Characterization, host bioassay, and in vitro culture of indigenous entompathogenic nematodes and their bacterial symbionts

摘要

The prevailing use of chemical pesticides has generated several problems includingudinsecticide resistance, outbreak of secondary pests, safety risks for humans anduddomestic animals, contamination of ground water and decrease in biodiversity amongudother environmental concerns (Webster, 1982). These problems and the nonsustainabilityudof control programs based mainly on conventional insecticides haveudstimulated increased interest in the development and implementation of costeffective,udenvironmentally safe alternatives to chemical pesticides for insect pestudcontrol. One of the most promising strategies to help minimize dependence onudchemical pesticides has been the recent application of entomopathogenic nematodesud(EPNs) as biocontrol agents. EPNs in the families Steinernematidae andudHeterorhabdidae have been shown to have considerable potential as biological controludagents. As a natural process, biological control has the potential to play an importantudrole in the suppression of field crop pests in agriculture. EPNs as biocontrol agentsudhave the following advantages: high virulence, safety to non target organisms, abilityudto search for hosts, high efficacy in favourable habitats, high reproductive potential,udease of mass production, ease of application (Ferron & Deguine, 1996).udTo isolate the EPNs in South African soil, 200 soil samples were randomly collectedudfrom 5 locations in the agricultural research council (ARC) Pretoria, Gautengudprovince in April 2006; and 5 locations in Brits, North West province in March,ud2006. At the different collection sites, soil samples were obtained from soilsudassociated with various types of vegetation. The nematodes were collected fromudsandy soil by the insect-baiting technique and maintained successfully in vivo for 12udmonths on Galleria mellonella (G. mellonella), 4 months on Tenebrio molitor (T.molitor); 2 months Pupae and in vitro (lipid agar) for 2 weeks in the laboratory. Outudof a total of 200 soil samples that were baited, 2 were found to be positive for EPNs.EPNs.udIVudIn addition to completing Koch’s postulates, the colour of cadavers infected by theudputative EPNs were also used as a diagnostic characteristic for categorizing theudnematode isolates. Characterization and identification of the EPN isolates were basedudon morphological characters, as well as on a molecular marker (18S rDNA).udOn the basis of the morphological and molecular data that was obtained both of theudEPNs isolates were placed in the family Heterorhabdidae: Heterorhabditisudbacteriophora (H. bacteriophora) and Heterorhabditis zealandica (H. zealandica).udAlso from the phylogenetic trees generated from the 18S rDNA sequence, theudindigenous putative H. bacteriophora was shown to be closely related to H.udbacteriophora (accession number EF690469) and indigenous putative H. zealandicaudto H. zealandica (accession number AY321481). The two EPNs were foundudassociated with Gram negative rod-shaped bacteria. The bacterial symbionts of theudtwo isolates were isolated and a region of the 16S rDNA gene was sequenced.udNational Center for Biotechnology Information (NCBI-BLAST) results of the 16SudrDNA sequence obtained showed the endosybiotic bacteria to be Photorhabdusudluminescens laumondii (P. laumondii) (H. bacteriophora) and Photorhabdus sp (H.udzealandica). Results of the tree showed that isolates from H. bacteriophora appearedudto be closely related to P. luminescens subsp laumondii strain TT01 Ay 278646. Theudisolates from H. zealandica appeared to be most closely related to Photorhabdus sp Accession number: Q 614 Ay 216500).udBioassays were used to determine the infectivity of the two EPNs. In this experimentuddifferent infective juvenile (IJs) concentrations (5, 10, 25, 50, 100,200 400 and 500)udof the two EPNs were applied per G. mellonella; T. molitor larva and pupae. Theudbioassay was carried out in two parts. In the first part, mortality data was collected forudH. bacteriophora and H. zealandica. The results showed that the degree ofudsusceptibility of G. mellonella, T. molitor larvae and pupae to each nematode speciesudwas different. When 24 h post-exposure mortality data for larvae exposed to the IJs ofudH. bacteriophora and H. zealandica were analyzed, ANOVA showed no differences udVudin mortality between insects exposed to different H. bacteriophora IJ doses (Fig: 8.1udABC). However, there were significant differences in mortality between insectsudexposed to different IJ doses of H. zealandica such as 5 and 500 IJs/insect (Fig: 8.2udABC) Therefore, no differences were noted when mortality data was comparedudbetween IJ doses at both 72 h and 96 h following IJ application to the insects. Theudhighest susceptibility was observed with G. mellonella followed by T. molitor pupaeudand then T. molitor larvae. According to Caroli et al., (1996), the total mortality ofudinsect such as G. mellonella and other lepidopterans, was reached within 24-72 h ofudexposure to nematodes at concentrations such as those tested here. In this studyudsimilar results were observed with high concentration of nematodes (100, 200 andud500). In the second part of the dose response bioassay, the number of progeny IJsudemerging from EPN-infected cadavers was determined for all two EPNs.udThe results indicate that IJ progeny production differed among the three insect hostsudused, the IJ doses they were exposed to, as well as the EPN species (Figs 8.3 & 8.4).udThe highest number of emerged IJs of H. zealandica was produced by G. mellonellaud(mean ± SEM: 220500 ± 133933 IJs), followed by T. molitor larvae (mean ± SEM:ud152133 ± 45466 IJs) and the lowest then T. molitor pupae (mean ± SEM: 103366 ± 56933 IJs).