FIELD OF THE INVENTION

The present invention pertains generally to methods and compositions and formulation related to the control of plant parasitic nematodes in the soil. The invention especially concerns methods and compositions for producing and desiccation-tolerant Paecilomyces lilacinus strain PI-1 (MTCC 5175) spores encapsulated with some of polymers for the control of root knot nematode.

BACKGROUND OF THE INVENTION
Chemical Nematicides in particular have been used extensively to control root knot nematodes for many years. An awareness of recent problems associated with the use of chemical Nematicides such as adverse effects on man and the environment has led to a focus on biological control alternatives to chemical Nematicides. For instance, certain fungi have been identified to be pathogenic to different pests. Importantly, many fungi, unlike pathogenic bacteria, viruses and protozoa need not necessarily be ingested by the target insect to initiate disease, but instead usually invade through their host's cuticle. The insect rapidly develops resistance to chemical Nematicides and is not adequately controlled with current pest management practices. Various strains of the fungus Paecilomyces lilacinus have been proposed as
a possible biological control agent for root knot nematode.

A significant constraint to the development of this fungus and other like fungi as biocontrol agents, however, is the availability of low-cost methods for producing infective propagules. Solid-substrate methods of producing spores from such fungi as Paecilomyces lilacinus have proven too costly for commercial consideration. Hence, liquid culture methods for producing spores are preferred.

Harmon et al. has described in U.S. Patent No. 5 288 634 a liquid fermentation media permitting the production of high level of Trichoderma harzianum conidia (and spores) that are resistant to desiccation.

Eyal et al. has described in U.S. Patent No. 5 360 607 a submerged culture technique for growing the mycelium of Paecilomyces fumosoroseus. The grown mycelial biomass is harvested and then formed into dry prill which can be used directly on plants and soil or the prill can be used as a carrier for sporulation of conidia spores.

Previous attempts to produce Paecilomyces lilacinus spores directly using liquid culture fermentation methods have yielded unstable spores which are desiccation intolerant and hence readily perish during drying. Clearly, there is a need to develop a method to rapidly produce high volumes of spores which are desiccation tolerant and have high survival rates after drying and storage. In particular it would be useful to produce desiccation tolerant spores of Paecilomyces lilacinus along with polymeric additives.

Six different polymeric additives studied (polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), Xanthan gum, and sodium alginate) for their ability to support growth and promote survival of Paecilomyces lilacinus during storage.

OBJECT OF THE INVENTION

The present invention addresses the need to effectively produce high volumes of desiccation tolerant spores of fungal species such as Paecilomyces lilacinus. The present invention accomplishes this by providing a liquid culture media optimally enriched in nitrogen and carbon and formulation with some polymers as an aqueous formulation with longer shelf life.

A frequent observation is that in carrier based formulations, the number of viable probagules decreases from 109 to 107 colony forming units (cfu) per g after 90 days of storage. The most consistent feedback received from the farmers is the concern about the shelf life of carrier based formulations, since they have shorter shelf life, which hardly extends beyond three to four months under normal storage conditions. The development of adequate liquid based formulations, which would ensure survival, protection of the strain which would allow timely, easy and precise delivery in the field could be a major step towards this goal.
SUMMARY OF THE INVENTION
The present invention addresses the need to effectively produce high volumes of desiccation tolerant spores of fungal species such as Paecilomyces lilacinus. Specifically, the invention provides a liquid culture medium which allows for the rapid production of a high volume of desiccation resistant fungal spores and a method of producing the same.

In a first general embodiment, the invention provides the use of a liquid culture medium for producing a high concentration of desiccation resistant fungal spores, whereby the medium comprises a nitrogen source and a carbon source. The nitrogen source is preferably selected from the group consisting of hydrolyzed casein, soy peptone, yeast extract, hydrolyzed soy protein, hydrolyzed cottonseed protein, hydrolyzed corn gluten protein, and other nitrogen sources. The optimal sporulation medium for P. lilacinus MTCC 5175 contained soy peptone as nitrogen source at 10 gm /lit and sucrose or maltose as the carbon source at 20 g/L, with ZnSO(4)-7H(2)0 at 250 mg/L, CuSO(4)-5H(2)0 at 10 mg/L, H(3)BO(4) at 5 mg/L, and Na(2)MoO(4)-2H(2)0 at 5 mg/L.

The present invention further contemplates a method of producing a high concentration of desiccation tolerant fungal spore, comprising the steps of inoculating liquid culture medium comprising a nitrogen source with fungal propagules, whereby the nitrogen source is between 5-10 gm; incubating the propagules for a sufficient time to allow for maximum sporulation; and coating them with polymeric additives like polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), xanthan gum and sodium alginate for their ability to support growth and promote survival of Paecilomyces lilacinus during storage. The additives have the stabilization property. This protective property is known as colloidal stabilization. The improvement of survival is analogous to the protective colloid effect where fungal spores represent one colloid and the suspension the other. The polymer is absorbed in a thin molecular layer on the surface of the individual colloidal particles resulting in a stabilized suspension that prevents coalescence of cells, which might block the 02 and nutrient diffusion from media to cells.

The nitrogen source is selected from the group consisting of hydrolyzed casein, yeast extract, hydrolyzed soy protein, hydrolyzed cottonseed protein, and hydrolyzed corn gluten protein.

In a preferred embodiment the nitrogen source is present at a concentration greater than 5grams/liter and optimally, at a concentration between 10 grams /liter. In an especially preferred embodiment, the liquid culture medium further comprises a carbon source greater than 15 grams/liter. In a most preferred embodiment the liquid culture medium comprises a nitrogen source greater than 13.2 grams/liter and a carbon source greater than or equal to 80 grams/liter combination of different trace elements starting from 5 mg to 250 mg per lit

Deposit of Microorganisms
Isolated and purified (microbially pure) samples of strain stanes pl-1 Paecilomyces lilacinus (MTCC 5175) as disclosed herein were deposited at the Institute of Microbial technology, Sector 39A, Chandigarh 160036 , India for patent purposes under the terms of the Budapest Treaty. The deposit was made on 16-7-2007

DETAILED DESCRIPTION OF THE INVENTION
Fungi have recently been recognized as a valuable potential source for natural insect control specifically, spores of certain fungi have been shown to contain metabolites that exert adverse physiological effects on insects. While spores from certain fungi hold great promise as bio control agents, a particular problem with such natural agents is the ability to rapidly produce a high volume of stable spores at low cost and which can be dried and stored without loss of activity.

A particularly serious plant parasitic nematode is root knot nematode has been reported to attack over 500 plants in warm climates worldwide. Resistance to chemical Nematicides and a lack of natural enemies has made the nematode is a serious pest in horticultural crops. Fungi, which penetrate the cuticle of the insect, such as Paecilomyces lilacinus, appear to offer the best opportunity for biological control.

The feasibility of using Paecilomyces lilacinus as bio control agent against nematodes dependent on numerous biological constraints, including the ability to produce high concentrations of stable propagules at a reasonable cost (Jaronski, 1985; Latge et al., 1985). On solid substrates, Paecilomyces fumosoroseus and Beauveria bassiana strains produce abundant aerial conidia which are amenable to storage as dry preparations. In submerged culture, Paecilomyces fumosoroseus, Paecilomyces farinosus, and Beauveria bassiana are reported to produce high concentrations of spores (Bidochka, et al 1987 ). Spores produced in liquid culture by various Entomopathogenic fungi are typically larger than aerial conidia, are not amenable to simple drying techniques, and tend to perish more rapidly during storage (Hegedus et al. 1992).

The present invention has focused on developing liquid culture techniques for producing desiccation tolerant spores such as spores from Paecilomyces lilacinus as well as other species. Various liquid culture medium were evaluated for producing Paecilomyces lilacinus spores The Spores were produced by inoculating liquid culture medium with fungal propagules;. Propagules are any living cells from a fungus that will propagate such as spores, mycelium or other fungal biomass. These propagules were coated with polymeric additives like polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), Xanthan gum, , and sodium alginate for their ability to support growth and promote survival of Paecilomyces lilacinus during storage based on spore yield and stability as aqueous formulation.

A liquid culture medium was identified which supported the rapid production of high concentrations of desiccation tolerant Paecilomyces lilacinus spores which were amenable of infecting and killing the nematodes.

OPTIMAL COMPOSITION OF LIQUID CULTURE MEDIUM FOR SPORE PRODUCTION AND ITS FORMULATION

The impact of nutrition on submerged culture production of desiccation tolerant Paecilomyces lilacinus spores was evaluated by growing the fungus in media with differing carbon concentrations. The optimal sporulation medium for P. lilacinus MTCC 5175 contained soy peptone as nitrogen source at 10 gm /lit and Glucose /sucrose or maltose as the carbon source at 20 g/L, with ZnSO(4)-7H(2)0 at 200 mg/L, CuSO(4)-5H(2)0 at 10 mg/L, H(3)BO(4) at 5 mg/L, and Na(2)MoO(4)-2H(2)0 at 5 mg/L . This media is amended with vitamin solution at a rate of 20 ml comprising of Thiamine 5 mg Biotin 2 mg, Folic acid 2 mg and pyridoxine 10mg.

The Spores were produced by inoculating liquid culture medium with fungal propagules;. Propagules are any living cells from a fungus that will propagate such as spores, mycelium or other fungal biomass. These propagules were coated with polymeric additives like polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), Xanthan gum, and sodium alginate for their ability to support growth and promote survival of Paecilomyces lilacinus during storage.

The impact of nutrition on submerged culture production of desiccation tolerant Paecilomyces lilacinus spore was evaluated by growing the fungus in media with differing carbon and nitrogen concentrations. Spores were produced by inoculating liquid culture medium with fungal propagules;. Propagules are any living cells from a fungus that will propagate such as spores, mycelium or other fungal biomass.

Effect of carbon sources on the spore production
Table -1 Example -1

Table-2 Example 2
As shown in Table 1 and 2, significantly higher concentrations of Paecilomyces lilacinus spores were produced in media which contained a high concentration of carbon and nitrogen. Under the conditions of this study, optimal production occurred at a carbon level of 20 grams glucose/liter and a nitrogen level of 10.0 grams of peptone /liter. Media having 20 grams glucose/liter and 10 grams Peptone /liter will hereinafter be referred to as SMP media.

Effect of Nitrogen sources on the spore production Table -3 Example -1

Table -4 Example 2

Effect of amendment of polymeric additives Table -5 Example -1

Table-6 Example -2
Upon experimentation, maximum sporulation was achieved with a PVP at a concentration of 10 grams/liter and Glycerol 5 ml /lit and xanthan gum 5 gm/lit and as shown in Table -5.

The survivability of the fungal propagules was studied under ambient room temperature up 16 months. The propagules were viable up to up to 480 days. The aqueous liquid formulation of Paecilomyces lilacinus has twin advantage of longer shelf life and tolerance to adverse conditions such as temperature and desiccation.

Example - Survival of fungal propagules in the proposed culture medium/ aqueous formulation after storage

The specification covers embodiments and examples for purpose of understanding and shall not limit the scope of the invention.

WE CLAIM:

1. A method of producing a high concentration of desiccation tolerant fungal spores comprising the steps :

a. Inoculating a liquid culture medium comprising of a combination of trace elements which is a nitrogen source and a carbon source with fungal propagules,

b. incubating the propagules to allow for maximum sporulation,

c. coating the propagules with selective polymeric additives

d. adding selective amino acids in the culture medium

e. collecting the resulting spores and

f. drying the spores

2. A liquid culture medium for producing a high concentration of desiccation resistant fungal spores, comprising of a nitrogen source at a concentration between 5.0 to 10 gm/ liter

3. The liquid culture medium as claimed in claim 1 may further include a carbon source.

4. The liquid culture medium as claimed in claim 1 wherein the nitrogen source is preferably selected from a group consisting of Peptone , yeast extract, and corn extract or combination thereon.

5. The liquid culture medium as claimed in claim 1 wherein the nitrogen source concentration is 10 grams/liter.

6. The liquid culture medium as claimed in claim 1 wherein the nitrogen source concentration is between 5grams/liter and 10 grams/liter

7. The liquid culture medium as claimed in claim 1 wherein the selective polymeric additives include PVP 10 gm/lit and Glycerol 5ml /lit and xanthan gum 5 gm /lit.

8. The liquid culture medium as claimed in claim 3 wherein the carbon source is greater than 15 grams / liter.

9. The liquid culture medium as claimed in claim 1 wherein the addition of selective amino acid may be at a concentration of 20 ml per lit, selective polymeric additives is PVP 10 gm/lit and Glycerol 5ml /lit and xanthan gum 5 gm/lit

10. The liquid culture medium as claimed in claim 1 wherein the addition of selective trace elements at a concentration of 5.0 mg to 200 mg ml per lit