media broth; phosphorus solubilization activity by using BRIP media; potassium solubilization activity by using Aleksandrov media; zinc solubilization activity by using Bunt and Rovira media possessing ZnO as insoluble Zinc sources; silica solubilization activity by using Bunt and Rovira media possessing Magnesium trisilicate as insoluble silica source; Indole acetic acid (IAA) production by using MS media supplemented with Tryptophan; Gibberlic acid (GA) production; (1- Aminocyclopropane-l-Carboxylate) ACC deaminase Activity, by using the protocol given by Glick (2003); Salt and temperature tolerance assays; Antagonistic activity against selected phytopathogens and insecticidal bioassays.

In a further embodiment, the present invention provides a bio-fertilizer composition comprising endophytes or microbial consortia isolated by the present process.

The composition can be formulated as in the liquid, powder or granule form and can be applied through direct broadcasting on soil, foliar application and drip method or through fertigation. Further, the bio-fertilizer composition further comprises the in-situ incubation medium specific for the plant part isolated from as a carrier.

Advantages of the invention:

• The assembled isolation chip of the present invention allows microencapsulation of microbes and simultaneously inhibits environmental contamination owing to 0.03 μπι pore membrane.

• Isolation and cultivation of endophytes by the present process from commercially important crop plants can indicate their efficiency in plant growth promotion (biopotential)/biotic and abiotic tolerance

• Cultivating endophytes by the present method can help study the mechanisms by which they benefit host plants and their potential application in sustainable agriculture. • The selection of target uncultivable microbes i.e. endophytes confer an additional novel advantage in isolation strategy wherein source of microbes i.e. plant materials are naturally enriched with selective microbes.

• The present process addresses a specific niche including host plant selective microbial endophyte pool. This gives an advantage with respect to target uncultivable microbes and avoids non-target microbes from isolation process.

• Surface sterilization process prior to Ichip protocol mentioned herein, ensures removal of non-target microbes and thus more selectivity towards endophytes.

• The present process is superior to metagenomics approach (WGS/amplicon) wherein in addition to uncultivable endophyte identification Ichip also allows its isolation.

Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1: Surface sterilization /Autoclave of isolation chip

Prior to isolating the assembling the isolation chip for microbial incubation each of the components were surface sterilized in ethanol, air dried in a Laminar Flow unit and rinsed in particle-free DNA-grade water. The isolation chip was manufactured using Polypropylene (PP) thereby making it autoclavable and used further as mentioned below.

Example2: Preparation of plant extract comprising liquid agar based medium

The surface of the leaves/stem/root/flower/seed and fruit of an healthy plant sample were sterilized by first immersing the leaves/stem/root/flower/seed and fruit in 1% Sodium hypochlorite for 10 min followed by 70% ethanol for 2 minutes. Subsequently, leaves were washed vigorously 2-3 times with autoclaved sterile distilled water. The plant parts were washed vigorously with particle-free DNA- grade sterile water. Surface sterilization step ensured effective removal of non- target microbes thus giving selectivity for isolation of endophytes. Plant materials were also processed without surface sterilization protocol. Samples without surface sterilization were processed to evaluate utility of surface sterilization by using chemicals. In order to prepare inoculum for Ichip, 1-10 % of surface sterilized leaves/stem/root/flower/seed and fruit(w/v) were crushed in sterile PBS and different dilutions (10 ^"2 to 10 ^"6 ) were plated on nutrient agar plate to enumerate the CFU from used plant sample. Same procedure was employed to process root, stem, seed, flower and fruit samples of the plant. Based on CFU count the same inoculum was diluted with 0.8% melted agar with minimum growth media, so as to achieve CFU of countable numbers. This was done so that cultivable endophyte can be obtained for later comparison with uncultivable endophytes after in-situ incubation via Ichip.

Example3: Process for isolation and cultivation

The central plate of the isolation chip was immersed into a suspension comprising surface sterilized plant extract in a liquid agar-based medium for 5 minutes (Fig. 2 a). Each through-hole of the central plate captures a volume of suspension containing a definite number of cells (Fig. 2 b). Microbial cells were immobilized inside small agar plugs which were formed once the agar solidifies. The central plate of the isolation chip inserted with the microbial cells was removed from the agar based medium in sterile environment so as to avoid environmental contamination. The central plate with microbial cells trapped in its pores was carefully assembled by first placing Ο.ΟΒμπι-ροΓε-βίζε, 47-mm polycarbonate membranes onto each array of through-holes from both sides of the central plate to prevent cell migration from in and out of the agar plugs. Finally, the top and bottom plates were applied and aligned and screws were tightened to provide pressure. Subsequently the assembled Ichip was transferred to a liquid plant extract suspension in order to provide in situ incubation. It provides the immobilized cells with their naturally occurring nutrients and growth factors. (Fig. 2d). The entire assembly of the isolation chip was incubated for 3-4weeks at a temperature of about 27°C.The incubation of Ichip was carried out in presence of specific chemicals/stimulants/elicitors so as to achieve selectivity for particular group of endophytes. In order to selectively isolate endophytes which can utilize ACC as sole energy source, Ichip incubation media was supplemented with ACC. Similarly, for salt tolerant endophytes, the incubation was carried out at higher salinity in the medium (supplemented with NaCl). Tryptophan was supplemented in incubation media to selectively enrich IAA producing endophytes in Ichip. After incubation, Ichips were washed vigorously in particle-free DNA-grade water and disassembled.

The incubation of inoculum in in-situ conditions was also carried out without using Ichip. Herein, the conventional enrichment protocol was employed in addition to use of in-situ conditions in presence of specific chemicals/stimulants/elicitors as mentioned above for Ichip incubation. As shown in figure 9A and 9B, the in-situ incubation without Ichip also yielded uncultivable endophyte, although less in number as compared to the number of uncultivables from Ichip corresponding to same inoculum". This shows efficiency of Ichip design to enrich uncultivable endophytes from an environmental sample viz. endophytic community.

Example 4: Isolation and cultivation of endophytes from seeds

(a) Preparation of inoculum from seeds

Seeds for isolation of endophytes were selected from healthy crop plants (standing crop). For the purpose of isolation of endophytes from seeds, either of the following methods was used;

(i) Surface sterilized/washed crushed seeds were used as inoculums for Ichip followed by in-situ incubation in powdered seed suspension or a synthetically prepared composition, or

(ii) Surface sterilized seeds were allowed to germinate in sterile conditions followed by preparation of a slurry/suspension preparation of 8-10 days old saplings. This suspension was used as an inoculum and subsequently as amedium for in-situ incubation in Ichip protocol.

(b) In-situ incubation medium when seeds are source of isolation of endophytes The medium used for in-situ incubation of the seed endophytes was as mentioned in Example 4(a) for preparation of inoculum. A powdered seed suspension can also be used as a general incubation medium for multiple crop seeds. Furthermore, an artificial medium was optionally also used having a similar constituent make up to seed extract used for endophyte isolation. The artificial medium contains sugars not limited to glucose, fructose, sucrose, fructan, and xylose; amino acids not limited to arginine, histidine, leucine, isoleucine, and methionine and minerals not limited to Ca, Mg, P, K, Na, Fe, and Mn.

After 4 week in-situ incubation, 11 colonies were isolated from Ichip followed by DNA extraction and PCR using RAPD primer no. 60 [(CAG) ₄ ].

Based on RAPD profile of endophytes isolated from red gram seed as inoculum and Ichip as in-situ incubation device, 2 endophytes were found to be different than group of cultivable endophytes (Fig. 3). Thus, based on genetic variance these 2 endophytes can be considered as previously being uncultivable, which transformed into cultivable due to in-situ incubation. Remaining 9 endophytes showed RAPD profile homology with the control cultivable endophytes. It shows that Ichip could also support growth of cultivable endophytes.

The use of artificial plant tissue based media for in-situ incubation could not support enrichment of uncultivable endophyte. This was validated by RAPD profile based data, wherein the endophytes which grew in artificial media showed genetic homology with the control cultivable endophytes (from same source of inoculum). The reason can be attributed to chemical composition differences between actual plant part juice and the artificial media (constituted using different chemicals). However, this artificial media could support growth of cultivable endophyte, thus can be used in development of plant based growth media for endophyte isolation.

Example 5: Isolation and cultivation of endophytes from fruit pulp

(a) Preparation of inoculum from fruit pulp Fresh fruits were selected from plant at fruiting stage for isolation of endophytes. The epidermis of the fruits was surface sterilized in 1% Sodium hypochlorite for 10 min followed by 70% ethanol for 2 minutes. Washed but unsterilized fruit sample was also processed. Subsequently, they were washed vigorously 2-3 times with autoclaved sterile distilled water. The fruit pulp was extracted and used for further steps.

(b) In-situ incubation medium constituents when fruit is a source of isolation of endophytes

The medium used for in-situ incubation of the fruit endophytes was as mentioned in Example 5(a) for preparation of inoculum. The fruit pulp of a plant species can also be used as general incubation medium for isolation of endophytes from different fruits.

After 4 week in-situ incubation, 22 colonies were isolated from Ichip followed by DNA extraction and PCR using RAPD primer no. 60 [(CAG ]. Based on RAPD profile of endophytes isolated from red gram fruit (pod) as inoculum and Ichip as in-situ incubation device, 4 endophytes were found to be different than group of cultivable endophytes (Fig. 4). Thus, based on genetic variance these 2 endophytes can be considered as previously being uncultivable, which transformed into cultivable due to in-situ incubation.

Example 6: Isolation and cultivation of endophytes from stem sap

(a) Preparation of inoculum from stem sap

Stems for isolation of endophytes were selected from healthy crop plant (standing crop). The stem sample was surface sterilized in 1% Sodium hypochlorite for 10 min followed by 70% ethanol for 2 minutes. Subsequently, they were washed vigorously 2-3 times with autoclaved sterile distilled water. In parallel, unsterilized but washed samples were also processed. The stem sap was extracted by crushing it in sterile mortar and pestle and used for further steps. (b) In-situ incubation medium constituents when stem is a source of isolation of endophytes

The medium used for in-situ incubation of the stem endophytes was as mentioned in Example 6(a) for preparation of inoculum. The stem sap from one crop plant can also be used as a general incubation medium for isolation of endophytes from multiple crop stem samples.

After 4 week in-situ incubation, 30 colonies were isolated from Ichip followed by DNA extraction and PCR using RAPD primer no. 60 [(CAG ]. Based on RAPD profile of endophytes isolated from red gram stem as inoculum and Ichip as an in- situ incubation device, 6 endophytes were found to be different than group of cultivable endophytes (Fig. 5). Thus, based on genetic variance these 2 endophytes can be considered as previously being uncultivable, which transformed into cultivable due to in-situ incubation.

Example 7: Isolation and cultivation of endophytes from root and leaf

(a) Preparing inoculum from root and leaf sap

Plant parts for isolation of endophytes were selected from healthy crop plant (standing crop). The samples were surface sterilized in 1% Sodium hypochlorite for 10 min followed by 70% ethanol for 2 minutes. Subsequently, they were washed vigorously 2-3 times with autoclaved sterile distilled water. In parallel, unsterilized but washed samples were also processed. The plant tissue sap was extracted by crushing it in sterile mortar and pestle and used for further steps.

(b) In-situ incubation medium constituents when root and leaf are source of isolation of endophytes

The medium used for in-situ incubation of the root and leaf endophytes was as mentioned in Example 7(a) for preparation of inoculum.

After 4 week in-situ incubation, 16 and 31 colonies were isolated from Ichip from root and leaf respectively. Subsequently, DNA was extracted from each culture and PCR performed using RAPD primer no. 60 [(CAG) ₄ ]. Based on RAPD profile of endophytes isolated from seed as inoculum and Ichip as in-situ incubation device, 4 endophytes each from root and leaf, were found to be different than group of cultivable endophytes (Fig. 6 and 7). Thus, based on genetic variance these 4 endophytes can be considered as previously being uncultivable, which transformed into cultivable due to in-situ incubation.

Additionally, in order to check effect of long period in-situ incubation, one Ichip with endophyte inoculum from leaf was incubated for 8 weeks (i.e. two times than regular protocol). Based on RAPD profile, it was found out that longer incubation promoted isolation of more number of uncultivable endophyte (Fig. 8)

Furthermore, in order to evaluate whether uncultivable endophytes can be isolated without using Ichip, plant extract inoculum was incubated in in-situ condition without Ichip. Here, enrichment was paralleled with control experiment with same inoculum but incubated via Ichip. Fig 9 (A) & (B) illustrates RAPD profile of control cultivable and uncultivable endophyte from Hibiscus leaf sample. When incubated via Ichip, a double number of uncultivable endophytes were obtained. Although less in number, enrichment protocol also yielded uncultivable endophytes.

Example 8: Isolation and cultivation of endophytes from flower

(a) Preparation of inoculum from flower sample

Flower for isolation of endophytes were selected from healthy crop plant (standing crop). The flower samples was surface sterilized in 1% Sodium hypochlorite for 10 min followed by 70% ethanol for 2 minutes. Subsequently, they were washed vigorously 2-3 times with autoclaved sterile distilled water. In parallel, unsterilized but washed samples were also processed. The flower sap was extracted by crushing it in sterile mortar and pestle and used for further steps.

(b) In-situ incubation medium constituents when flower is a source of isolation of endophytes The medium used for in-situ incubation of the flower endophytes was as mentioned in Example 8(a) for preparation of inoculum. The flower sap from one crop plant can also be used as general incubation medium for isolation of endophytes from multiple crop flower samples.

After 4 week in-situ incubation, 30 colonies were isolated from Ichip followed by DNA extraction and PCR using RAPD primer no. 60 [(CAG ]. Based on RAPD profile of endophytes isolated from seed as inoculum and Ichip as in-situ incubation device, 5 endophytes were found to be different than group of cultivable endophytes (Fig. 9). Thus, based on genetic variance these 5 endophytes can be considered as previously being uncultivable, which transformed into cultivable due to in-situ incubation.

Example 9: Purification and identification of microbial colony

The Agar plugs from the central plate of the isolation chip were extracted with unwound and sterile no. 1 gauge paper clips for further sub-culturing in solid agar media plates. Sub-culturing was done to ensure the purity of the microbial colonies that were isolated. On identifying a pure culture, the micro-organisms were processed for DNA extraction. The PCR compatible genomic DNA was extracted using a DNA isolation kit. All the isolates obtained after in situ incubation via Ichip were subjected to RAPD [Rapid Amplified Polymorphic DNA] analysis to check variance in genetic makeup i.e. to remove redundancy among isolates. At the same time these Ichip isolates are checked against control cultivable endophytes comprising other strains of Bacilli and Microbacterium from the same source of isolation.

If the RAPD profiles of endophytes from Ichip were observed to be similar with that of cultivable endophytes, those endophytes were discarded. However, the endophytes with RAPD profile which is totally dissimilar to the control cultivable endophytes were retained for further DNA sequencing based identification. The differentiation of endophytes was based on RAPD profiling (qualitative method). 16S rRNA/ITS gene sequencing was done in order to differentiate and validate quantitatively the novelty of uncultivable endophytes as compared to cultivable endophytes. The phylogenetic analysis based on 16S rRNA/ITS gene sequences of isolated endophytes revealed difference at species level among the cultivable and uncultivable endophytes. One such uncultivable endophyte was characterised at taxonomic level by sequencing 16S rRNA gene.

Uncultivable endophytes were characterised for bio potential with respect to nutrient solubilisation and phytohormone producing activities. Uncultured Pseudomonas sp. EndoE was found to produce Indole Acetic Acid (IAA) at 85 mg/L cone, being highest among all the isolated endophytes (including cultivable and uncultivable). The same culture was also found to possess biocontrol activity against phyto pathogen Pyricularia oryz e. (Figure. 10)

Example 10:

(a) Determining biocompatibility among screened endophytes

The best endophytes for respective activities were selected for use in consortia designing. However, prior to preparation of a single microbial formulation it was important to determine the bio compatibility amongst microbes. The microbial members in a given formulation were determined to not have antagonistic activity against each other. At the same time they were determined to grow simultaneously in a single environment.

(b) Consortia designing/efficacy checks

Post determining the biocompatibility of the screened endophytes, the microbial consortia was designed based on its attributes. A biofertilizer formulation was indicated to harbor the best endophytes responsible for nutrient supply and phytohormone production. Similarly, an efficient biocontrol product would possess endophytes showing antagonistic effect against phyto-pathogens. Insecticidal endophytes would comprise separate microbial consortia.

(c) Determining stability and efficacy of formulation The formulated products were evaluated for their viability /stability in accelerated temperature in order to establish the shelf life. Efficacy in terms of plant growth promotion and amelioration of biotic/abiotic stress conditions based on its compositions were determined. The evaluation of growth promotion efficacy was done via field trials with multiple crops and locations. The crops selection for evaluation of biofertilizer formulations was done (i) based on source of endophytes present in consortia, thus checking product for crop specificity and (ii) one formulation for multiple crops to see versatility in product compatibility, thus checking whether endophyte from one plant can work efficiently for different plants.