WO1999008534A1

WO1999008534A1 - Novel compositions and methods for controlling nematodes

Info

Publication number: WO1999008534A1
Application number: PCT/US1998/016952
Authority: WO
Inventors: Zhongxiao Chen; Donald W. Dickson
Original assignee: University Of Florida
Priority date: 1997-08-15
Filing date: 1998-08-14
Publication date: 1999-02-25
Also published as: AU9105498A

Abstract

The subject invention concerns pest-control compositions and advantageous methods of using these compositions. More specifically, this invention relates to the use of bacteria to control plant-parasitic nematodes.

Description

DESCRIPTION

NOVEL COMPOSITIONS AND METHODS FOR CONTROLLING NEMATODES

Cross-Reference to a Related Application This application claims priority from U.S. provisional patent application serial number 60/055,842, filed August 15, 1997.

Background of the Invention

This invention relates to a method for utilizing bacterial endospores from Pasteuria, or Pasteuria-like, bacteria for the control of nematode pests. These bacteria are able to produce endospores that have the unique and useful property of being able to attach to, infect and kill phytopathogenic nematodes. Damage to plants caused by nematodes is a prevalent and serious economic problem. Nematodes cause wide-spread and serious damage in many plant species. Plant-parasitic nematodes cause a $6 billion monetary loss to United States agriculture each year (Sasser, J.N., D.W. Freckman [1987] "A world perspective on nematology: The role of the society," in Vistas on Nematology: A Commemoration of the Twenty-Fifth Anniversary of the Society of Nematologists (J.A. Veech and D.W. Dickson, Eds.),

Society of Nematologists, Hyattsville, MD, pp. 7-14). Many genera of nematodes are known to cause such damage. Plant-parasitic nematodes include members of the Phylum Nematoda, Orders Tylenchida and Dorylaimide. In the Order Tylenchida, the plant- parasitic nematodes are found in two super-families: Tylenchoidea and Criconematoidea. There are more than 100,000 described species of nematodes.

Efforts to eliminate or minimize damage caused by nematodes in agricultural settings have typically involved the use of soil fumigation with materials such as chloropicrin, methyl bromide, and dazomet, which volatilize to spread the active ingredient throughout the soil. Such fumigation materials can be highly toxic and may create an environmental hazard. Various non-fumigant chemicals have also been used, but these too create serious environmental problems and can be highly toxic to humans. Peanut (Arachis hypogaea L.), one of the most important crops grown in the southern United States, is host to a virulent pathogenic nematode, Meloidogyne arenaria (Neal) Chitwood race 1 (Minton, N.A., P. Baujard [1990] "Nematode parasites of peanut," in Plant-Parasitic Nematodes in Subtropical and Tropical Agriculture (M. Luc, R.A. Sikora, J. Bridge, Eds.), CAB International, Wallingford, UK, pp. 285-320). In

Texas, initial population densities of 8.8 to 16.6 second-stage juveniles (J2) of M. arenaria per 100 cm³ soil were sufficient to cause 10% suppression of yield (Wheeler, T.A., J.L. Starr [1987] Peanut Science 14:94-96), whereas the damage threshold in Florida was determined to be as low as 1.0 J2/100 cm³ soil (McSorley, R., et al. [1992] J. Nematology 24:193-198). The management of M. arenaria relies largely on a combination of methods that includes nematicides and crop rotation (Dickson, D.W., T.E. Hewlett [1989] Supplement to J. Nematology 21:671-676; Minton & Baujard, supra). Economic considerations and potential environmental hazards of nematicides, however, have created a climate of uncertainty regarding their continued use on peanut. It is also documented that nematicides do not provide desired levels of control when nematode population densities are high (Dickson, D.W., T.E. Hewlett [1988] Ann. Appl. Nematol. 2:95-101), and some successful rotations must remain in place for more than four years (Dickson & Hewlett [1989], supra).

The regular use of chemical toxins to control unwanted organisms can select for drug-resistant strains. The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action. Also, nematicides routinely used for control of plant-parasitic nematodes are rapidly being pulled from the market as concern for environmental safety increases. In the year 2001, Methyl Bromide, a mainstay in the control of such parasites, will no longer be marketed in the United States. Therefore, less harmful control agents are clearly needed.

Biological control of nematodes offers an alternative or supplemental management tactic to chemical or cultural control of nematode pathogens. A small number of research articles have been published concerning the effects of δ-endotoxins from B. thuringiensis species on the viability of nematode eggs. See, for example, Bottjer, Bone and Gill ([1985] Experimental Parasitology 60:239-244); Ignoffo and

Dropkin (Ignoffo, CM., Dropkin, V.H. [1977] J. Kans. Entomol. Soc. 50:394-398); and Ciordia, H. and W.E. Bizzell ([1961] Jour, of Parasitology 47:41 [abstract]). Several patents have issued describing the control of nematodes with B.t. See, for example, U.S. Patent Nos. 4,948,734; 5,093,120; 5,281,530; 5,426,049; 5,439,881; 5,236,843; 5,322,932; 5,151,363; 5,270,448; 5,350,577.

The pesticidal activity of avermectins is well known. The avermectins are disaccharide derivatives of pentacyclic, 16-membered lactones. They can be divided into four major compounds: A_la, A_2a, B_la, and B^; and four minor compounds: A_lb, A_2b, B_lb, and B_2b.

The organism which produces avermectins was isolated and identified as

Streptomyces avermitilis MA-4680 NRRL-8165). Characteristics of the avermectin producing culture and the fermentation process are well documented and known to those skilled in the art. The isolation and purification of these compounds is also described in

U.S. Patent No. 4,310,519, issued January 12, 1982.

Another family of pesticides produced by fermentation are the milbemycins, which are closely related to the avermectins. The milbemycins and their many derivatives are also well known to those skilled in the art and are the subject of U.S. patents. See, for example, U.S. Patent No. 4,547,520.

Fatty acids are a class of natural compounds which occur abundantly in nature and which have interesting and valuable biological activities. Tarjan and Cheo (Tarjan,

A.C., P.C. Cheo [1956] "Nematocidal Value of Some Fatty Acids," Bulletin 332, Contribution 884, Agricultural Experiment Station, University of Rhode Island,

Kingston, 41 pp.) report the activity of certain fatty acids against nematodes.

Fatty acids were also examined by Malik and Jairajpuri (Malik, Z., M.S. Jairajpuri

[1977] Nematol. medit. 12:73-79), who observed nematode toxicity at high concentrations of the fatty acids. Stadler et al. (Stadler, M., A. Mayer, H. Anke, O. Sterner [1994] Planta Med. 60:128-132) studied fatty acids and other compounds with nematicidal activity which could be obtained from cultures of Basidiomycetes.

Endospore-forming bacteria of the Pasteuria group have been recognized as potential biorational control agents of plant-pathogenic nematodes (Sayre, R.M. [1980]

Plant Disease 64:527-532; Stirling, G.R. [1984] Phytopathology 74:55-60). The first report of a Pasteuria-like organism infecting nematodes was by N. A. Cobb (2nd ed.

Hawaiian Sugar Planters Assoc, Expt. Sta. Div. Path. Physiol. Bull, vol. 5, pp. 163-195, 1906), who observed numerous retractile spores in nematodes of the species Dorylaimus bulbiferous.

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite of Meloidogyne spp. Endospores of P. penetrans attach to the cuticle of Meloidogyne spp. J2 in soil. Infection occurs after the J2 enters a plant root, where parasitized nematodes mostly develop into females, but are incapable of reproduction (Bird, A.F. [1986] Parasitology 93:571-580). The females, and sometimes males (Hatz, B., D.W. Dickson [1992] J. Nematol. 24:512-521), become filled with endospores, which are eventually released into the soil upon host disintegration. Pasteuria penetrans (Mankau) Sayre and Starr, has shown potential for biological control of root-knot nematodes (Chen et al. [ 1996] J. Nematol. 29:159-168; Chen et al. [1997] J. Nematol. 29:1-8; Dickson et al. [1994] "Control of plant-parasitic nematodes by biological antagonists," in Pest Management in the Subtropics, Biological Control - A Florida Perspective (D. Rosen, F.D. Bennett, J.L. Capinera, Eds.), Intercept, Hampshire, UK, pp. 575-601; Mankau, R., N. Prasad [1977] J. Nematol. 9:40-45; Sayre,

R.M., M.P. Starr [1985] Proc. of the Helminthological Society of Washington 52:149- 165; Stirling, G.R. [1984] Phytopathology 74:55-60: Stirling [1991] Biological Control of Plant Parasitic Nematodes: Progress, Problems, and Prospects, CAB International, Wallingford, UK; Brown et al. [1985] Soil Biology and Biochemistry 17:483-486; Chen et al, 1996, 1997, supra; Oostendorp et al. [1991] J. Nematol. 23:58-64; Stirling, 1984, supra).

R.M. Sayre et al. (Sayre, R.M., M.P. Starr, A.M. Golde, W.P. Wergin, and B.Y. Endo [1988] Proc. Helminthol. Soc. Wash. 55(l):28-49) suggested that Pasteuria penetrans is a specific parasite of the root-knot nematode Meloidogyne incognita and proposed that other bacteria of the "Pasteuria group" deserve separate species designations. Starr et al. (Starr, M.P., R.M. Sayre [1988] Ann. Inst. Pasteur /Microbiol. 139:11-31) assigned the species name Pasteuria thornei sp. nov. to the endospore- forming bacterium that is primarily parasitic on the root-lesion nematode Pratylenchus brachyurus, and the species name Pasteuria penetrans sensu stricto emend, to the endospore-forming bacterium which is primarily parasitic on the root-knot nematode

Meloidogyne incognita. Although bacteria of the Pasteuria group have a recognized potential for use as biorational control agents against phytopathogenic nematodes, their widespread use in commercial agriculture will depend on the availability of reliable methods to utilize these bacteria. The major obstacle for the practical application of P. penetrans to soil infested with root-knot nematodes is the lack of a sufficient production of endospores. Currently, the parasite must be produced in vivo in Meloidogyne spp., which, in turn, must be reared on susceptible plants (Stirling, G.R., M.F. Wachtel [1980] Nematologica 26:308-312), or in nematode-excised root systems (Verdejo, S., B.A. Jaffee [1988] Phytopathology 78:1284-1286). Neither procedure provides production of sufficient inoculum for large- scale applications.

Brief Summary of the Invention The subject invention concerns compositions and methods for the control of plant pests. More specifically, this invention relates to the use of Pasteuria bacteria to control plant-parasitic nematodes. In a specific embodiment, Pasteuria penetrans can be used to suppress parasitic nematodes, including root-knot nematodes, on peanuts. The increased yields and reduced galls resulting from adding P. penetrans to plots containing M. arenaria demonstrates that P. penetrans reduces the damaging effects of M. arenaria on peanut. In accordance with the teachings of the subject invention, effective control of phytopathogenic nematodes can be achieved by applying, in a first year, a relatively low level of inoculum to soil. Surprisingly and advantageously, it has been found that, within three years, an initially low concentration of bacterial endospores will increase to yield a highly effective Pasteuria endospore concentration. The initial endospore concentration may be, for example, 1,000 endospores/g of soil. Within three years, the endospore concentration will greatly exceed the 30,000-40,000 endospores/g of soil needed for effective nematode control. In a preferred embodiment, the Pasteuria endospore concentration will be at about 100,000 to about 135,000 three years after inoculation. The ability of Pasteuria penetrans for suppressing Meloidogyne arenaria on peanut (Arachis hypogaea) was demonstrated over a 3-year period in a field microplot experiment. Pasteuria penetrans population established and amplified during the 3-year period. The equilibrium density of P. penetrans in field microplots was determined to be 133,000 endospores/g of soil.

Endospores were inoculated into the top 20 cm of soil in microplots at rates of 0; 1,000; 3,000; 10,000; and 100,000 endospores/g of soil. The microplots were previously infested with M. arenaria race 1 and tomato had been planted to increase the inoculum.

One peanut seedling was planted in each microplot. In the first year, root gall indices and pod galls per microplot were significantly reduced by 60% and 95% at 100,000 endospores/g of soil, respectively. In the second year, root and pod gall indices were significantly reduced by 81% and 90%, respectively, at 100,000 endospores/g of soil, and by 61 % and 82%, respectively, at 10,000 endospores/g of soil. Pod yields were increased by 94% at 100,000 and by 57% at 10,000 endospores/g of soil. In the third year, there were no galls observed on peanut pods at 100,000 endospores/g of soil. Root and pod gall indices were reduced in all microplots inoculated with P. penetrans. Pod yields were increased by 183%, 294%, 225%, and 275% at 1,000; 3,000; 10,000; and 100,000 endospores/g of soil, respectively. Reduction of root and pod galls correlated with increase of percentage of second-stage juveniles with endospores attached before planting over the 3-year period.

A further aspect of the subject invention is a method for producing Pasteuria endospores which comprises growing plants in the presence of Pasteuria and nematodes such that the number of Pasteuria endospores increases over time as described herein. Then, at harvest, the roots may be harvested and ground up to obtain a composition comprising a high concentration of the Pasteuria endospores together with the root material. This composition can then be used to effectively inoculate a field for growing a new crop. In a preferred embodiment, the plant from which the ground root composition is made can be a tomato plant.

In a further embodiment of the subject invention, Pasteuria can be combined with additional nematode control agent(s). The additional control agent used according to this embodiment of the subject invention can be any known chemical or biological nematicidal agent, or any such agent which may be developed in the future. Preferably, the nematicide is non-toxic to the environment or to humans, and does not kill bacteria. The compositions of the subject invention can be formulated in various forms in order to provide an effective concentration of bacteria. Such formulations include, for example, granules, water-soluble sprays, or pellets.

Detailed Description of the Invention

The subject invention concerns pest-control compositions and advantageous methods of using these compositions. More specifically, the subject invention relates to the use of Pasteuria, or related bacteria, to control plant-parasitic nematodes. The use of bacteria as an agent to control plant-parasitic nematodes can be very valuable and helps to avoid a negative impact on the environment which can result from the normal use of synthetic chemical pesticides.

In one embodiment, the ability to control nematodes with Pasteuria has been demonstrated on peanut plants. In accordance with the subject invention, it was found that P. penetrans consistently suppressed M. arenaria race 1 on peanut over 3 years in field conditions.

The suppression of root-knot nematodes correlates to the incidence of P. penetrans; the correlations were consistent for both root gall and pod gall reduction over 3 years. This indicates that P. penetrans was the primary factor suppressing root and pod galls. Peanut has a long growth duration in field, up to 125 days. Several generations of root-knot nematodes may be developed on peanut in Florida. Juveniles that hatch from egg masses on root surfaces in mid-season may quickly enter adjacent roots without being exposed to endospores in soil.

Microplots were inoculated with endospores of P. penetrans the first year only. Advantageously, it was found that the suppressiveness of the soil increased over the years. This indicated that the P. penetrans population established and amplified in the plot. The amplification of P. penetrans was evidenced by the increasing percentage of J2 with endospores attached and the number of endospores attached per J2 over the 3- year period. The fate of endospores in soil was unknown, but leaching and dissipation were inevitable. Our results provided evidence that P. penetrans had a net gain in population density in field microplots. The amplification of population densities of P. penetrans was more remarkable in low inoculum levels than in the high inoculum level of 100,000 endospores/g of soil. In the third year after inoculation, there were few nematodes in microplots treated with 100,000 endospores/g of soil. Thus, further amplification of P. penetrans in these microplots was negligible. Moreover, decreasing of endospore density was expected, because of leaching and dissipation of endospores in the environment. The longevity of the soil remaining suppressive to root-knot nematodes and the rate of dissipation of endospores were therefore suφrising and advantageous.

At the third year, the endospore densities in microplots treated with P. penetrans were high, ranging from 99,000 to 137,000 endospores/g of soil. The mean endospore density in microplots treated with 100,000 endospores/g of soil was 133,000 endospores/g of soil, which was the maximum or equilibrium endospore density in soil. Pasteuria penetrans requires host nematodes to complete its life cycle. Without host nematodes, P. penetrans cannot amplify itself in soil. Further increase of endospore density was negligible in microplots at 100,000 endospores/g of soil because of lack of host nematodes. Therefore, 133,000 endospores/g of soil was considered as the observed value of equilibrium density of P. penetrans in soil. The equilibrium density estimated from regression analysis was 132,900 endospores/g of soil. Thus, the equilibrium density of P. penetrans in field microplots under continuous cropping of peanut was around 133,000 endospores/g of soil. Equilibrium density of P. penetrans was obtained at the second year of experiment in microplots treated with 100,000 endospores/g of soil, and at the third year of experiment in microplots treated with 1,000 and 10,000 endospores/g of soil. The rapid increase of endospore density in field microplots provided a very promising option for biological control of arenaria. Field application at 1,000 endospores/g of soil is equivalent to 2 x 10¹² endospores/h.a. in the top 20 cm of soil. One thousand endospores/g of soil only requires about 1-2 kg of root powder/h.a.

Various pest-control compositions of the subject invention can be made according to known methods and techniques. The pest-control compositions of the subject invention may vary in the form in which they are produced and applied. Similarly, the pest-control compositions can vary in their chemical composition, most importantly in the concentration of bacteria that is present. One skilled in the art having the benefit of the instant disclosure can readily optimize the efficacy of the subject invention for a desired application. For example, selecting the best concentration and the form of application will depend on the intended use of the subject invention, and will be readily recognized by ordinarily skilled artisans have the benefit of the teachings provided herein.

Materials and Methods The experimental protocol and isolates of M. arenaria race 1 and P. penetrans (designated P-20) were the same as previously reported (Chen et al, 1996, supra). Briefly, the microplots were infested with M. arenaria race 1 and planted in tomato to further increase the inoculum. Pasteuria penetrans was then incoφorated into microplots at five levels, 0, 1,000, 3,000, 10,000, and 100,000 endospore/g of soil, with 10 replicates each. Both M. arenaria and P. penetrans were inoculated in the first year only. Ten-day-old peanut seedlings were harvested in 125 days. The microplots were planted with wheat as a winter cover crop. Soil samples consisted of three cores (2.5 -cm-diameter x 20-m-deep) diagonally arranged in a microplot. Juveniles were extracted from 100 cm³ soil using centrifugal flotation (Jenkins, W.R. [1964] Plant Dis. Reporter 48:692) and counted using a compound inverted light microscope. Root galling was indexed on a 0 to 10 scale based on the percentage of roots galled (0 = no galls on roots; 1 = 1 to 10% roots galled; 2 = 11 to 20% roots galled; ... 10 = 91 to 100% roots galled) (Barker, K.R. et al. [1986]

"Determining nematode population responses to control agents," in Methods for Evaluating Pesticides for Control of Plant Pathogens (K.D. Hickey, Ed.), APS Press, St. Paul, MN, pp. 283-296). Pod galls were counted individually in year 1 and indexed on the 0 to 10 scale in years 2 and 3. Dry weights of foliage and pods were determined by drying at 60 °C for 1 week.

Before data analysis, percentage were transformed by

arc sin if) Root and pod gall indices were transformed by

arcsin(Jx/10)

Nematode densities and numbers of endospores per J2 were transformed by log₁₀ (x+l). One microplot in the control was apparently contaminated by P. penetrans and thus excluded in statistical analysis. Data were subjected to analysis of variances and Duncan's multiple-range test. In regression analyses ofdata sets of paired observations, we followed Ferris' (Ferris, H. [1984] J. Nematol 16:1-9) approach to reduce variability and improve the goodness-of-fit. Fifty microplot observations of percentages of J2 with endospores attached were divided into seven groups, each individual group having six to eight observations. Consequently, field observations of root and pod gall indices were grouped as they were paired to the percentages of J2 with endospores attached. The observations in each group were then averaged; the paired means were subjected to regression analysis (Ferris, supra).

Results Suppression of root and pod gall indices. Pasteuria penetrans suppressed root and pod galls induced by root-knot nematodes over a three-year period (Table 1). In the first year, root gall index and pod galls per plot were reduced by 60% and 95%, respectively, at 100,000 endospores/g of soil (P < 0.05). In the second year, root and pod gall indices, respectively, were reduced by 81% and 90% at 100,000 endospores/g of soil, and 61% and 92% at 10,000 endospores/g of soil (P < 0.05). In the third year, there was not a single gall observed on peanut pods at 100,000 endospores/g of soil. Pod gall indices were reduced by 72%, 92%, and 95% at 1,000, 3,000, and 10,000 endospores/g of soil, respectively (P < 0.05). Root galls were not observed in eight of the ten root systems at 100,000 endospores/g of soil, and root gall indices were reduced by 30%, 69%, 73%, and 99% at 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively (P < 0.05). The suppression of root and pod galls was increased year after year.

"Data show significance levels of analysis of variance; ns = not significant at P < 0.05; — = data not available. Percentage were transformed by arcsin(square root of x). Root and pod gall indices were transformed by arcsin(square root of x/10). Population density of second-stage juveniles (J2) in soil and number of endospores per J2 data were transformed by log₁₀( +l).

Yield increase. Foliage and pod yields were not different among the treatments in year 1 (P < 0.05). Increase of foliage and pod yields was observed in years 2 and 3. In year 2, pod yields at 10,000 and 100,000 endospores/g of soil, and foliage yield at 100,000 endospores/g of soil were different from the control (P < 0.01) (Table 1). In year 3, foliage and pod yields in all the treatments inoculated with P. penetrans endospores were different from the control (P __ 0.05).

Compared to the control, foliage yield was increased by 52% at 100,000 endospores/g of soil, whereas pod yields were increased by 57% and 94% at 10,000 and 100,000 endospores/g of soil, respectively, in year 2 (P < 0.05). In year 3, the foliage weight was increased by 187%, 240%, 189%, and 229%, while the pod weight was increased by 183%, 294%, 225%, and 275%, at 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively (P < 0.05).

Suppression of final population density of J2. Pasteuria penetrans did not suppress final population densities of J2 in soil in years 1 and 2; there were no differences among the treatments (P < 0.05) (Table 1). In year 3, population densities of J2 in soil were lower in 10,000 and 100,000 endospores/g of soil than in the control (P < 0.05). The suppression of population densities of J2 in soil correlated well to the percentage of J2 with endospores attached (P < 0.01). Population densities of J2 were reduced exponentially with increasing percentage of J2 with endospores attached.

Correlation of endospore attachment of J2 to inoculum levels of P. penetrans. The percentage of J2 with endospores attached and number of endospores attached per J2 were increased with increasing inoculum levels of P. penetrans (Table 1). When the control was excluded from the calculation, percentage of J2 with endospore attached correlated well with the inoculum levels of P. penetrans in years 1 and 2. The regression equations were: ^, = - 117.3+41.4 log ;*: (r = 0.997, P < 0.01), before planting, year \; y = -72.0+28.6 \og x (r = 0.966, P < 0.05), at harvest, year 1; and y = -86.9+35.3 log x (r = 0.998, P < 0.01), at harvest, year 2; where; is the percentage of J2 with endospores attached and x is the number of endospores inoculated per gram of soil (1,000 ≤ x ≤ 100,000 endospores/g of soil). When numbers of endospores attached per J2 were used for regression, the regression equations were: logy = -4.25+1.09 log x (r = 0.985, P < 0.01), before planting, year 1; logy = -3.86+0.981 log x (r = 0.989, P < 0.01), at harvest, year 1; and log v = - 1.33+0.47 log x (r = 0.980, P < 0.01), at harvest, year 2; where y is the number of endospores attached per J2 and x is the number of endospores inoculated per gram of soil (1,000 < x ≤ 100,000 endospores/g of soil). In year 3, the percentage of

J2 with endospores attached and number of endospores attached per J2 at harvest were not correlated with the inoculum levels of P. penetrans (P < 0.05).

Population establishment and amplification of P. penetrans. Population densities of P. penetrans in field microplots, in terms of number of endospores attached per J2 and percentage of J2 with endospores attached, tended to increase over the years. Increase of endospore population was more remarkable in 1,000, 3,000, and 10,000 endospores/g of soil than in 100,000 endospores/g of soil. Based on the regression equation of percentage of J2 with endospores attached with the inoculum levels of P. penetrans before planting in year 1, endospore densities reached 137,000, 99,000, 136,000, and 133,000 endospores/g of soil at harvest, year 3, at 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively. Endospore densities in soil in year 3 increased 136, 33, 13.6, and 1.33-folds at 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively, compared with the imtial inoculum levels in year 1 (Table 2). A linear regression was obtained between the logarithm of increasing folds and the logarithm of initial inoculum levels: logy = 5.02-0.98 log x (r = 0.997, P < 0.01); where y was the increasing folds and x was the initial inoculum levels of P. penetrans before planting in year 1. The anti-logarithm of the equation was =105000x^"0-⁹⁸. The equilibrium endospore density, here defined as no further increasing of endospore density (y = 1), was 132,900 endospores/g of soil.

^aMicroplots were inoculated with P. penetrans endospores before planting in year 1. ^bPopulation densities of P. penetrans microplots were estimated based on a regression equation; the percentages of second-stage juveniles with endospores attached at harvest were used.

Increasing folds = (Population density at harvest)/(Inoculum levels).

Although the foregoing invention has been described in some detail by way of illustration and example, it will be understood that the present invention is not limited to the particular description and specific embodiments described, but rather may comprise a combination of the above elements and variations thereof. It should be understood that the examples and embodiments described herein are for illustrative puφoses only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Claims

1. A method for the control of plant pests wherein said method comprises applying Pasteuria to said plant pests or their situs.

2. The method, according to claim 1, wherein said Pasteuria is Pasteuria penetrans.

3. The method, according to claim 1, wherein said pests are parasitic nematodes.

4. The method, according to claim 3, wherein said parasitic nematodes are root- knot nematodes.

5. The method, according to claim 1, wherein said method is used to protect peanut plants from damage caused by parasitic nematodes.

6. The method, according to claim 1 , wherein said Pasteuria are applied to soil in which said plant pathogens are to be controlled.

7. The method, according to claim 6, wherein said Pasteuria are applied to said soil at a concentration of at least about 1 ,000 endospores/g of soil.

8. The method, according to claim 7, wherein said Pasteuria are applied to said soil at a concentration of at least about 10,000 endospores/g of soil.

9. The method, according to claim 7, wherein said Pasteuria exist in the soil at a concentration of at least about 100,000 endospores/g of soil within three years of application of the endospores to said soil.

10. The method, according to claim 9, wherein said Pasteuria are Pasteuria penetrans and said method is used to protect peanuts from parasitic nematodes.

11. A method for producing Pasteuria endospores wherein said method comprises growing plants in the presence of Pasteuria and nematodes such that the number of Pasteuria endospores increases over time, and wherein said method further comprises harvesting the roots of said plants and collecting endospores from said roots.

12. The method, according to claim 11, which comprises grinding said roots to obtain a composition comprising Pasteuria endospores and root material.

13. The method, according to claim 11, wherein said plants are tomato plants.

14. The method, according to claim 11, wherein said Pasteuria are Pasteuria penetrans.

15. A composition for controlling plant pests, wherein said composition comprises Pasteuria endospores and plant root material.

16. The composition, according to claim 15, wherein said Pasteuria are Pasteuria penetrans.

17. The composition, according to claim 16, wherein said plant root material is from tomato plants.