GURURAJ KATTI, J.S. PRASAD, A.P. PADMAKUMARI and M ...

38Table I. Effect of storage periods on pathogenicity and multiplication of Rhabditis (Oscheius) sp. on Galleria mellonella andCorcyra cephalonica.Host speciesTreatmentStorage period (days)Time for host larval mortality(hrs)No. of EPNs recovered from hostlarvaMRG. mellonella 110 57.6 b 39955 b 399.6G. mellonella 50 65.6 b 48464 b 484.6G. mellonella 5 61.6 b 54482 a 544.8C. cephalonica 110 24.4 a 16566 c 165.7C. cephalonica 50 26.4 a 14646 c 146.5C. cephalonica 5 65.2 b 19019 c 190.2MR - Multiplication rate.Figures within a column followed by different letters are significantly different at P = 0.05, following DMRT.abad, India. Both Rhabditis (Oscheius) sp. and S. thermophilumwere maintained in vivo on two insect hostsviz., G. mellonella, reared on artificial diet as describedby Singh (1994) and C. cephalonica, reared on coarseSorghum bicolor (Ganguly, 2000) at the Directorate ofRice Research (DRR), Rajendranagar, Hyderabad. Theinfective juveniles of each EPN were harvested separatelyusing a modified White trap method (White,1927), surface sterilized with 0.1% formalin and storedin water (2000 IJs/ml) at room temperature (27-30 0 C).Effect of storage on survival of EPNs at room temperature.Freshly harvested IJs of Rhabditis (Oscheius) sp.and S. thermophilum were washed three times in tap waterand stored in 100 ml distilled water in 250 ml conicalflasks at a concentration of 2000 IJs/ml. Formalin(0.1%) was added to avoid contamination and the flaskswere plugged with non-absorbent cotton. Treatmentswere storage periods for 50, 80, 100, 135 and 150 daysat 28 ± 2 0 C, arranged according to a completely randomizedblock design, with five replicates per EPNspecies and storage period. The survival of IJs was monitoredby counting active nematodes at the end of eachstorage period (Krishna Prasad and Rao, 1980). The percent survival of EPNs was then calculated.Effect of storage on the infectivity of EPNs. After storageat room temperature for varying periods, the twospecies of EPNs were evaluated for their infectivity,pathogenicity and multiplication rate on final instar larvaeof G. mellonella and C. cephalonica.Nematode samples were maintained at 28 ± 2 0 C infive batches for storage periods of 5, 50 and 110 daysfor Rhabditis (Oscheius) sp. and of 5, 50, 100 and 150days for S. thermophilum. Suspensions of infective juveniles(100 IJs/ml) were pipetted onto moist filter paperplaced in 2.5-cm-diameter Petri-plates. A single larva ofthe host was then placed in each Petri-plate for exposureto each EPN for 12 h. After exposure, the larvaewere washed with formalin (0.1%) and transferred tofresh Petri-plates containing food. Partially groundsorghum grains were provided to C. cephalonica (25g/larva) and solid artificial diet was provided to G. mellonella(25 g/larva). Observations on infectivity weremade at six-hour intervals until 100% larval mortalitywas observed in all the treatments. Only the time takenfor 100% mortality was considered in analysis of the data.The dead insect larvae were carefully placed onWhite traps and the IJs that emerged (White, 1927)were counted by dilution count under a stereozoom microscope.The data were analysed by ANOVA andmeans were compared by Duncan’s Multiple Range Test(DMRT).RESULTSEffect of storage on survival of EPNs at room temperature.The survival of IJs of EPNs under storage variedbetween the two species. There was 100% survival ofboth isolates up to 50 days of storage at room temperature.Thereafter, survival of Rhabditis (Oscheius) sp. decreasedto 75%, 50%, 30% and zero after 80, 100, 135and 150 days of storage, respectively. Survival of S. thermophilumwas 80%, 75%, 50% and 10% after 80, 100,135 and 150 days of storage, respectively (Fig. 1).Effect of storage on the infectivity of EPNs. The timetaken by Rhabditis (Oscheius) sp. to kill the insectsranged from 57.6 to 65.6 h for G. mellonella and from24.4 to 65.2 h for C. cephalonica, after periods of storagevarying from 5 to 110 days (Table I). Significantly moreRhabditis (Oscheius) sp. IJs were recovered from G.mellonella (39956 to 54482/larva) than from C.cephalonica (14646 to 19019/larva).Steinernema thermophilum also took more time tokill G. mellonella (20.8 to 74.4 h) than C. cephalonica(19.6 to 57.2 h), after periods of storage varying from 5

Gururaj Katti et al. 39DISCUSSIONFig. 1. Survival of the EPNs under different storage periods inthe laboratory. Bars represent standard errors.to 150 days (Table II). The IJs stored for 100 days tookleast time (20.8 h) to kill G. mellonella, while storage ofIJs for periods as long as 150 days or as short as 50 or 5days required significantly more time (36.8 to 74.4 h).The IJs stored for 50 or 100 days required the least time(19.6 to 25.6 h) to kill C. cephalonica followed by IJsstored for 150 days (50.4 h) and 5 days (57.2 h). The recoveryof S. thermophilum was greater from G. mellonella(36033 to 49298/larva) than from C. cephalonica(24161 to 28154/larva).The multiplication rate of both nematode species wassimilar on G. mellonella except for Rhabditis (Oscheius)sp. stored for 110 days. When compared to Rhabditis(Oscheius) sp., S. thermophilum had a slightly highermultiplication rate on C. cephalonica.Steinernema thermophilum survived better thanRhabditis (Oscheius) sp. at room temperatures. Selvan etal. (1993), studying the shelf life of Heterorhabditis bacteriophoraPoinar and S. glaseri (Steiner) Wouts,Mracek, Gerdin et Bedding, which survived for 7 and36 weeks, respectively, deduced that poor storage stabilityin entomopathogenic nematodes could be due to anincrease with storage of unsaturated fatty acids in thefreshly emerged infective juveniles. The differences insurvival and infectivity of the two EPNs could also bedue to difference in activity of the IJs under storage(Hussaini et al., 2005).There was no significant effect of the storage periodof IJs on time taken by Rhabditis (Oscheius) sp. to killG. mellonella, while the time necessary to kill C.cephalonica decreased with increase in storage time. TheIJs of S. thermophilum stored for 50 and 100 days weresignificantly more effective than those stored for 5 and150 days in killing both hosts. Jung (1996) reported lowinfectivity of EPN isolates during the first week ofemergence. Lewis et al. (1997) also observed that IJs ofS. carpocapsae became more mobile with age, whileO’Leary et al. (1998) found that the host finding abilityof IJs of H. megidis improved with increased durationof storage.Galleria mellonella larvae yielded significantly moreIJs of S. thermophilum than the larvae of C. cephalonica,but storage periods did not affect the recovery of IJs.Rajkumar et al. (2003) also observed greater recovery ofIJs of Heterorhabditis sp. from G. mellonella in comparisonto Steinernema sp., while Zaki et al. (2000) observedno differences in recovery of IJs of Steinernemasp. and Heterorhabditis sp. from Bombyx mori.The survival of Rhabditis (Oscheius) sp. and S. thermophilumdeclined after 50 days of storage. AlthoughTable II. Effect of storage periods on pathogenicity and multiplication of Steinernema thermophilum on Galleria mellonella andCorcyra cephalonica.Host speciesTreatmentStorage period (days)Time for host larval mortality(hrs)No. of EPNs recovered from hostlarvaMRG. mellonella 150 74.4 c 48977 a 489. 8G. mellonella 100 20.8 a 49298 a 493.0G. mellonella 50 36.8 b 36033 a 360.3G. mellonella 5 60.8 c 41575 a 415.7C. cephalonica 150 50.4 b 24161 b 2416.1C. cephalonica 100 25.6 a 24744 b 247.4C. cephalonica 50 19.6 a 27432 b 274.3C. cephalonica 5 57.2 c 28154 b 281.5MR - Multiplication rate.Figures within a column followed by different letters are significantly different at P = 0.05, following DMRT.

40the infectivity of Rhabditis (Oscheius) sp. increased afterlonger storage, in C. cephalonica there was no significanteffect of storage period on the recovery of nematode. InS. thermophilum, the IJs took less time to kill whenstored for 50-100 days, irrespective of the host. The recoveryof IJs was more in G. mellonella than C.cephalonica. However, within each host the storage periodhad no significant effect on the recoveries of S. thermophilum.As both EPNs are amenable to quick andeasy multiplication, studies to further improve their shelflife could contribute to the development of appropriatedelivery systems for field management of rice pests.Awareness of hazards caused by the usage of chemicalsin agriculture, is increasing the demand for pesticide-freeproduce in India, particularly in northern areaswhere quality rices and basmati varieties are grownmainly for export purposes. In these areas, stem borerand leaf folder are the major insect pest problems andthe farmers are being encouraged to resort to non-pesticidaland organic inputs for their management. Withthe identified potential of the EPNs and the added advantageof organic rice attracting premium prices, developmentof EPNs as a component in rice pest managementwill be economically beneficial to the farmers.LITERATURE CITEDCRRI, 1975. Annual Report, Central Rice Research Institute,Cuttack, India, 384 pp.CRRI, 1977. Annual Report, Central Rice Research Institute,Cuttack, India, 265 pp.Ganguly S., 2000. Life cycle of an indigenous strain of entomopathogenicnematode, Steinernema sp. National NematologySymposium on Integrated Nematode Managementfor Sustainable Agricultural in the Changing Agri-ecologicaland Economic Scenario in the New Millenium. November23-24, 2000. Nematological Society of India,Bhubaneswar, Orissa, India, pp. 9-10.Georgis R., 1987. Nematodes for biological control of urbaninsects. 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