Patent Application: US-70205310-A

Abstract:
this invention relates to methods of prolonged storage of the species of entomopathogenic nematodes belonging to the genera of steinernema and heterorhabditis for use as bio - pesticides wherein storage in surfactant solution or antimicrobial solution on sterilized polyurethane foam allows maintaining infectivity of at least 50 % for six months at 10 - 15 c storage conditions .

Description:
the monoxenic solid artificial medium ( chicken offal ) proves useful for large - scale production of these nematodes and is being used globally . the techniques described by bedding ( 1984 ) for the production of nematodes ijs per unit time were followed . with bedding &# 39 ; s method , normally 5 - 7 million infective juveniles of steinernema were produced in a single 500 ml flask containing 80 g of chicken offal medium and stored in flasks containing distilled water at 10 - 15 ° c . for 1 - 12 months . ijs as the infective stage juveniles reared in above mentioned media , kept their infectivity for up - to 8 months . the infective juveniles of steinernematids and heterorhabditids were stored on moist autoclaved polyether polyurethane foam . the foam was coated with ijs more than 10 times of foam placed in sterilized container and stored in growth chamber at 10 - 15 ° c . the infectivity of the above mentioned seven species during prolonged storage was analyzed by a three - way anova . the analysis of variance indicated that there were significant differences between infectivity of fresh to 12 months storage ijs ( f = 23 . 2 ; df = 7 ; p = 0 . 05 ) but among species , there were no significant differences ( f = 0 . 05 ; df = 6 ; p = 0 . 05 ) that means all seven nematode species were highly infective during fresh to eight months storage . seven entomopathogenic nematode isolates viz ., s . pakistanese shahina et al ., 2001 ; s . asiaticum anis et al ., 2002 ; s . feltiae filipjev , 1934 ; steinernema abbasi elawad et al ., 1997 ; steinernema siamkayai stock et al ., 1998 ; heterorhabditis indica poinar et al ., 1992 and h . bacteriophora poinar , 1976 were reared in monoxenic culture on chicken offal medium described by bedding ( 1984 ). the third infective stage juvenile collects from medium , washed with tap water and were kept in distilled water with a drop of triton x - 100 ( a wetting agent that prevents nematodes from striking to the side of the container ) or 0 . 1 % formalin and stored on moist autoclaved polyether polyurethane foam . the foam was coated with ijs more than 10 times of foam weight , placed in sterilized container and stored in growth chamber . the effect of prolonged storage on infectivity of epn isolates s . pakistanese shahina et al ., 2001 ; s . asiaticum anis et al ., 2002 ; s . feltiae filipjev , 1934 ; steinernema abbasi elawad et al ., 1997 ; steinernema siamkayai stock et al ., 1998 ; heterorhabditis indica poinar et al ., 1992 and h . bacteriophora poinar , 1976 at 10 - 15 ° c . were tested by using the galleria sand bioassay ; wherein , 25 g . mellonella larvae were placed in a 9 cm petri dish then covered with a layer of moist sterile sand . moist sand was obtained by mixing water in the ratio of 10 - 18 % w / w with dried sterile soil . infective juveniles were suspended in 200 μl of sterilized distilled water and distributed evenly on the top of the sand . six different age groups of infective stage juveniles viz ., freshly hatched , 1 - 12 month old ijs were used . 500 ijs were inoculated to check the mortality of g . mellonella or infectivity of different age group of infective stage juveniles . for each treatment three replicates were used . freshly hatched infective stage juvenile of epn isolates viz ., s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora had maximum infectivity and showed 100 % mortality of g . mellonella larvae . epn isolates s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora were tested and two months old storage of infective stage juveniles of above mentioned isolates showed 85 , 81 , 83 , 80 , 79 , 83 and 79 % mortality of g . mellonella larvae , respectively . epn isolates s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora were tested and four months old storage of infective stage juvenile of above mentioned isolates showed 73 , 69 , 70 , 67 , 63 , 70 and 65 % mortality of g . mellonella larvae , respectively . epn isolates s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora were tested and six months old storage of infective stage juvenile of above mentioned isolates showed 55 , 54 . 5 54 , 51 , 49 , 53 and 48 % mortality of g . mellonella larvae , respectively . epn isolates s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora were tested and eight months old storage of infective stage juvenile of above mentioned isolates showed 44 , 45 , 43 , 39 , 31 , 40 and 32 % mortality of g . mellonella larvae , respectively . epn isolates s . pakistanese ; s . asiaticum ; s . feltiae ; steinernema abbasi ; steinernema siamkayai ; heterorhabditis indica and h . bacteriophora were tested and ten months old storage of infective stage juvenile of above mentioned isolates showed 30 , 29 , 28 , 23 , 19 , 29 and 23 % mortality of g . mellonella larvae , respectively . epn isolates steinernema pakistanese ; s . asiaticum ; s . feltiae ; s . abbasi ; s . siamkayai ; heterorhabditis indica and h . bacteriophora were tested and twelve months old storage of infective stage juvenile of above mentioned isolates showed 14 , 14 , 14 , 11 , 09 , 13 , and 11 % mortality of g . mellonella larvae respectively . it could be concluded that epn can be successfully cultured on a large scale and could be used under field condition to provide protection to the crops from insects . akhurst , r . j . & amp ; 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