Title of Invention

"A NEW LABORATORY METHOD FOR PRODUCTION OF PASTEURIA PENETRANS"

Abstract

This invention relates to a method of large scale production of multiplying bacterial parasite Pasteuria penetrans for root knot nematodes characterizing in that infecting the plant growing in moss with nematode, Meloidogyne incognita and Meloidogyne javanica attached with bacteria Pasteuria penetrans; wrapping of the roots in moss followed by incorporation of the same in pipe and watering in growth room on tissue culture rack; drying of the moss bearing the roots followed by grinding to obtain a powder containing PASTEURIA PENETRANS.

Full Text

FIELD OF INVENTION The invention relates to a method of mass multiplying bacterial parasite Pasteuria penetrans for root knot nematodes. BACKGROUND OF INVENTION Pasteuria penetrans have undoubtedly been proven to be a very promising bioligical control agent of root-knot nematodes. Consequently, efforts were made to devise means to culture it on in vitro systems. Initial attempts at in vitro culture (Reise et.al., 1988; Bishop 85 EUar, 1991; Raise et al., 1991) were not successful and investments and interest in Pasteuria spp. As nematode control product were greatly reduced. These attempts to culture Pasteuria typically used vegetative cells from infected nematodes released into selected culture media through mechanical disruption of surface sterilized nematodes. Culture media employed included an extensive array of carbon and nitrogen sources, commercial growth media for insect and other cells, extracts of other microrganisms or fungi, or cultured nematode cells. Significant growth of Pasteuria was not observed in these media and in one study the Pasteuria cells lysed after a few days (Bishop 8B EUar, 1991). A patent was obtained for an in vitro cultivation system that involved adding ex-planted tissues from Ascaris suum, but this work was never published (Pervic 8B COX, 1992). Another method of in vitro cultivation of Pasteuria has been reported recently (Gerber 8B White, 2001) which involves co-culture of P. penetrans with another bacterium Enterobactor cloacae. However, the spore yields of P. penetrans are reportedly very low and improvements are desired to make it commercially viable. Current methods of mass producing P. penetrans are based on in vivo systems and rely on its multiplication on nematode host on greenhouse grown plants (Stirling 85 Wachtel, 1980). A hydroponic system has also been reported (Serracin et al., 1994). Some alternative methods of culturing P. penetrans on excised or transformed root cultures were also reported (Verdejo &, Jaffee, 1988; Verdejo 86 Mankau, 1986.) But all these systems are of academic interest only and are not commercial savvy. The inventive features of the present invention have been depicted in the principal claim and the advantageous features have been indicated in the subsidiary claims. OBJECTS OF THE INVENTION An object of this invention is to propose a process for the mass production of Pasteuria Penetrans. Another object of this invention is to propose a process for the mass production of Pasteuria Penetrans on a semi commercial scale. Still another object of this invention is to propose a process for the mass production of Pasteuria Penetrans which is a soil-less system thus alleviating contamination by other pathogens.Objects of Invention Yet another object of this invention is to propose a process for the mass production of Pasteuria Penetrans which is suitable for seed treatment and nursery bed application. A further object of this invention is to propose a process for the mass production of Pasteuria penetrans which does not require application of any nutrients. Further objects and advantages of the invention will be more apparent from the ensuing descriptive. STATEMENT OF INVENTION According to this invention there is provided a method of large scale production of multiplying bacterial parasite Pasteuria penetrans for root knot nematodes characterizing in that: a) infecting the plant growing in moss with nematode, Meloidogyne incognita and Meloidogyne javanica attached with bacteria Pasteuria penetrans; b) wrapping of the roots in moss followed by incorporation of the same in pipe and watering in growth room on tissue culture rack; c) drying of the moss bearing the roots followed by grinding to obtain a powder containing PASTEURIA PENETRANS. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawing and wherein: Figure .1 shows influence of plant hosts on the spore yield of Pasteuria penetrans in aeroponic system. Figure .2 shows effect of auxins and cytokinins on the fresh root wt. (mg) of black gram. Figure .3 shows effect of different concentrations of lAA and IBA on the fresh root weight of black gram. Figure .4 shows effect of IBA on the fresh root weight (mg) of black gram. Figure .5 shows effect of IBA on the spore yield of Pasteuria penetrans on black gram in aeroponic system. Figure .6 shows effect of split inoculation of nematodes on the spore yield of Pasteuria penetrans on black gram in aerophonic system. Figure .7 shows black gram seedlings raised in seedling trays filled with river sand. Figure .8A shows roots wrapped in moss. Figure .8B shows roots and moss fixed in pipe. Figure .9 shows several pipes stacked in baskets and placed on tissue culture rack shelf. DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO ACCOMPANYING DRAWING. According to this invention there is provided a process for mass production of Pasteuria Penetrans comprising the steps of : a) influence of plant hosts on the spore yield of Pasteuria penetrans. b) Standardizing plant growth regulators to promote rooting so as to increase the nematode penetration sites. c) Inoculating spore-encumbered nematodes. d) Standardizing watering and photo period requires. The process is based on an in vivo system involving aeroponics. The method involved several steps to standardise the best plant host ideal for aeroponic conditions, use of plant growth regulators to enhance root growth so as to achieve maximum nematode penetration, method of nematode inoculation, and finally the use of tissue culture racks to standardize the photo and thermal regimes to obtain good plant growth in a growth room. The method has been designed for a,growth room where optimum thermal and photoperiod can be regulated and it is possible to multiply P. penetrans throughout the year. The end product is a root powder containing spores of P. penetrans which can directly be used as a bio-nematicide against root-knot nematodes, Meloidogyne spp. A step-wise development of the method along with the data is provided hereinafter only as an example: STEP-1- Influence of Plant Hosts The experiment was conducted in a screen-house. Six plant hosts, viz., tomato, brinjal, black gram, green gram, okra and pigeon pea were tested. Seeds were sown in plastic trays filled with river sand. Freshly hatched J2s of M. javanica encumbered with 4-5 spores of P. penetrans were inoculated @ 1000/plant. One week after inoculation, plants were transferred to plastic pipes with their roots wrapped in moss (Sphagnum sp.). Observations were recorded 40 days after inoculation. Roots of ten plants from each host were dried, milled and spore count taken in the root powder using a haemacytometer. Black gram supported maximum multiplication of P. penetrans, significantly more as compared to other plant hosts. This was closely followed by green gram. No multiplication of nematode was recorded on pigeon pea (Fig. 1 of the accompanying drawings). Based upon these results, black gram was selected for further experimentation. STEP 11- Influence of Plant Growth Regulators A series of pilot experiments were conducted to standardize qualitatively and quantitatively the plant growth regulators which promote rooting so as to increase the nematode penetration sites leading to enhanced spore yields of the bacterium. In these experiments no nematode/bacterium were inoculated. Expt.l; Two auxins viz., lAA and IBA each at 25,50 and 100 ppm, and two cytokinins viz., 6 Benzyl adenine and Kinetin each at 0.25, 0.5 and 1.0 ppm each were tested as seed dip. Seeds of black gram were dipped in the respective concentrations of growth regulator for 2 hrs before sowing in trays. Ten days after germination, the plants were uprooted and observations recorded on fresh root weight (Fig.2). In general. cytokinins failed to promote root growth. Arhong the two auxins, IBA was more effective; 50 ppm concentration gave maximum root weight. Expt.2; In the second experiment, only lAA and IBA were further tested at five concentrations each i.e., 10,20,30,40 and 50 ppm. The fresh root weights of black gram were recorded ten days after sowing. The data are presented in Fig.3. IBA at 50 ppm enhanced maximum root wt. As compared to other concentrations and lAA. Expt.3; only IBA was tested at 5 concentrations i.e., 20, 40, 60, 80 and 100 ppm and fresh root of black gram was recorded after 10 days. Maximum root weight was observed at 80 ppm, however it was on par with 60 ppm. Therefore, based upon the earlier and this experiment, the concentration of 50 ppm was selected for main experiment to study effect of IBA on the multiplication of P. penetrans. The data are presented in Fig. 4. Expt.4; After standardization of growth regulator and its concentration, IBA 50 ppm was selected for further experimentation on nematode invasion and spore production by P. penetrans on black gram in aeroponic systems. Seeds were dipped in IBA (50 ppm) or nutrient solution or water only for 2 hrs. Further details are same as explained earlier. The results are presented in Table 1 and Fig.5. Seed dip in IBA resulted in 42.6 millions spores per plant which is about 1.4 times higher compared to seed dip in nutrient solution or water alone. Table 1. Effect of IBA as seed dip on the nematode penetration and P. penetrans spore production on black gram (Table Removed) STEP- 111. Influence of Split Inoculation After standardization of host and use of plant growth regulator for mass multiplication of P. penetrans in aeroponic systems, further optimization of bacterial spore yield was attempted by testing different methods of inoculating spore-encumbered nematodes. Nematodes were inoculated 4 times (500 + 500 + 500 an alternate days) or 2 times (1000 + 1000 at two days interval), or only once- 2000. Other experimental details are same. Split inoculation of nematodes in four instalments increased spore production tremendously (91.4 millions per plant) than conventional single inoculation (15.4 millions), which is almost six times (Fig.6.). STEP-IV- Standardisation of Watering and Photoperiod regimes Tissue culture racks bearing four shelves were used. Black gram plants were raised in seedling trays (96 holes, Fig. 7). Other protocols i.e., use of IBA, split nematode inoculation were followed as standardized in earlier experiments. The plants were transferred to pipes with their roots wrapped in moss (Fig. 8). The pipes bearing the plants were put in plastic baskets and placed on the shelves (Fig. 9). Each shelf (was fixed with four fluorescent tubes of 40 W) accommodated 500 pipes easily. The tubes were switched on for 6, 12, 18 and 24 hours for respective shelves. The plant growth was best at 24 hrs photoperiod. Watering the plants twice a day was sufficient. The ambient temperature was maintained at 25-300 C. The plants were removed after 25 days of last inoculation. After detaching the shoots, the roots along with the moss plug was allowed to dry till it was fit for grinding. The plugs were finally milled to a fine powder using a Wiley grinding mill. The coarse material was removed by passing the powder through a 100 mesh sieve. Each pipe/plant yielded on an average 100 million spores. Projected production estimates with the technology Parasite preparation yield from one pipe = 3kg Yield from 2000 pipes or one rack = 6 kg Yield from a growth room of 10 racks = 60 kg in a cycle of one month Yield per year (x 12) = ,720 kg Amount of powder required for treating 1 sq.m. nursery area = 60g @ Ix l04 per g soil). The flowchart of the process for the mass production of Pasteuria penetrans is as follows: Dipping of seeds in plant growth regulator for 2 hrs ↓ Sowing the seeds in river sand in seedling trays ↓ Encumbering of the spores of P.penetrans on the Juvenile (larva) of Meloidogyne javanica/M. incognita ↓ Inoculation of 500 J2/plant four times on alternate days (in roots) ↓ One week after last inoculation, removal of the plants followed by wraping the roots in moss to form a plug ↓ Fixing of the plug to one end of plastic pipe ( for example: 15 cm long) ↓ Arrangement of the pipes in baskets and keeping the moss wet with water (once a day) ↓ Removal of the plants 25 days after last inoculation ↓ Detachment of the shoots, retaining the moss plug bearing the roots, and letting it dry ↓ Grinding of the roots along with plugs to make a powder ↓ Removal of coarse material by passing through for example 100 mesh sieve During the process, growth room is maintained at 28 ± 2°C, having tissue culture racks each with four shelves, each shelf bearing four fluorescent tube lights) It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims:- We Claim 1. A method of large scale production of multiplying bacterial parasite Pasteuria penetrans for root knot nematodes characterizing in that: a) infecting the plant growing in moss with nematode, Meloidogyne incognita and Meloidogyne javanica attached with bacteria Pasteuria penetrans; b) wrapping of the roots in moss followed by incorporation of the same in pipe and watering in growth room on tissue culture rack; c) drying of the moss bearing the roots followed by grinding to obtain a powder containing PASTEURIA PENETRANS. 2. A method as claimed in claim 1, wherein the plants are selected from Tomato (variety selection 7), Brinjal (variety Br-112), Black gram (variety T-9), Green gram (variety Pusa Baisakhi), Okra (variety Varsha uphaar) and Pigeon Pea (variety T-21). 3. A method as claimed in claim 1, wherein the bacteria is spore of Pasteuria penetrans. 4. A method as claimed in claim 1, wherein the inoculation is carried out 4 times on alternate days or 2 times at the interval of two days or once in which for times on alternate days is preferred. 5. A method as claimed in claim 1, wherein the pipe is made of plastic. 6. A method as claimed in claim 1, wherein the plants are removed 25 days after last inoculation.

Documents:

1953-DEL-2004-Abstract-(10-05-2010).pdf

1953-DEL-2004-Abstract-(20-09-2010).pdf

1953-del-2004-abstract.pdf

1953-DEL-2004-Claims-(10-05-2010).pdf

1953-DEL-2004-Claims-(13-03-2009).pdf

1953-DEL-2004-Claims-(20-09-2010).pdf

1953-DEL-2004-Correspondence-Others-(10-05-2010).pdf

1953-DEL-2004-Correspondence-Others-(13-03-2009).pdf

1953-DEL-2004-Correspondence-Others-(20-09-2010).pdf

1953-del-2004-correspondence-others.pdf

1953-del-2004-correspondence-po.pdf

1953-DEL-2004-Description (Complete)-(13-03-2009).pdf

1953-DEL-2004-Description (Complete)-(20-09-2010).pdf

1953-del-2004-description (provisional).pdf

1953-DEL-2004-Drawings-(13-03-2009).pdf

1953-del-2004-drawings.pdf

1953-DEL-2004-Form-1-(10-05-2010).pdf

1953-del-2004-form-1.pdf

1953-del-2004-form-18.pdf

1953-DEL-2004-Form-2-(10-05-2010).pdf

1953-DEL-2004-Form-2-(13-03-2009).pdf

1953-del-2004-form-2.pdf

1953-DEL-2004-Form-3-(10-05-2010).pdf

1953-DEL-2004-Form-5-(13-03-2009).pdf

1953-DEL-2004-GPA-(10-05-2010).pdf

1953-del-2004-gpa.pdf

1953-del-2004-petition-138.pdf