4. DESCRIPTION (description shall start from the next page)

The field of this invention is agriculture, biological control of plant parasitic nematodes in integrated pest management, more specifically, scale-up production, down-stream processing and development of formulation of biological control agents (antagonistic fungi, more specifically the beneficial fungus, Pochonia chlamydosporia var. chlamydosporia) used therein.

Growing awareness among farming community and the demand from consumers for organically produced farm products (vegetables, fruits, mushrooms, beverages, food grains etc.) with minimum or pesticide residue-free products strengthened the cause of biological control as a component of Integrated Pest Management. Biological control agents are ecologically viable alternatives to conventional chemicals for management of plant diseases & nematodes.

Among several plant parasitic nematodes reported globally and within the country, root- knot and cyst nematodes are serious problems in several crops. Root-knot nematodes (Meloidogyne spp.) are widely distributed with wide host range and cause serious crop losses to field, horticultural, ornamental and medicinal & aromatic crops. Important susceptible crops are rice, cowpea, pigeon pea, tobacco, cotton, ginger, grapevine, citrus, banana, pomegranate, guava etc., cucurbits (gherkin, cucumber, melon), tomato, potato, brinjal, okra, beetroot, capsicum, carrot, palak, carnation, gerbera, gladioli, rose, chrysanthemum etc. Cyst nematodes cause significant yield losses in pigeon pea, potato, wheat, barley, etc. Damage due to nematodes increase multiple fold when they are associated with wilt bacterium (Ralstonia), and wilt fungi (Fusarium, Rhizoctonia, etc.) in infested soil, which can cause total crop losses by breaking the natural wilt-tolerance in plant germplasm. The overall average yield losses in different crops due to nematodes were estimated to be 10% amounting to US$100 billion.

Due to the phase out of effective but ecologically harmful fumigant nematicides for the control of plant parasitic nematodes, a potential market for a biological nematicides (based on antagonistic fungi) has opened up. Soil-borne beneficial fungi belonging to Hyphomycetes/Deuteromycales viz., Paecilomyces lilacinus, Pochonia chlamydosporia etc. have already demonstrated efficacy against root-knot, cyst, reniform nematodes and other important groups of plant parasitic nematodes. Antagonistic fungi may be introduced into a plant's environment in a number of different ways, including addition to soil, application directly to a plant's foliage, and the like (Whipps et ai, 1993). Biopesticides occupy 3-5% of the pesticide market (Mukhopadhyay, 1996). It has been estimated that annual requirement of Trichoderma powder formulation for seed treatment of pulses and oil seeds alone is 54,000 tonnes for the country, keeping aside the requirement for soil application for other soil-borne pathogens and
nematodes (Jeyarajan and Angappan, 1998).

The use of antagonistic fungi has now become an integral part of crop protection. Several antagonistic and entomopathogenic fungi are already available in the market as formulations in the world, while the availability of efficacious, cost-effective and registered formulations with long shelf-life is limited to a few in India. Talc formulations of Trichoderma spp. are the only widely proclaimed antagonistic fungus-based bioagent products available in the Indian market, opening a wide scope for expansion of the market for bio-nematicides and bio-insecticides. The production systems evolved for Trichoderma were suitable under liquid and submerged conditions, which to a large extent, did not suit other fungi leading to switching over to other systems of production as in case of another very successful fungal production system, namely, LUBILOSA. Recently, in USA, Australia and some parts of S-E Asia Paecilomyces lilacinus 251 has successfully been registered as a bionematicide. Paecilomyces lilacinus is a soil-borne fungus and well-known egg parasite which attacks in some extend also juveniles and sedentary females of several species of nematodes such as Meloidogyne spp., Globodera rostochiensis, Radopholus similis, Pratylenchus spp., Heterodera spp., Rotylenchulus reniformis, Tylenchulus semipenetrans. A formulation with a strain selected in Australia, P. lilacinus strain 251, is used in banana, tomato, tobacco, potato and other crops in South Africa and the Philippines. Mediterranean trials in tomato and cucumber are promising and show the interest for use as a biocontrol agent in both integrated crop management and organic farming (Laffranque, J. P., Pi, M. O., Decroos, Y., Aertens, F. Biological control of nematodes with the soil-borne fungus Paecilomyces lilacinus strain 251. Colloque international tomate sous abri, protection integree - agriculture biologique, Avignon, France, 17-18 et 19 septembre 2003).

Literature on Pochonia chlamydosporia as a biological control agent against plant parasitic nematodes is extensive (Bourne et al., 1996; Kerry, 1985; Kerry and Bourne 1996 and 2002; Leij et al., 1992; Leij et al., 1993; Biological Control of Plant Parasitic Nematodes ed. Stirling, G.R. CAB International (1991)) and is now widely accepted natural control agent against root-knot and cyst nematodes in a wide variety of crop rhizospheres and offers immense scope for commercial production and large scale use in field as alternative to chemical control. Recent developments in solid-state fermentation and formulation technology provide the opportunity to successfully commercialize these fungal biological control agents and introduce them into a world wide market (Kiewnick, S. Biological control of plant parasitic nematodes with Paecilomyces lilacinus, strain 251. Bulletin OILB/SROP, 2004 (Vol. 27) (No. 1) 133-136).

In India, several native germplasm of Pochonia chlamydosporia, a beneficial fungus belonging to Hyphomycetes have been isolated and characterized as an effective biological control agent of cyst, root-knot, and reniform nematodes which are serious soil-borne biotic limiting factors of several crops (Nagesh and Janakiram 2004a; Nagesh et al., 2006b; Nagesh et a/., 2007a and 2007b, Reddy and Nagesh, 2002, Rao et al., 2002, 2005).

Objective of the invention

• To develop authentic silicates-mixture based amorphous formulation of beneficial fungus Pochonia chlamydosporia var. chlamydosporia with the chlamydospores of the fungus as active ingredients and defined conditions for formulation for long shelf-life for application in biological (non-chemical) means of control of plant parasitic nematodes (more specifically root-knot and cysts nematodes) as a component in integrated pest management practices.

• To develop a complete set of replicable protocols, from production of chlamydospores in di¬phasic system (with increased spore productivity per production cycle and shorter production cycles) to the development of amorphous formulation of P. chlamydosporia through down¬stream processing, for solid-state production system and down-stream processing for scale- up and commercialization.

• To devise semi-synthetic solid media which support higher and consistent chlamydospore production.

Existing state-of-art.

Prior -art search is based on published literature and patents available internationally and within India. The search was done primarily on pathogenicity, mass production, formulations developed, technologies for scale-up production and down-stream processing, bioefficacy and use of Pochonia chlamydosporia.

Literature search

1. Proven pathogenicity/benefit of Pochonia chlamydosporia -

a. International: Pochonia chlamydosporia as a biological control agent against plant parasitic nematodes is extensively studied and is now widely accepted natural control agent against root- knot and cyst nematodes in a wide variety of crop rhizospheres and offer immense scope for commercial production and large scale use in field as alternative to chemical control. (Bourne et a/., 1996; Kerry, 1985; Kerry and Bourne 1996 and 2002; Leij et al., 1992; Leij et al., 1993; Biological Control of Plant Parasitic Nematodes ed. Stirling, G.R. CAB International., 1991; Wang et al.2005; Hirsch etal., 2000; Stirling, 1983).

b. National, NBAII (PDBC), AICRP Biological Control of Insect Pests, Diseases, Weeds and Nematodes trials (Khan et al., 2000, 2005; Rao et al., 2003, 2004, 2005; Nagesh et al., 2002; Nagesh et Reddy, 2005; Nagesh et Janaki Ram, 2005).
c. AICRP Biocontrol of Insect Pests, Diseases, Weeds and Nematodes trials - NBAII multi- location trials at AAU, Anand, TNAU, MPKV, Pune 2006-10.

2. Mass production & formulations— There are few published papers about Solid-state fermentation (Solid-state fermentation- Mini Review, 1998, Agro-Food-Industry Hi-Tech, March/April, pp.29-36).

As per the published literature, PDA, PDB, CMA, sugar solution, molasses, leaf material, bagasse, grains, organic manures, De-oiled cakes etc., have been found to support the mycelial growth of P. chlamydosporia under laboratory conditions. Similarly, talc, granules, organic mix formulations of P. chlamydosporia were developed with its mycelial biomass in laboratory studies (Rodriguez-Kabana et al. 1984.; De Leij and Kerry,1992; Kerry and Bourne, 2002a., IOBC/OILB research manual. Milled barley or wheat as solid media.Kerry and Bourne, 2002b., On CMA (corn meal agar) for laboratory use; Sankaranarayan et al., 2001, 2002; Sasikala, 2003; Rao et al., 2005; Nagesh et al., 2007a).

(In mass production studies, maximum spore yield obtained in different media was 104 to 107 spores/g solid substrates; 104 spores/ml, Kerry et Bourne, 2002)

3. Production containers: Containers used generally were polythene bags, conical flasks of different capacities, liquid fermentors for batches.

Patent search

A. the production of chlamydospores of the beneficial fungus, Pochonia chlamydosporia is mainly using

a. Solid media as explained in the IOBC/OILB research manual (Kerry and Bourne, 2002) and International Patent application no. PCT/GB90/01237 (1990) by Kerry and De Leij). The invention relates to Verticillium chlamydosporium (synonimized as Pochonia chlamydosporia) strain AC, a sample of which has been deposited at the Culture Collection of the CAB International Mycological Institute, or a derivative, variant or mutant there of having nematicidal activity, and its use in the control of nematodes. The invention claims a nematicidal composition of V. chlamydosporium strain AC including a gum, sticker, adhesive and/or surfactant along with chlamydospores, conidia/or hyphal fragments for the control of Meloidogyne species viz., M. incognita, M.javanica, M. arenaria and M. hapla. The specific strain was shown to yield 4.7 X 107 chlamydospores/g of milled wheat or barley at 25°C 3 weeks after inoculation. It also claims a method of application comprising of application to seeds, root, soil, compost or as post plant.

b. In liquid media as explained by Antunes Franco, International Application no.
PCT/IB2006/053257 in 2006.

c. Mass production of P. chlamydosporia (V. chlamydosporium) on a solid substrate like bran or grain or formulated as alginate granules and applied to soil for nematode control was established by Rodriguez-Kabana et al. 1984. Nematropica, 19, 155-170; De Leij F. and Kerry, B. R. 1992. Revue de Nematologie, 14, 157-164; Patent Specification PCT/GB90/01237 or International Publication Number WO 91/01642.

d. Mass production in liquid media, down stream processing and subsequent development of granular formulation with cfus of P. chlamydosporia, Dactylella Candida and Hirsutella rhossiliensis were also well defined in Patent Specification WO 94/28725 or PCT/AU94/00332.

e. Patent Specification No. US 2009/0169518 A1 refers to isolation and utilization of the fungus Pochonia chlamydosporia var. chlamydosporia strain PcMr in biological control of nematodes, characterized in that it has a high virulence against Meloidogyne spp. In in vitro microbiological tests, pot tests and field tests. This strain can be utilized as part of the biological control methods that effectively control Meloidogyne species and this invention refers also to production and utilization of nematicides based on Pochonia chlamydosporia var. chlamydosporia strain PcMR and any organisms derived from this strain. Novelty in this invention refers more to the efficacy of specific strain of P. chlamydosporia against root-knot nematodes under different assay conditions, while the production of the chlamydospores of the fungus (2.2x107 chlamydospores/g of barley) is based on the barley:sand (10:1) as production medium as in the case of IOBC methods (Kerry & Bourne, 2002). Chlamydospores were further extracted through wet method as per the International Patent request PTI04/000009) and formulated with sand at 10:1 proportion and stored at 4°C for further utilization.

B. Down-stream processing:

No clear information on Down-stream processing including drying of media, spore harvest, spore drying to ideal water activity and water content for spore viability and shelf-life, identification of ideal silicate mineral mixture for developing the formulation with desired and defined features, homogenization and examination of the formulation developed for desired shelf- life.

Patent No Inventor Title Abstract: This invention refers to the W0/2007/031949 Antunes Liquid media for creation of liquid media to grow Franco, ospore P. chlamydosporia designed to 1562/DELNP/2008 Carlos production of produce conidia denominated Manuel P. chlamydosporia. chlamydospores. USPA 2009 Antunes P. chlamydosporia isolation & utilization of P 0169518 Franco, strain pcmr and chlamydosporia var. chlamydosporia Carlos method to use it in strain PcMR in biological control of Manuel biological control nematodes, characterized in that it has of the root-knot- a high virulence nematode.against Meloidogyne spp. in in-vitro microbiologic tests, pot tests and field tests. USPA 2006 Khan et Novel process for process for producing biopesticides0292124 al commercial based on T. harzianum, P. chlamydo-production of sporia, P. fluorescens comprising 1621/DEL/2005 biopesticides. preparing mass or stock culture of biocontrol fungi and bacteria on sawdust, soil and molasses mixture, and then immobilizing the bioagents in a flyable base carrier.

Patent No Inventor Title Abstract: This invention refers to PCT/GB90/01237 Kerry et Biological nematicidal composition of V~ (1990) De Leij composition of chlamydosporium strain AC including a gum sticker, adhesive and/or surfactant along with chlamydospores, conidia/or hyphal fragments for control of Meloidogyne spp. With yield 4.7 X 107 chlamydospores/g of milled wheat or barley at 25°C 3 weeks after inoculation. Also a method of application to seeds, root, soil, compost or as post plant.
417/CHE/2006 Nagesh A simple design a simple design for a container for small¬er al of container for scale production of antagonistic fungi through small-scale solid-state production and the methods solid-state thereof. It comprises of a tiered plates production and (multilayered) and a fixing frame assembly in amorphous an air tight stainless steel container with lid. formulations of antagonistic fungi

Patent sites such as www.uspto.gov/patft;www.european-patent-office.org; www.freepatents.com,www.ipo.go.jp were searched but there is no patent applied anywhere in the world for the claims on

i. Development of silicates mix-based amorphous formulation (wettable powder) of Pochonia chlamydosporia var. chlamydosporia as bionematicide and methods thereof for scale-up production and down-stream processing for commercial use.

ii. Semi-synthetic media for increased chlamydospore productivity (yield/cycle) and shortened production cycle.

iii. Partitioning of events of production cycles thus facilitating automation and mechanization for scale-up.

iv. Amorphous wettable formulation of P. chlamydosporia var. chlamydosporia with defined physical parameters and 12 months shelf-life.

Drawbacks in the existing state-of-art.

Several beneficial soil fungi and their natural variants were reported to be highly antagonistic to several soil-borne plant pathogens including plant parasitic nematodes. Some of these beneficial fungi have reached the status of formulations as commercial products with variable shelf-life, quality parameters and bio-efficacy. Paradoxically, most formulations are talc based with insufficiently defined physical, biological and chemical features. The active ingredients in the formulations are mostly described as CFUs (colony forming units) of a fungus with no specific reference to spores/conidia/chlamydospores/blastospores, which only implies that the active ingredient is a mixture of fungal structures/mycelial biomass.

Ironically, with specific reference to the beneficial fungus under report, Pochonia chlamydosporia, a valid formulation with the desired features of biologically active, contaminant- free and at least 12 months shelf-life is yet to be available in the market for farmers to use in the world including India.

According to the literature available, the production of chlamydospores of the fungus, Pochonia chlamydosporia, is mainly using

a. Solid media as explained in the IOBC/OILB research manual (Kerry and Bourne, 2002) and International Patent application no. PCT/GB90/01237 (1990) by Kerry and De Leij). The solid media defined here are natural grain which can yield 107 spores per g substrate and take a minimum of 21 days (three weeks) for completion of sporulation. However, for commercialization and scale-up of spore production, critically chlamydospore yield per g substrate (productivity) should be higher and production cycle to be shorter.

b. On CMA (corn meal agar) for laboratory use (Kerry and Bourne, 2002). CMA is an ideal medium for lab studies and cannot be used for mass production.

c. In liquid media as explained by Antunes Franco, International Application no. PCT/ IB2006/ 053257 in 2006. As mentioned elsewhere in the text, blastospores/aleurospores are the primary spores produced in liquid media although conidia and chlamydospores are also obtained. Blastospores are physiologically weak, poor in survival in harsh environments and in formulations. Further, the time taken per batch in fermentor is longer and chlamydospore yields are low.

Further, protocols for well-defined package of technologies for down-stream processing of the mass produced chlamydospores especially spore separation (harvesting), spore drying (to a desired range of moisture content), suitable inert material or their combinations for amorphous formulations, moisture content of the formulation and packaging with long shelf-life (12 months), are seriously lacking.

How these drawbacks have been overcome by our invention.

Essentially we have considered the following factors as critical for achieving the above stated objectives of this invention and addressed them for developing the amorphous formulation of Pochonia chlamydosporia var. chlamydosporia NBAII PC55.

> Selection of biologically active isolate (with genetic and morphological distinct identity) which is responsive to media & growth conditions modifications, proven pathogenicity & identified virulence factor

> production media: definition of semi-synthetic solid media that enhance spore productivity per cycle and production cycles in a given time period
>post-production partitioning for easy and clear down-stream processing and automation: Down¬stream processing including drying of media, spore harvest, spore drying to ideal water activity and water content for spore viability and shelf-life,

> Identification of production media factors and post production media factors that enhance spore viability or shelf-life and minimize contamination

> Physical parameters of formulation for better shelf-life with biological activity: identification of ideal silicate mineral mixture for developing the formulation with desired and defined features, homogenization and examination of the formulation developed for desired shelf-life.

Novelty of our claim

• Biologically active formulation of chlamydospores of Pochonia chlamydosporia var. chlamydosporia strain NBAII PC55, and combination of mixture of mineral silicates as carrier
material and defining specific physical properties of the formulation

• Development of semi-synthetic media for enhanced solid-state production of chlamydospores (109 chlamydospores/g semi-synthetic media i.e.,100 fold increase in chlamydospores of P. chlamydosporia)

• Identification of physical conditions of mycelial growth and sporulation that become essential for consistent and reproducible chlamydospore production and post-production processing events (100 fold increase in chlamydospores of P. chlamydosporia in shorter production cycle)

• The protocol that improves/enhances mass production of such spores by identifying ideal physical conditions for mycelial growth and spore production.

• The protocol that necessarily partitions/clearly demarcates the mycelial growth vs spore production stages facilitating scale-up of production, mechanization, down-stream processing and developing a formulation with biologically active product and shelf-life

• The invention that necessarily delinks and obtains active ingredient (fungal spores) in relatively pure form for development of any formulation with high shelf-life.

• Essentially the present claim defines creation of amorphous formulation of P. chlamydosporia with chlamydospores as active ingredient with defined physical parameters of the formulation (moisture content, water activity, pH etc.) and the complete set of procedures there of for production, post-production (down-stream processing) and packaging; and the effective control of plant parasitic nematodes, Meloidogyne incognita (root-knot nematode), Heterodera cajani (pigeon pea cyst nematode) and Globodera rostochienis and G. pallida (potato/Golden cyst nematodes).

• In the present claim, maximization of chlamydospore production was addressed and identified suitable semi-synthetic solid media/substrates for solid-state production, method of production (di-phasic); identified that provision of maximum surface area for fungal biomass and sporulation critical for P. chlamydosporia and small-scale chlamydospore production in container with trays (Indian Patent Application No. CHE-417, 2006) or specially identified trays for scale-up; moisture (water to substrate ratio), water activity (aw), incubation temperature, method of inoculation to stabilize water content in medium.

• The claim includes increased spore yield per cycle and shortened production cycle (shorter duration from inoculation to spore harvest), which ensured productivity of active ingredient,
chlamydospores of P. chlamydosporia for scale-up and commercialization.

• Further, the claim also describes a specific combination of inert silicates as carrier material in developing the amorphous formulation aided in retaining the shelf-life for 12 months with a spore viability of 90% with minimum contamination (Fig. 11).

Search Examination Report

Patent sites such as www.uspto.gov/patft;www.european-patent-office.org; www.freepatents.com.www.jpo.go.jp were searched but there is no patent applied anywhere in the world for the claims on

i. Development of silicates mix-based amorphous formulation (wettable powder) of Pochonia chlamydosporia var. chlamydosporia as bionematicide and methods thereof for scale-up production and down-stream processing for commercial use.

ii. Semi-synthetic media for increased chlamydospore productivity (yield/cycle) and shortened production cycle.

iii. Partitioning of events of production cycles thus facilitating automation and mechanization for scale-up.

iv. Amorphous wettable formulation of P. chlamydosporia var. chlamydosporia with defined physical parameters and 12 months shelf-life.

Description of protocols developed for the claims

• The method of claim wherein the Organism and strain: Pochonia chlamydosporia var. chlamydosporia strain NBAII PC55 isolated from infected egg masses of root-knot nematodes (Nagesh et al., 2001, Nagesh et al., 2007b) (Fig. 1); characterized based on its morphological features (Nagesh et al., 2006b) according to the original description by Gams (1988). The particular strain under reference was deposited with MTCC & Gene Bank National Facility, Institute of Microbial Technology, Chandigarh, and the accession number is MTCC5583. Further, the strain was characterized based on the (3-tubulin gene sequence as described by Zare et al. (2001) (Fig. 2) and the sequence was deposited in NCBI genbank with the accession number AY593965.

• Virulence of the specific strain of P. chlamydosporia var. chlamydosporia, strain NBAII PC55 against nematodes was characterized by detection of serine protease gene in the genome of the fungus (Fig. 3) and production of proteases and chitinase in medium (detected in the medium as zone of clearance) (Annual Report, NBAII, 2009-10).

• The bio-efficacy of the strain NBAII PC55 was examined and reported (Nagesh et al 2005a and 2005b) (Figs. 4 & 5).

• Multi-location trials of the efficacy of the formulation developed with the chlamydospores of P. chlamydosporia var. chlamydosporia, strain NBAII PC55 (AICRP Biocontrol Reports 2007-08, 2008-09, 2009-10).

Preparation of starter culture: Prepare 100ml of potato dextrose broth (Himedia) in 250ml conical flask and plug it with cotton plug; autoclave the same at 15psi for 20 minutes; inoculate a loopful of fresh culture of Pochonia chlamydosporia var. chlamydosporia, strain NBAII PC55 in the medium in a laminar hood; grow the culture by placing the inoculated conical flask in orbital shaker at 30°C and 180rpm for 72-96 hours.

Production of chlamydospores
Table 1. Composition of semi-synthetic production media (100g):

Medium 1 Medium 2 Medium 3 Medium 4
Barley grain Rice grain (whole): Corn (milled): 88g Ragi (Finger millet)
(milled): 98g 88g (whole): 88g
Yeast autolysate: Yeast autolysate: Yeast autolysate: Yeast autolysate: 1g
1g IS 13
Maltose: 1g Maltose: 1g Maltose: 1g Maltose: 1g
Barley Barley Barley flour/powder: flour/powder: 10g flour/powder: 10g 10g

Soak grain or milled grain in luke-warm water for 2 hours, followed by draining of water on a sieve. Weigh barley flour/powder, maltose and yeast autolysate as given in Table 1 and mix thoroughly with respective grain (whole or milled). Fill bulk of the semi-synthetic media thus prepared in to autoclavable polythene bags and autoclave for 60 minutes at 121 °C or at 15psi.

a. Small scale as per the novel design of mass production unit (Nagesh et al., Indian Patent Application No. 417/CHE, 2006).

• Take 30 grams of each semi-synthetic media (Table 1) and spread as thin layer in each stainless steel plate individually.

• Arrangement of plates and frame-stand: Place five plates with media in five slots of the frame-stand and shift the frame to close position in order to hold 5 plates in proper position.

• Place the frame-stand with plates and media carefully in the stainless steel container and secure the airtight lid.

• Keep the whole container in 20-litre pressure cooker; shut the top lid and autoclave the container for 45 minutes.

• Let the cooker cool to room temperature and take out the container carefully from it, and keep that the container outside for 30 minutes in order to ensure the media to attain ambient temperature.

• Treat the worker's hands with alcohol or rectified spirit to disinfect before handling the trays, media or inoculation. Open the lid of the container under laminar hood, disembark the frame and dismantle the plates individually.

• Inoculate the fungus Pochonia chlamydosporia by spreading 5 ml of the starter liquid culture (72-96 hours old culture) on the autoclaved semi-synthetic media per plate.

• Place the inoculated plates carefully back in to the slots in frame-stand, adjust the frame to close position, place the stand back in to the steel container and secure the container with lid.

• Place the container in an incubator (incubation temperature of 28 °C) for 7 days or in a room with controlled temperature at 28 °C for 9 days.

• Take out the plates from the container after 7 days or 9 days depending on the incubation method suggested.

• Use clean plastic trays (size in cm 42x31x7.5, Ixbxh) swabbed with rectified spirit, for further sporulation and media drying.

• Unload the sporulated media from each plate to these plastic trays and uniformly spread it in each tray.

• Close the trays containing spore-laden media tightly with cling (Saran) wrap and leave the trays for 2 days in shelf in order to complete the residual sporulation.

• After 2 days, make as many small holes as possible in saran wrap by poking a needle without tearing the wrap and leave the trays for drying under a fan in a room/ laminar hood at ambient temperatures and R.H. or dryer where R. H. and temperature are regulated.

• At 24-37°C ambient temperatures and an R.H. of 60-90%, drying to 8-11% will take 3-4 days.

• Harvest chlamydospores from the substrate by dry method (explained in the text).
b. Protocol for Scale-up production of P. chlamydosporia var. chlamydosporia strain NBAII PC 55 chlamydospores

• Trays: Pre-fabricated stainless steel trays (size in cm: Ixbxh 42x31x5.5) or plastic trays readily available in the local markets (size in cm: Ixbxh 42x31x7.5).

• Preparation of trays for chlamydospore production: Wash thoroughly the trays with soap water followed by fresh tap water, air dry to remove moisture followed by hot air drying in hot air oven at 80-90°C for 45 min to 1 hour in case of stainless steel trays, and at 45-50°C for 45min to1 hour in case of plastic trays. This is followed by cleaning the trays with a swab of formaldehyde in well-ventilated area or under fume hood. The operator is invariably to wear a hood or mask protecting eyes and nostrils and a pair of gloves while handling formaldehyde. Alternatively, clean the trays with 98% ethanol or rectified spirit swabs inside the laminar hood followed by addition of autoclaved production media (Table 1).

• Autoclave composite grain media (Table 1) in bulk in autoclavable polythene bags at 15 psi for 20 minutes, cool the media and use them in laminar hood for mycelial growth and sporulation in trays. Record the parameters of solid media and semi-synthetic media before and after autoclaving (Table 2).

• Place about 350g of Mediuml, 300g of Medium2 or 350g of Medium3 per tray (stainless steel/plastic) under the laminar hood and inoculate 100 ml of starter liquid culture of P. chlamydosporia (72-96 hours old) per tray and mix the medium/substrate with the inoculum with a wooden ladle or hand (use autoclaved gloves) in order to ensure uniform spread of starter inoculum.

• Spread the media + inoculum mixture uniformly in trays and cover the trays with cling wrap/saran wrap to prevent contamination and flies. Place the trays in racks in a controlled temperature room and incubate the trays at 30 °C for 12 days.

• Facilitation of production of chlamydospores and drying of the substrate laden with mycelia and chlamydospores:

Option 1. Shift the trays incubated at 30 °C for 12 days to a hot air oven set at a temperature of 32 °C (±1°C) and incubate them for 2 days.

Option 2. After incubation of the trays at 30°C for 12 days in controlled temperature room, set the temperature to 32°C and continue incubation for 2 days.

• Record the moisture content of the substrate for desired moisture level of 6-8% using Sartorius Moisture analyzer (Model MA150).

• Transfer the dried substrate laden with spores (Fig. 6) to a mechanical clod breaker or in to thick polythene bag, secure the bag air tight with rubber band and rub the clods or using a mallet make the clods small and dislodge spores as much as possible.

• Separation of chlamydospores from substrate/nutrient particles or Harvest of chlamydospores by dry method:

Option 1. Transfer the material into a mycoharvester marketed by CABI, UK model (MH5), follow the manufacturer's protocol in user's manual and collect the harvested spores.

Option 2. Place the spore -laden substrate particles in to a stainless steel mesh sieve 60 mesh) (Fig. 6), place a slightly smaller sieve (with a difference of 1cm in diameter) over the first sieve and a collection plate/polythene cover fitting to the bottom to collect the spores. Roll the second sieve over the substrate in the first sieve ensuring maximum dislodging of the spores.

• Collect the spores through option 1 or option 2, weigh the harvested chlamydospores/spore concentrate (Fig. 7) in a sensitive balance and calculate the spore yield per unit weight of initial substrate. Further, record the spore number per 10mg of harvested spore concentrate by serial dilution and Hemocytometer (Fig. 8) and corroborated with plating the serial dilutions on potato dextrose agar plates at 30°C. Calculate chlamydospore yield (spores/g substrate and spores/g spore concentrate from each mass production medium) and number of days for completion of sporulation (duration of production cycle) from the data recorded (Table 3).

• Record the moisture content and water activity of the harvested spores and depending on the necessity, dry the spore concentrate in hot air oven at 40 °C for 6 hours to bring the moisture content to less than 4% before using the same for developing its formulation or for long-term storing in sterile conditions.

"Values in parentheses are log10 values of number of spores per g substrate.
**We have adopted a simple method for rating the caking and clumping based on operational convenience of the solid media + water + fungal inoculation, growth and sporulation at harvest (as per Nagesh et al., Indian Patent Application No. CHE-417, 2006). The rating scale was:

i. Media with fungus remains as a single mass or ball, the caking and clumping rating =100.

ii. Media with fungus remains as 2-3 major tenacious clumps, the rating = 75-99.

iii. Media with fungus remains as more than 3 major tenacious clumps, the rating = 50-74.

iv. Media with fungus remains as more than 3 major tenacious clumps with fallout powder, the rating = 25-49.

v. Media with fungus remains as small clumps with fallout powder, the rating = 10-24.

vi. Media with fungus remains as thin tenacious flakes with fallout powder, the rating = 5-10.

vii. Media with fungus remains as powder/flakes or small loose clumps which disintegrate when touched with a needle or brush, the rating = <5.
This scale was devised specifically for the specific initial moisture as it was observed that the rating for the same media could differ with the initial moisture contents (Nagesh et al., Indian Patent Application No. CHE-417, 2006).

Development of silicate mineral mix-based amorphous formulation:

Inherent physical and chemical properties of the inert material used before and after formulation with chlamydospores were recorded and given in Tables 4, 5 and 6. Standard protocol followed for suspensibility and wettability is as follows:

Suspensibility

Suspensibility of spray-dried formulations was determined by a procedure described by Lisansky et al. (1993). A sample (3 g) was added to 97 ml of distilled water in a 100-ml measuring cylinder. The cylinder was inverted for 30 times and a 1-ml sample was taken from the top 10 ml, dried to constant weight at 105 °C in a hot air oven (Memmert, Schwabach, Germany). The determination was repeated three times. This "control" experiment gave a result for a fully suspended product. The above procedure was repeated and the cylinder was allowed to stand for 1 h before sampling (treatment experiment). The suspensibility was reported as mg of formulation
obtained per ml of sample on drying on immediately after shaking and after I hour of shaking.

Wettability

The procedure of Lisansky et al. (1993) was followed by transferring 100 ml of standard hard water (CIPAC, MT18; CIPAC 1970) into a 250-ml beaker. A representative sample of the powder (0.1 g) was added at once by dropping it on the water via glass funnel held in a ring stand. The bottom of the funnel was 10 cm from the surface of the water. The stopwatch was started and the time taken (to the nearest second) for it to become completely wetted was recorded. The experiment was repeated three times and the result was an average.

• Preparation of inert mix for formulations using three silicate minerals: Mix pre-autoclaved talc, kaolinite and red earth whose properties are recorded (Table 4) at 1.0:1.0:0.25 proportion in a mechanical blender (Fig. 9).

• Take a known weight of spore concentrate with known spore concentration (number of chlamydospores/g spore concentrate) and use them for blending with a definite proportion of autoclaved inert mix of silicates to arrive at the desired spore load per g of formulation (106 8 spores per g).

• Blend the spore-laden inert silicates in a mechanical blade blender without generating excess heat of talc formulation.

Table 5. Details of properties of pure spore concentrate of P. chlamydosporia var. chlamydosporia, strain NBAII PC55 and formulated chlamydospores on amorphous inert silicate material.
Properties Spore concentrate of Talc + kaolinite Talc + kaolinite + red earth + P. chlamydosporia + red earth P. chlamydosporia spore (1:1:0.25) Ph 6.5-7.0 6.5-7.0 Color Pale yellow Dirty white/ Dirty white/ pale yellow pale yellow Water activity.4620.4570.514 Water content 3^57 062 1^08 Suspensibility - 21.50*24.70*
5.36** 4.73**

Wettability - 26.98 minutes 35.06 minutes *weight of material suspended per ml water immediately after shaking (initial mixture of material 3g/100ml water)

** Weight of material suspended per ml water after 1 hour of shaking (initial mixture of material 3g/100ml water).

Table 6. Shelf-life studies of the P. chlamydosporia var. chlamydosporia, strain NBAII PC55 formulation developed [Months of ISpore viability (%) [Moisture content Water activity storage
Initial 96^6 1.4 -2.2 0.45-0.65
3 96.2 -96.5 1.6 -2.2 0.45-0.65
6 93.4-94.0 1.6 -2.0 0.44-0.64
9 92.2-93.4 1.6-2.0 0.45-0.60
12 90.6-91.1 1.6-1.9 0.43-0.60

The summary of complete set of protocol developed is presented in flow chart Fig. 12.

Selected references

Annual Report, 2009-2010. National Bureau of Agriculturally Important Insects (ICAR), P.B. No. 2491, H.A. Farm Post, Bellary Road, Bangalore 560024.

Proceedings of the XVII Biocontrol Workers Group Meeting AICRP on Biological Control of Insects, Diseases, Nematodes and Weeds Annual Workshop meetings, 2007-08, 2008-09, 2009-2010. National Bureau of Agriculturally Important Insects (ICAR), P.B. No. 2491, H.A. Farm Post, Bellary Road, Bangalore 560024.

Bourne, J. M., Kerry, B. R. and Leij, F. A. A. M. de 1996. The importance of the host plant on the interaction between root-knot nematodes (Meloidogyne spp.) and the nematophagous fungus, Vertcillium chlamydosporium Goddard. Biocontrol Science and Technology, 6, 539-548.

de Leij, B.R. Kerry and J.A. Dennehy, Verticillium chlamydosporium as a biological control agent for Meloidogyne incognita and M. hapla in pot and micro-plot tests, Nematologica 39 (1993), pp. 115-126.

Domsch K. H., Gams W. & Anderson T. (Eds) 1980 Compendium of Soil Fungi. Academic Press, London, pp 529-532.

Gams W, A contribution to the knowledge of nematophagous species of Verticillium, Netherlands Journal of Plant Pathology 4 (1998), pp. 123-148.

Godoy, G. et al., 1983. Fungal parasites of Meloidogyne arenaria eggs in Alabama soil. A mycological survey and greenhouse studies. Nematropica, 13: 201-213.

Jatala, P. 1986. Biological control of plant-parasitic nematodes. Annual Revue of Phytopathology, 24, 453-489.

Jatala, P., Kaltenbach, R. and Bocangel, M. 1979. Biological control of Meloidogyne incognita acrita and Globodera pallida on potatoes. Journal of Nematology, 11, 303.

Jeyarajan, R. and Angappan, K. 1998. Mass production technology for fungal antagonists and field evaluation. In "Biological suppression of plant diseases, phytoparasitic nematodes and weeds" (S. P. Singh and S. S. Hussaini Eds.), P.D.B.C., P.B. No.2491, H.A. Farm Post, Hebbal, Bangalore 560 024. pp. 48-56.

Kerry, B.R. & Bourne, J.M.(1996) The importance of rhizosphere interactions in the biological control of plant parasitic nematodes - a case study using Verticillium chlamydosporium.. Pesticide Science 47, 669-675.

Kerry B. R. and Bourne, J. M. (ed.) 2002. A Manual for Research on Verticillium chlamydosporium, a Potential Biological Control Agent for Root-Knot Nematodes. IOBC/WPRS, Gent, Belgium, 85 pp.

Leij, F.A.A.M.de, Davies, K.G. and Kerry, B. R. 1992. The use of Verticillium chlamydosporium as a biological control agent for Meloidogyne incognita and M. hapla in pot and micro-plot tests. Nematologica. 39, 115-126.

Lisansky, S. G., R. J. Quinlan, and G. Tassoni. 1993. The Bacillus thuringiensis production handbook. CPL Press, Newbury, UK.

Lomer, C. J., Prior, C. and Kooyman, C. 1997. "Development of Metarhizium spp. for the control of grasshoppers and locusts". Memoirs of the Entomological Society of Canada 171: 265-286.

Mukhopadhyay, A. N. 1996. Recent innovations in plant disease control by eco-friendly biopestcides. Sectional Presidential Address, Agricultural Sciences, 83rd Indian Science Congress, 1996, Patiala, India, pp.16.

Nagesh, M. Hussaini, S. S. Chidanandaswamy, B. S. 2005a. Incidence of root-knot nematode, Meloidogyne incognita on Gherkin, Cucumis sativus and yield losses. Indian Journal of Plant Protection, 33; 309-311.

Nagesh, M. Hussaini, S. S., Chidanandaswamy, B. S. and Biswas, S. R. 2006a. Studies on simple mass production systems of nematophagous fungus, Arthrobotrys oligospora. International Journal of Nematology. 16, 58-64.

Nagesh, M. Hussaini, S. S. Chindandaswamy, B. S. Shubha, M. R. Ruby, K. M. 2007a. Relationship between initial water content of the substrate and mycelial growth and sporulation of the nematophagous fungi, Paecilomyces lilacinus and Pochonia chlamydosporia. Nematologia Mediterranea 35, 57-60.

Nagesh, M., Hussaini, S.S. Ramanujam, B. and Chidanandaswamy, B. S. 2006b. Management of Meloidogyne incognita and Fusarium oxysporum f. sp. Lycopersici wilt complex using antagonistic fungi in tomato. Nematologia Mediterranea 34, 63-68.

Nagesh, M., Hussaini, S.S. Ramanujam, B. and Rangeswaran, R. 2007b. Molecular identification, characterization, variability and infectivity of Indian isolates of the nematophagous fungus Pochonia chlamydosporia. Nematologia Mediterranea 35, 47-56.

Nagesh M, and Janakiram T. 2004. Root-knot nematode problem in polyhouse roses and its management using dazomet, neem cake and Pochonia chlamydosporia (Verticillium chlamydosporium). Journal of Ornamental Horticulture, 7, 147-152.

Nagesh, M., Parvatha Reddy, P. and Rama, N. 2001. Pathogenicity of selected antagonistic soil fungi on Meloidogyne incognita (Kofoid and White) eggs and egg masses under in vitro and in vivo conditions. Journal of Biological Control, 15, 63-68.

Nagesh, M. Reddy, P. P. 2005b. Management of carnation and gerbera to control the root-knot nematode, Meloidogyne incognita, in commercial polyhouses. Nematologia Mediterranea, 33,157- 162.

Nagesh, M,, Singh, K. P. 2004. Bio management of Meloidogyne incognita on Polianthes tuberosa using Glomus mosseae and Pochonia chalmydosporia as bulb dressing in combination with neem cake. Journal of Ornamental Horticulture, 7, 45-151.

Proceedings of the Brighton Crop Protection Conference, Pests and diseases -1988. Vol.2., November 20-24 November 1988. Two microorganisms for the biological control of plant parasitic nematodes, 603-607.

Samson, R. A. 1974 Paecilomyces and some allied Hyphomycetes. Studies in Mycology No 6. Centralbureau voor Schimmelcultures, Baarn. pp 119.

Stirling, G. R. and West, L. M. 1991 Fungal parasites of root-knot nematode eggs from tropical and sub¬tropical regions of Australia. Australasian Plant Pathology, 20, 149-154.

Stirling, G. R. 1991. Biological Control of Plant Parasitic Nematodes. CABI Publishing, UK. pp 282.

Whipps, J. M., McQuillken, M. P. and Budge, S. P. 1993. Use of fungal antagonists for biocontrol of damping-off and Sclerotinia diseases. Pesticide Science, 37: 309-13.

Zare,R. and Gams, W. 2001. A revision of Verticillium sect. Prostrata. III. Generic classification. Nova Hedwigia. 72. 329-337.

5. CLAIMS (not applicable for provisional specification. Claims should start with the preamble - "I/We Claim" on separate page)

We claim:

Claim 1 includes - Creation of amorphous formulation of the beneficial fungus, Pochonia chlamydosporia Var. chlamydosporia strain NBAII PC55 (accession number MTCC 5583) comprising of chlamydospores mixed with a specific proportion of a combinatorial amorphous powders of talc, kaolinite and red earth silicate minerals, wherein at least 90% spores are viable 12 months of storage at a temperature of between 25°C and 38°C for the control of root- knot and cyst nematodes with a spore load of 107 per g of formulation.

Claim 2 includes - A specific di-phasic (liquid-solid phases) method of production of aerial chlamydospores and the specific semi-synthetic solid substrates which ensure chlamydospore production/yield of 109 chlamydospores/g substrate and 1400-1600 mg of spores/100g substrate in 14-15 days per production cycle (100 fold increase in chlamydospores of P. chlamydosporia in shorter production cycle of 14-15 days).

Claim 3 includes - Protocol for mycelial growth and sporulation (chlamydospores) with optimal physical parameters identified as, a temperature range of 30-32°C and a relative humidity ranging between 60-80%, Substrate moisture content and water activity (aw) were 40-42% and 0.9-0.95, respectively.

Claim 4 includes - Down-stream processing: spore drying for 2-3 days at ambient temperature and relative humidity. Spore moisture content and water activity before drying i.e., immediately after completion of sporulation and after drying (3-5% and 0.3-0.5aw respectively).

Claim 5 includes - A spore yield of 162 mg/g substrate i.e. equivalent to 2.1x109 spores per mg of spores with a spore aw and moisture content of 3.57% and 0.46aw in 14-15 days by these protocols.

Claim 6 includes - Definition of the combination of amorphous ingredients for developing the formulation. Talc (mineral composition- talc:kaolinite:red earth at 1.0:1:0.25) of following physical and chemical properties.

Claim 7 includes - Blending, mixing or homogenization of ingredients with spore concentrate including the proportion of talc + kaolinite + red earth mixture and spore concentrate, water activity and water content, suspensibility and wettability.

Claim 8 includes - Amorphous formulation of fungal spores (Pochonia chlamydosporia) (Fig. 10) comprising of conidia or spores with substrate mixed with a specific proportion of amorphous talc + kaolinite + red earth mix powder, wherein at least 90% conidia or spores (Fig. 11) are viable after twelve months of storage at a temperature of between 25°C and 37°C.

Claim 9 We claim that the method of mass production of P. chlamydosporia on semi-synthetic solid media have the advantages of increasing the surface area for mycelial growth, facilitation of robust chlamydospore proliferation, uniform growth of mycelium and sporulation, decreasing the time taken for sporulation, conservation of space and post-harvest processing. The time taken for growth and sporulation of the fungus under study, Pochonia chlamydosporia, was reduced by 13-15 days compared to the conventional solid state fermentation in polythene bags and conical flasks and in liquid media of existing methods of production. For instance, P. chlamydosporia sporulated in 13-15 days of incubation at 30-32°C and an RH of 60-80%, while, it took 20 to 25 days to complete sporulation on the same medium either in polythene bags or in glass conical flasks incubated at room temperature (26-34 °C).

The claim includes increased spore yield per cycle and shortened production cycle (shorter duration from inoculation to spore harvest), which ensured productivity of active ingredient, chlamydospores of P. chlamydosporia for scale-up and commercialization.

We also claim that the spore yield was significantly higher on the novel semi-synthetic media at same conditions under this (streamlined) complete set of protocols compared to other methods in practice. Further, inherent problems in solid-state mass production system like contamination of the media, handling of the process, caking, substrate drying for processing, contamination by flies, etc. were minimized.

Claim 10 The above set of standardized protocols and the series of processes from fungal spore inoculation, development of formulation through down-stream processing to packaging of P. chlamydosporia var. chlamydosporia strain NBAII PC55 (MTCC5583) formulation thus have practical utility and scope for mechanization, automation and commercial use. It has immense potential to be used commercially as the scale of operation can be increased.

There is no report of such silicate minerals-mix based amorphous formulation of chlamydospores of Pochonia chlamydosporia and its predominantly solid-state production on semi-synthetic media developed within the country and outside the country so far. Therefore the product and processes as per the claims have potential market in developing nations, SAARC countries, world over etc.