WO2005055724A1

WO2005055724A1 - Biopesticidal compositions

Info

Publication number: WO2005055724A1
Application number: PCT/IN2004/000304
Authority: WO
Inventors: Raj Kamal Bhatnagar; Raman Rajagopal; Nagarjuna G. V. Rao
Original assignee: International Centre For Genetic Engineering And Biotechnology; Deshmukh, Panjabrao, Krishi, Vidyapeeth
Priority date: 2003-12-15
Filing date: 2004-10-01
Publication date: 2005-06-23

Abstract

Biopesticidal compositions based on Photorhabdus luminescens is disclosed. The composition as a spray is extremely effective in absence of its symbiont nematodes. Also disclosed are methods for treatment of crops using the novel compositions of the present invention.

Description

BIOPESTICIDAL COMPOSITIONS Field of invention The present invention relates to biopesticidal compositions. More particularly, the present invention relates to biopesticidal compositions for controlling inter alia, agricultural, horticultural and forestry pests. In particular, the present invention relates to biopesticidal compositions based on the bacterium Photorhabdus luminescens for controlling and eradicating various agricultural, horticultural and forestry pests. Background of the invention The motile Gram-negative bacterium Photorhabdus luminescens of family Enterobacteriaceae is a symbiont of the entomopathogenic nematode Heterorhabditis sp. This bacterium is a potent insect pathogen and is normally found associated with its symbiont nematode [(Boemare, N.E., R.J. Akhurst and R.G. Mourant. 1993. DNA relatedness between Xenorhabdus spp (Enterobacteriaceae); (Kaya, H.K. and R. Gaugler. 1993. Entomopathogenic nematodes Annu. Rev. Entomol. 38: 181-206., symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int. J. Syst. Bacteriol. 43: 249-255.)]. Studies on the insecticidal complex produced by these bacterium have revealed that several extracellular macromolecules such as proteases, upases, broad spectrum antibiotics produced during its growth contribute towards insecticidal activity [{Forst, S and K. Nealson. 1996. Molecular biology of the symbiotic pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol Rev. 60: 21-43.); Forst, S., B. Dowds, N. Boemare and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp. : bugs that kill bugs. Annu. Rev. Microbiol. 51: 47-72)]. During growth Photorhabdus secretes insecticidal toxin proteins into the culture medium which have been purified [(Bowen, D.J. and J.C. Ensign. 1998. Purification and characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64: 3029-3035; (Rajagopal, R. and R.K. Bhatnagar. 2002. Insecticidal toxic proteins produced by P. luminescens akhurstii the symbiont of Heterorhabditis indica. J Nematol. 34: 23-27.] and their genes cloned [(Bowen, D.J., T.A Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia andRH jfrench- Constant. 1998. Novel insecticidal toxins fi'om the bacterium Photorhabdus luminescens. Science 280: 2129-2132)]. The inability to isolate Photorhabdus from the environment without its symbiont nematode is suggestive of a critical symbiotic relationship between them. Since the bacterium has not been isolated in a free living form from the environment, its ability to survive, multiply and to infect insects in soil on its own has been questioned. Gotz et al (Gotz, P., A. Boman andH G. Boman. 1981. Interactions between insect immunity and an insect pathogenic nematode with symbiotic bacteria. Proc. R. Soc. Lond. B. 212: 333-350.) have shown that the nematode symbiont helps in overcoming the host's defense by secreting an immune inhibitor. Poinar et al. (Poinar, G. 0., Jr., G. Thomas, H. Haygooa landK. H. Nealson. 1980. Growth and luminescence of the symbiotic bacteria associated with the nematode Heterorharbditis bacteriophora. Soil Biol. Biochem. 12: 5-10.) studied the persistence of Photorhabdus in sterile soil and were unable to recover any cells even on the day following inoculation of soil with the bacteria. Morgan et al (Morgan, J. A. W., V. Kuntzelmann, S. Tavernor, M. A. Ousley and C. Winstanley. 1997. Survival of Xenorhabdus nematophillus and Photorhabdus luminescens in water and soil. J. Appl. Microbiol. 83: 665-670.) transformed Photorhabdus bacteria with a plasmid conferring resistance to kanamycin, for ease of monitoring, followed their survival and observed a rapid decline in their population upon release in soil. Lately, Bleakley and Chen (Bleakley, B. H. andX. Chen. 1999. Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil. Can. J. Microbiol. 45: 273-278) reported the successful survival of these bacteria, up to one month, in sterile (autoclaved) acidic soil augmented with calcium carbonate and gelatin or cosamino acids, but did not evaluate its insect pathogenic potential under these conditions. Several reports referred to above assessed the insecticidal potential of bacteria Photorhabdus by injecting the live bacterial cells into the insect. The results were not encouraging. On the contrary, the prior art studies indicated that the insecticidal properties of Photorhabdus diminished considerably when it was employed without its symbiont nematodes. The entomopathogenic nematodes of the genus Heterorhabditis are effective biological control agents against insect pests belonging to different orders. These nematodes provide better control of insects in the soil environment when compared to chemical insecticides. Since nematodes require a thin layer of water to be active, they have not been successful as an aerial spray on crop canopy to control foliage insect pests. Efforts to use them as foliar spray have been unsuccessful as they poorly survive in the desiccated aerial environment. It is known that Heterorhabditis indica has a symbiotic association with the bacterium Photorhabdus luminescens, particularly, Photorhabdus luminescens sub sp akhurstii. Till date, it has been maintained that the Photorhabdus bacteria does not have the ability to infect an insect host on its own and the nematode helps the bacteria to enter into the insect haemocoel. Upon entering the insect, Photorhabdus multiply and rapidly kill the insect. The nematode requires the bacteria for quickly killing the insect and also for proper multiplication. Axenic nematodes do not reproduce properly and have poor multiplication effeciency. Under such a scenario, the entomopathogenic nematodes can only be used as a soil insecticide and aerial application of Photorhabdus is considered impractical due to its inability to infect insect host on its own. P. luminescejis has been reported to secrete insecticidal toxin proteins which are very effective in controlling insect pests. Though the secretion of the toxin proteins by P. luminescens has been known for the last few years, there is a serious doubt about its ability to survive in an environment other than its specific nematode host. Earlier studies had shown that this bacteria is not stable in the soil environment. Thus, the prior art reports suggest that while the entomopathogenic nematodes can be employed with some degree of success along with its symbiont, P. luminescens as a soil insecticide, prior art continued to rely upon chemical insecticides for aerial application on crop plants. In addition, the prior at reports suggest that P. luminescens without its symbiont is unstable and non-efficacious either as a soil or aerial insecticide. Apart from general agriculture pests, which infest the cash crops, there are also several pests, which are specific to certain specific genera of plants. Common examples of these are gall midge, which infest rice, white woolly aphids, which affect sugarcane etc. Ceratovacuna lanigera Zehn , commonly known as white woolly sugarcane aphids thrive in warm humid atmosphere and fluctuating climate, mainly cloudy weather lead to their fast development and spread. Severe incidence of these pests have been observed in several parts of India and neighbouring countries. The White woolly sugarcane aphids belong to Family Pemphigidae and Order Homoptera. It is normally found in Asian region. Ceratovacuna lanigera is found in Philippines, Indonesia, Thailand, Taiwan, China, Japan, Korea, India, Pakistan. C. graminum is found in India while C. japonica is found in Japan and Korea. These pests primarily affect sugarcane crops although Bamboo crops are known to act as secondary hosts. This pest has the following characteristics: Nymph : • Both sides of midrib at lower surface of leaves • A newly emerged nymph yellowish or greenish yellow and very active • At the fifth segment, a pair of hollow tubes present • At third and fourth instar, white coloured woolly filaments are noticed on dorsal side of the body

Adult : • Adult emerge after fourth moulting • Black, with two pairs of transparent wings • Wing venation is clearly visible • Wing, 2.71 mm length and 1.21mm width • Can fly up to some distance • Possesses a pair of compound eyes • Nymph and adult possesses a pair of protuberances NATURE OF DAMAGE • Nymphs and adults suck the cell sap at lower side of leaves (phloem) with piercing and sucking type of mouth parts • Extra sugar is excreted as honeydew on upper side of lower leaf • Sooty mold colonizes on it and leaf turns black affecting photosynthesis • Leaf turns dull and margins dry • Plant becomes weak, growth hampered • Loss in cane yield and sugar recovery LOSSES • In Indonesia, 26 % reduction in cane yield and 24 % reduction in sugar content (Farina, 1994) • In Taiwan, young plants killed (Hill, 1993)

In Maharashtra, India • Loss in sugarcane yield -12 to 20 % (> 75 % infestation) • Loss in sugar recovery - 0.52 to 1.20 unit • Loss in sugar content in leaf- 0.38 gm/100 gm • Loss in chlorophyll content in leaf- 16.64 gm/100 gm SPREAD OF THE PEST • Winged female can travel up to 1.5 to 2 km. with wind currents • Honeydew secretion attracts the ants which carry the early nymphal stages to the next destination • Transport of infested canes • Infested seed (through leaves) • Planting of susceptible varieties Various chemical compounds have been tried in the art to control the spread of pests as well as treat sugarcane crops infested with these pests. However, considering the speed with which the infestation of the pests take place as well as the extent of damage can be caused in a short period of time, there has been a strong need for an alternative pesticide for the control inter alia of sugarcane pests Ceratovacuna lanigera. It is well established that the application of chemical insecticides results in environmental damage. These concerns have led the search of alternative, safer and economical pesticides. The use of biological pesticides to control the insect pests with minimal deleterious effect on the environment is being pursued vigorously. Of the various candidate biological control agents identified and tested thus far, the bacterial insect pathogen Bacillus thuringiensis has proved to be an effective agent for agricultural pests. Bacillus thuringiensis formulations occupy 95% of the total biological insecticide market. The major reason for the success of Bacillus thuringiensis spores in the field is because of the ease of its application with commonly used agricultural equipments like sprayers and dusters. None of the other biological insecticide has this character to an extent as comparable to Bacillus thuringiensis spores and hence their low acceptance by the farmers. Accordingly, there has been a log-felt need for alternative insecticidal compositions, which are effective against a wider spectrum of agricultural, horticultural and forestry pests and which do not have the usual disadvantages of the known chemical formulations. Objects of the invention It is, therefore, an important object of the present invention provide a pesticidal composition, which overcomes the disadvantages of the prior art. It is another object of the present invention to provide a pesticidal composition, which is environment friendly. It is yet another object of the present invention to provide a pesticidal composition, which is a better alternative to the conventional chemical compositions. It is yet another object of the present invention to provide a biopesticidal which effective for both soil and aerial applications. It is still another object of the present invention to provide a pesticidal composition, which are capable of being applied with commonly used agricultural equipment like sprayers and dusters. Summary of the invention The above and other objects of the invention are achieved by the novel biopesticidal composition of the present invention based on the bacterium Photorhabdus luminescens for controlling and eradicating various agricultural pests. The invention is based on the surprising and unexpected finding that when Photorhabdus luminescens is formulated as a sprayable composition, it is highly efficacious as a pesticide both for soil and aerial application even in the absence of its symbiont, nematodes. It is all the more surprising since this finding is quite contrary to the prior art reports, which have more or less established (1) that Photorhabdus luminescens is able to retain its insecticidal activity only in the presence of its symbiont nematodes; (2) even in the presence of nematodes, it is efficacious only for soil applications and (3) it loses its efficacy when applied aerially. In another important embodiment, the present invention is based on the unexpected finding that a biopesticide based on bacterium Photorhabdus luminescence is extremely effective in controlling the spread of Ceratovacuna lanigera as well as in treating sugarcane crops infested with Ceratovacuna lanigera. In particular, a synergistic biopesticidal composition comprising essentially of bacterium Photorhabdus luminescens and chitinase from plant, insect and bacterial sources is extremely effective in controlling the spread of Ceratovacuna lanigera as well as in treating sugarcane crops infested with Ceratovacuna lanigera. Several laboratory and field testing programme were carried out to evaluate and assess the product. Elaborative and extensive field trials were conducted to study the efficacy of the product on the serious pest of sugarcane. The results obtained from these experiments have given a clear indication that the product is very effective and is novel based on the bacterium Photorhabdus luminescence and the plant, bacterial and insect chitinase purified to greatest extent and stabilized for longer shelf life. Accordingly, the present invention provides a sprayable biopesticidal composition comprising Photorhabdus luminescens, and conventional additives and/or adjuvants. In an embodiment, the additives are selected from surfactants, mineral oils, paraffin oil and the like. In another embodiment, the sprayable composition is in the form of aerosol. In a preferred feature, said Photorhabdus luminescens is present in said spray in a concentration of 10⁵ to 10¹⁵ CFU/ml. In an embodiment, Photorhabdus luminescens active cells are encapsulated in alginate beads. Preferably, the beads are sodium alginate beads. In another embodiment, the present invention provides a sprayable biopesticidal composition comprising Photorhabdus luminescens and chitinase and optionally, conventional additives and/or adjuvants. In an embodiment, said chitinase is of plant, insect or bacterial origin. In a preferred embodiment, said Photorhabdus luminescens is present in said spray in a concentration of 10⁵ to 10¹⁵ CFU/ml and said chitinase are employed in an amount of from 100 mg to 5 gm per litre. In a preferred feature, crops are selected from but limited to cabbage, cotton, pulses, peas and sugarcane, bamboo, grapes, citrus, mango and guava.

Definitions In the present invention, unless stated to the contrary, "agricultural pests" would include plant pests in general and agricultural, horticultural and forestry pests in particular. Unless repugnant to the context, the expression "pests" is deemed to include but is not limited to one or more of mealy bugs, termites, grubs, sucking pests, Plutella larvae of S. liture, Plutellaβarias vitella and Pectinophora gossypiella and also

Ceratovacuna lanigera and other insect pests of plants in general. "Crops" means any plant useful to humans and is preferably selected from and not limited to cabbage, cotton, pulses, peas and sugarcane, bamboo, grapes, citrus, mango and guava. "Plants" include plants, shrubs and trees. Detailed description of the invention The present invention for the first time successfully explores the insecticidal ability of P. luminescens without its symbiotic carrier nematode. In an embodiment of the present invention, actively growing cells of P. luminescens are encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads when mixed in sterilized soil and exposed to S. litura 6 instar larvae result in 100% mortality of the insects by 48 hours while the use of alginate encapsulated Heterorhabditis nematode result in only 40% mortality by 72 hours. This finding is quite surprising and contrary to the teachings of prior art. In accordance with the present invention, growing cells of P. luminescens were formulated into a sprayable liquid composition by the addition of standard adjuvants and surfactants and examined for their ability to infect various insect larvae. The diluted formulation was spread on cotton and cabbage leaves and neonate larvae of Pectinophora gossipiella and Plutella xylostella were released on the plants. The feeding larvae died in 48 hours indicating that the bacteria alone were sufficient to kill the insect. The bacterium was re-isolated from the insect cadaver thus proving Koch's postulates. Subsequently the sprayable composition of the present invention was applied on Cabbage crop in field during cropping season. This resulted in effective control of diamond back moth (DBM), Plutella xylostella damage in Cabbage. In addition, results of spraying Photorhabdus composition to control field infestation of another pest, sugarcane woolly aphid, Ceratovacuna lanigera Zehnt on sugarcane reveals that P. luminescens effectively controls incidence of this insect pest up to 15 days. The extent of control achieved by P. luminescens spray was equivalent to that obtained with commonly used insecticides. Mammalian toxicity analysis revealed that the bacterium is not toxic to higher mammals. P. luminescens formulation when sprayed with commonly used plant protection sprayers gives very effective control of the most serious pests of cotton and pigeon pea. The protection and yield offered by the P. luminescens spray is significantly better than the control plants and is comparable to the results given by currently deployed chemical insecticides and Bt spores. The present invention will now be described with reference to the accompanying drawings and following examples in order to illustrate the preferred embodiments. It will be apparent to a person skilled in the art that various modifications of the present invention are possible without departing from the spirit and scope of the invention and what is described hereinafter are only for illustrative purposes. In the accompanying drawings: Figure 1A: Laboratory bioassay of Photorhabdus on insects - Spodoptera litura 6^th instar larvae killed following (a) exposure to Photorhabdus in alginate beads and (b) larvae killed due to exposure to Heterorhabditis indica in alginate beads.

Figure IB: Plutella xylostella larvae killed (a) following four days exposure to

Photorhabdus spray and (b) healthy larvae. Figure 2A: Cabbage heads (a) damaged by Plutella xylostella and (b) protected by

Photorhabdus spray.

Figure 2B: Sugarcane (a) damaged by white wolly aphid Ceratovacuna lanigeraand

(b) infection reduced by Photorhabdus spray.

Figure 2C: Teak trees (a) infested by termites and (b) protected by Photorhabdus spray. In order to demonstrate the present invention, Photorhabdus luminescens akhurstii was formulated in a sprayable liquid formulation and assessed for its viability under laboratory conditions. Later, this bacterial formulation was sprayed on various crop plants such as cotton and pigeon pea plants grown in agricultural fields and assessed for its ability to control insect pests. In accordance with the following

Examples:

Example 1

A. Culturing of bacteria (P. luminescens) and preparation of alginate beads P. luminescens sub sp. akhurstii strain K-l, isolated from H. indica (Rajagopal, R. & Bhatnagar, R. K. (2002) J. Nematol 34, 23-27) was used in the present invention.

Cultures were grown overnight on Luria Bertani (LB) medium from starter cultures at

0.5%ι inoculum concentration. The cells were pelleted by centrifuging at 15,000 x g at

4°C. The supernatant was passed through a 0.22μ filter while the pellet was resuspended in an equal volume of fresh LB medium. Escherichia coli K-12 was also grown and processed similarly. The different treatments resulting in 6 different alginate beads were as follows: Tl hotorhabdus luminescens K-l, overnight cell suspension (50ml) containing approximately 1 x 10⁹cells/ml (The medium was used as such without centrifugation). T2. P. luminescens overnight cell suspension, centrifuged and pellet resuspended in fresh LB medium (50ml) resulting in approximately 1 x 10⁹ cells/ml. T3. P. luminescens overnight cell suspension, centrifuged at 12,000 rpm and supernatant passed through 0.22μ filter (50ml). T4. E. Coli K-12, overnight cell suspension (50ml) containing 1 x 10⁹cells/ml

(The medium is used as such without centrifugation). T5. Sterile water (50ml) T6. H. indica infective juveniles (IJ) in 50 ml sterile water @ 300 IJ/ml. Bacterial suspension or the culture supernatant (50ml) was mixed thoroughly with 50 ml of a solution containing 2% sodium alginate and 2% sucrose in sterile water and poured dropwise into 1.47% calcium chloride (CaCl ) solution resulting in the formation of uniform, 3-4 mm diameter, round beads. After 30 minutes, the beads were collected on a wire mesh filter, decanting the CaCl₂. They were washed/flushed repeatedly (5 times) with approximately 200 ml of sterile water each time, and allowed to fully decant. This material was used for further experiments described in Examples 2-5. B. Preparation of sprayable formulation of Photorhabdus: P. luminescens sub sp. akhurstii strain K-l, isolated from H. indica was used in this Example. For preparing the spray formulation, P. luminescens cultures were grown overnight on Luria Bertani (LB) medium from starter cultures at 0.5% inoculum concentration. One liter of fully grown broth cultures with an approximate cell density of 1 x 10⁹ cells / ml was supplemented with surfactants and emulsifiers like paraffin oil @ lOOμl/ml, Tween-20 @ 0.5μl/ml, sucrose 5%, Triton X-100 and Tween 80 to final concentration of 5%. This stock was further diluted with distilled water to a ratio of 450 liters per hectare and sprayed using standard knapsack sprayer. Commonly available chemical insecticides viz. Endosulfan 35 EC@ 0.07%, Qninalphos 25 EC @0.04% and Monocrotophos 36 WSC @ 0.04% were used as standards for comparison in pigeon pea. The commonly used biological insecticides like Bacillus thuringiensis (Delfin) and H. armigera nuclear polyhedrosis virus (Ha NPV) were used for comparison in cotton. For laboratory bioassay of this formulation the original stock was diluted with sterile distilled water to varying concentrations of bacterial cells by serial dilution. Example 2 Laboratory bioassa s of Photorhabdus alginate beads Alginate beads prepared from the six different treatments were tested immediately on S. litura larvae. All the insects (100%) tested with Treatment T2 and 94% insects in T-l alginate beads (both of which contained P. luminescens cells) died by 24 hrs, while there was no mortality in insects tested with T4 and T5 beads as shown in Tablel below:

Table 1

The dead insects turned reddish and luminesced strongly in dark. The bacteria were isolated from the cadaver of the insect (Kaya, H.K. & Stock, S.P. (1997) in Manual of techniques in insect pathology, ed. Lacey L.A. (Academic Press, San Diego, USA.) pp. 281-324 and genomic DNA prepared and PCR-RFLP analysis conducted on the 16S rDNA gene according to Rajagopal and Bhatnagar((Rajagopal, R. & Bhatnagar, R. K. (2002) J Nematol34, 23-27). The PCR-RFLP profile of the reisolated bacteria and the initial bacteria (K-l) were identical (results not shown) fulfulling the Koch's postulates and establishing fidelity of infection and proliferation. Treatment T- 3 was included to discriminate between the insect mortality as a consequence of the secreted insecticidal protein complex as compared with insect mortality obtained by the direct infection of the insect by P. luminescens cells. T-3 alginate beads, from the culture supernatant, resulted in 20% death of larvae by 48 hrs, suggesting that the secreted toxin protein retains its activity in alginate beads. To understand insect behaviour as a result of infection, the insects released on T 2 alginate beads were observed every 6 hours. The insects were active and ingesting the beads at the 6^th hours. By the 12^th hour approximately 40% of the beads were consumed and insect movement was greatly reduced. By 18 hours the insects were immobile and by 24 hours they were dead. They turned reddish black and emitted strong luminescens in dark by 48 hours. (Attempts to isolate Photorhabdus CFU from soil samples of this treatment failed at each of the above four intervals viz. 6, 12, 18 and 24 hours). Heterorhabditis indica infective juveniles (carrying Photorhabdus in their gut) encapsulated in alginate beads (T-6) did not result in mortality of S. litura till 48 hours, and by 72 hours only 40% of the insects had died when compared to 100% mortality in T-l and T-2 beads which contain only Photorhabdus cells. Comparing the cadaver of the larvae killed by Photorhabdus alone and those killed by H. indica (Figure 1A) revealed that in the former there was complete degradation of insect body with shriveling and shrinkage, while the integrity of the cadaver was retained in the latter treatment. Example 2 Laboratory bioassays of Photorhabdus sprayable formulation Laboratory bioassays were conducted by using the Photorhabdus cells against Earias vitella, Pectinophora gosypiella and Plutella xylostella by leaf coating method. For the assessment of doses, serial dilutions were made from the original stock solution prepared in Example 1 to very low (10¹ cells/ml), Medium (10⁴ Cells/ml) and very high doses (10⁶cells/ml) to determine the median lethal doses. Approximately, 15cm² of leaf surface was coated with 200 μl of the formulation at the respective dilutions and air dried. As a control, the leaves were coated with water containing all the other spray components except Photorhabdus cells. Each dose was replicated six times and ten larvae of each insect were released on each leaf disc. The mortality was recorded after every 24 hours the observations were recorded up to 96 hrs. After coating the Photorhabdus cells formulated as a liquid spray on cabbage leaves, ten Plutella xylostella larvae were released per 15 cm² cabbage leaf and almost all the larvae were killed by 24 hours at a bacterial load of 10⁶ and 10⁴ as shown in Table 2 below: Table 2. Mortality response of 4^th instar larvae of Plutella xylostella against spray formulation of Photorhabdus.

Even at the lowest dose of 10 cells per ml. approximately 50% mortality was observed and by 96 hours 80% of the Plutella larvae had died. From this it can be safely concluded that Photorhabdus cells when formulated as a spray retains its ability to infect insect hosts on its own (Figure IB). Further, the quality of cabbage heads obtained following spraying of the composition of the present invention was highly superior when compared to that of no control treatment (figure 2A). Independent experiments using cotton leaves with larvae of Earias vitella and Pectinophora gossypiella gave similar results as shown in Tables 3 and 4 below:

Table 3. Mortality response of 3 »rι'l instar larvae of Earias vitella against spray formulation of Photorhabdus.

Table 4. Mortality response of 3^rd instar larvae of P. gossypiella against spray formulation of Photorhabdus.

The dead insects turned reddish and luminesced strongly in dark. The bacteria were isolated from the body of the dead insect. Genomic DNA were prepared and PCR- RFLP analysis conducted on the 16S rDNA gene according to documented procedures. The PCR-RFLP profile of the reisolated bacteria and the initial bacteria (K-l) were identical fulfilling the Koch's postulates and establishing fidelity of infection. From the above, it can be safely concluded that Photorhabdus cells when formulated as a spray retains its ability to infect insect hosts on its own. Even very low doses gave effective control of the insects. Efficacy of the formulation having Photorhabdus was also assessed in parallel with delivery through its natural host nematode. Heterorhabditis indica infective juveniles (carrying Photorhabdus in their gut) did not result in mortality of insect larvae till 48 hours, and by 72 hrs only 40% of the insects had died as compared to 100% mortality in Photorhabdus formulation. Comparing the cadaver of the larvae killed by Photorhabdus alone and those killed by H. indica, (Figure 1) reveals that in the former there is complete degradation resulting in shriveling and shrinkage, while the integrity of the cadaver is still maintained in the latter. Example 3 Field evaluation of sprayable formulation on sugarcane Experiments to study the efficacy of Photorhabdus spray on sugarcane demonstrated that the formulation @ 5x10¹¹ bacterial cells per ha. gave good control (88%) of sugarcane woolly aphids as shown in Table 5 below: Table 5 Bioefficacy of P. luminescens after 15 days of spraying for control of sugarcane woolly aphid, Ceratovacuna lanigera on Sugarcane

The level of protection provided till 15 days after spraying by P. luminescens was comaprable to control obtained with the commonly used chemical insecticide (Figure 2B). Example 4 Field evaluation of sprayable formulation on cotton The liquid formulation prepared in accordance with Example 1 was tested under field conditions on the cotton and pigeon pea crop in the Akola region of Maharashtra, India during the Kharif growing season. The treatments on cotton crop included 1) P. luminescens 750 ml stock solution / ha 2) P. luminescens 1000 ml stock solution / ha 3) Bacillus thuringiensis (Delfin) 1 kg/ha 4) HaNPV 500 ml/ha and 5) Untreated as control The experiment was conducted in a randomised block design (RBD) with five replicatioris and the individual plot size was 10 x 5.4 mt2 containing 55 plants each of cotton variety PKV hybrid 2, planted at a spacing of 90 x 90 cm. Spraying of the insecticides were initiated at economic threshold level (ETL) and two more sprays were applied when the pest population again reached the ETL. Observation of infestation of bollworm damage (by Helicoverpa armigera, Earias vitella and Pectinophora gossypiella) to green fruiting bodies (GFB) i.e. bolls and squares were recorded from five randomly selected plants from each replication plot for statistical analysis. Damage to loculi was recorded at harvest. The yield of seed cotton was recorded from each plot after harvest and denoted on a per hectare basis. Data was transformed in to arc sine transformation and subjected to ANOVA. Results of the above mentioned experiment indicates that Photorhabdus spray formulation gave effective control to the green fruiting bodies till 10 days of spraying as shown in Table 6 below: Table 6. Bioefficacy of different biopesticides in comparison with P. luminescence in Cotton for control of Bollworms.

Figures in parentheses are arc sine values

The Photorhabdus formulation @ 1000ml per ha. gave consistently good results in the three different sprays, each initiated at ETL on the cotton crop spread over a period of 210 days. At the end of the experiment this treatment recorded the lowest loculi damage and the highest yield of 7.38 quintals, better results than the commonly used biopesticides - B. thuringiensis (Dipel) and H. armigera NPV.

Example 5 Field evaluation of sprayable formulation on pigeon pea The treatments on pigeon pea included 1) P. luminescens @ 500 ml stock solution / ha 2) P. luminescens @ 750 ml stock solution / ha

3) P. luminescens @ 1000 ml stock solution / ha

4) Endosulfan 35 EC @ 0.07%

5) Quinalphos 25 EC @ 0.04%

6) Monocrotophos 36 WSC @ 0.04% and

7) Untreated as control. The experiment was conducted in RBD with five replications and the individual plot size was 7.8 x 6 mt² containing 260 plants each of pigeon pea variety C - 11, planted at a spacing of 60 x 30 cm. Spraying, of the insecticides were initiated at economic threshold level (ETL) of 0.5 larvae / plant and two more sprays were applied when the pest population again reached the ETL. Five plants were selected randomly from each replication plot. Five pod bunches of each selected plants were used to record the damaged pods caused by Helcoverpa armigera and Exelastis automosa. also record the level of grain damage. The total grain yield was recorded at harvest. Data was transformed in to arc sine transformation and subjected to ANOVA. Observations of the experiment indicate that P. luminescens spray @ 1000 ml/ha gave the best results when compared to other treatments in terms of protection of grain and pod damage and also recorded the maximum yield. These results are shown in Table 7 below:

Table 7: Bioefficacy of different pesticides in comparison with P. luminescence on tur pod borers and grain yield

Figures in parentheses are arc sine values

Treatments involving P. luminesiens at 750 ml/ha or higher gave results comparable to those of the commonly used chemical insecticides. This clearly demonstrates that P. luminescens can effectively protect crop plants from insect damage resulting from increased yield. The results obtained with P. luminescens spray is comparable to that obtained presently by the farmers using other chemical and biological insecticides.

Example 6 Field evaluation of Photorhabdus sprayable formulation on white ants Fields were surveyed for live termitaria and thirty such termitaria were selected.

The insecticidal formulation of Photorhabdus was drenched into the termitaris without disturbing the upper strata by making a small hole, just enough for a 50 ml syringe nozzle to enter. 1000 ml of the formulation was applied in each termiatra. As a positive control, Chlorpyriphos 5EC was applied at 2ml / liter and water alone at one liter per termitaria acted as a negative control. Ten replications were done for each treatment. Forty eight hours after applying the treatments, the termitaria were observed and all the termites were dead in the colonies treated with Chlorpyriphos and

Photorhabdus formulation. Example 7

Field evaluation of biopesticidal composition containing Photorhabdus luminescens and chitinase The field testing of efficacy of Improved Biopesticide formulation with wider activity spectrum based on the bacterium Photorhabdus luminescens sub sp. akhurstii strain K-l, isolated from Heterorhabditis indica and chitinase from plant, insect and bacterial sources was carried out as shown in Table 8 below in the sugarcane growing areas of Maharashtra especially in the western region where the infestation was severe. The trails were conducted on the standing crop of sugarcane during October - November 2003. All the treatments were replicated three times and the design of Experiment was in Randomized Block (RBD). Location I : Vasnatdada Sugar Institute, Manjri, Pune (MS)

Table 8: Treatment details:

Observations:

The above table indicates that the treatment T4, T5 and T7 had a tremendous impact on the population and within three days there was total suppression of the white wooly aphids on sugarcane. The treatment T4 consists of P. luminescence with mineral oil and chitinase and the T5 contains only P. luminescence and chitinase however the treatment T7 is a chemical insecticide which is commonly used for the control of this pest. Our results indicate that the newly developed formulation based on P. luminescence along with chitinase of the plant, bacterial and insect sources has given excellent control of the pest at par with the chemical insecticide. Similar replicated trails were conducted at four more locations and the results are presented in Table 9 below: Table 9: Results

From the above results it is clear that the combination of P. luminescence with the chitinase is very effective in the management of the sugarcane white wooly aphids and similar results are anticipated on other soft bodied insects especially aphids on other important crops like cotton, mustard, safflower. It is also anticipated that the control of mealy bugs thrips, etc. can also be achieved with the use of Improved Biopesticide formulation with wider activity spectrum based on the bacterium Photorhabdus luminescens sub sp. akhurstii strain K-l, isolated from Heterorhabditis indica and chitmase from plant, insect and bacterial sources. According to the present invention, Photorhabdus luminescens strain K-l has been formulated into a sprayable aerial formulation. Approximately one liter of the formulation is enough to effectively protect an area of one hactare from insect pest damage. Preliminary analysis reveal that the formulation is effective in controlling white ants, termites, ants and cockroaches other than lepidopteran pests. This clearly demonstrates that the bacteria can pathogenise the insect host independent of the symbiont nematode. While the entomopathogenic nematode with the bacteria inside is able to kill insects only by 4 days, P. luminescens alone kills the insect larvae between 1-2 days. Tests with mammalian toxicity (on rats, mice and rabbit) of P. luminescens reveal that they are safe to these organisms. Organ, Organellar and standard biochemical analysis of Photorhabdus fed animals did not reveal any undesirable symptoms or deleterious effects. Though human clinical isolates of Photorhabdus have been isolated, PCR-RFLP studies of the 16S ribosomal RNA gene reveal that they share only 25 - 30 % similarity -with P. luminescens akhurstii. Based on these studies, the human clinical isolates of Photorhabdus have been classified as Photorhabdus atypica. This opens a new methodology to combat insects pests and protect crop plants from their damage.

Claims

Claims:

1. A sprayable biopesticidal composition including Photorhabdus luminescens.

2. A composition as claimed in claim 1 further including conventional additives and/or adjuvants.

3. A composition as claimed in claim 2 wherein said additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

4. A composition as claimed in any preceding claim in form of an aerosol.

5. A composition as claimed in any preceding claim wherein said Photorhabdus luminescens is encapsulated in alginate beads.

6. A composition as claimed in claim 5 wherein said alginate beads comprise sodium alginate beads.

7. A composition as claimed in any preceding claim wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml.

8. A biopesticidal composition comprising an effective amount of Photorhabdus luminescens and chitinase.

9. A composition as claimed in claim 8 further including conventional additives and/or adjuvants.

10. A composition as claimed in claim 9 wherein aid additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

11. A composition as claimed in any one of claims 7 to 9 wherein said chitinase is of plant, insect or bacterial origin.

12. A composition as claimed in any one of claims 8 to 11 wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml and said chitinase is present in amount of 100 mg to 5 gms per litre.

13. A method for treating crops to render them resistant to free of agricultural pests which comprises spraying on said crops an effective amount of a biopesticidal composition including Photorhabdus luminescens.

14. A method as claimed in claim 13 wherein said composition further includes conventional additives and/or adjuvants.

15. A method as claimed in claim 14 wherein said additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

16. A method as claimed in any one of claims 13 to 15 wherein said composition is in form of an aerosol.

17. A method as claimed in any one of claims 13 to 16 wherein said Photorhabdus luminescens is encapsulated in alginate beads.

18. A method as claimed in claim 16 wherein said alginate beads comprise sodium alginate beads.

19. A method as claimed in any one of claims 13 to 16 wherein said crops are selected from cabbage, cotton, pulses, peas, grapes, mango, guava, citrus, bamboo and sugarcane.

20. A method as claimed in claim 19 wherein said pests are selected from mealy bugs, termites, grubs, sucking pests, Plutella larvae of S. liture, Plutella,Earias vitella and Pectinophora gossypiella and also Ceratovacuna lanigera.

21. A method as claimed in any one of claims 13 to 20 wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml.

22. A method for treating crops to render them free of or resistant to pests which comprises applying to said crops a biopesticidal composition comprising an effective amount of Photorhabdus luminescens and chitinase.

23. A method as claimed in claim 22 further including conventional additives and/or adjuvants.

24. A composition as claimed in claim 22 wherein aid additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

25. A composition as claimed in any one of claims 22 to 24 wherein said chitinase is of plant, insect or bacterial origin.

26. A composition as claimed in any one of claims 22 to 25 wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml and said chitinase is present in amount of 100 mg to 5 gms per litre.

27. A method as claimed in any of claims 22 to 26 wherein said crops are selected from sugarcane and bamboo and said pest is Ceratovacuna lanigera.

28. Use of a composition containing Photorhabdus luminescens in the preparation of a biopesticidal composition for treatment of crops to render them resistant to or free of agricultural pests.

29. Use as claimed in claim 28 wherein said composition further includes conventional additives and/or adjuvants.

30. Use as claimed in claim 29 wherein said additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

3 1. Use as claimed in any preceding claim 28 to 30 in form of an aerosol.

32. Use as claimed in any preceding claim 28 to 30 wherein said Photorhabdus luminescens is encapsulated in alginate beads.

33. Use as claimed in claim 32 wherein said alginate beads comprise sodium alginate beads.

34. Use as claimed in any one claims 28 to 33 wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml.

35. Use of a composition containing Photorhabdus luminescens in the preparation of a biopesticidal composition for treatment of crops to render them resistant to or free of agricultural pests, said composition also containing an effective amount of chitinase.

36. Use as claimed in claim 35 wherein said composition further includes conventional additives and/or adjuvants.

37. Use as claimed in claim 36 wherein said additives are selected from one or more of surfactants, emulsifiers, mineral oils, paraffin oil, tween and sucrose.

38. Use as claimed in any one of claims 35 to 37 wherein said chitinase is of plant, insect or bacterial origin.

39. A composition as claimed in any one of claims 35 to 36 wherein said Photorhabdus luminescens is present in an amount of 10⁵ to 10¹⁵ CFU/ml and said chitinase is present in amount of 100 mg to 5 gms per litre.