| Beauveria bassiana is an important biological agent for control of Bemisia tabaci. Systemic researches on the immune response of insect to entomopathogenic and the cross-talk between insect and fungi have been extensively conducted in the fields of pest management with entomopathogenic fungi. B. tabaci-B.bassiana system was used as research targets in this study. High-efficient B.bassiana isolates were selected for control of the whitefly on the basis of their virulence, spore production, and thermotolerance under laboratory conditions. The sublethal effect of B.bassiana on subsequent generations of whiteflies was analysed using comparative life table method. The growth kinetics of three isolates within the whiteflies were determined by q RT-PCR technology. The immune-related genes in whiteflies were screened out by comparing the transcriptomes between fungi-treated whiteflies and control. The major results and conclusions are listed as follows:1. Selection of B.bassiana isolates for control of the whitefly B.tabaciTwenty-two B.bassiana isolates were evaluated for their virulence to fourth instars of B. tabaci at a concentration of 1×107 conidia?ml-1. Thirteen isolates, with mortality rates ranging from 70 to 88%, were selected for further analysis because they were more pathogenic to whitefly nymphs than the other isolates. Spore production of these isolates was evaluated on sabouraud dextrose agar plus 0.25%(w/v) yeast extract after 10 days of incubation at 26°C. Seven of these B.bassiana isolates produced signi?cantly more conidia than the others, with spore production ranging from 3.0×108 to 3.9×108 conidia?cm-2. The thermal tolerances of these seven isolates were then determined through examination of the germination rates of conidia after exposure to 45°C for 2 h. Two isolates, GZGY-1-3 and SCWJ-2, were found to be the most tolerant to this temperature regime, with germination rates of 60 and 79%, respectively. On the basis of their virulence, conidia production and tolerance to high temperature, these two isolates are promising candidates for development as mycoinsecticides for management of B. tabaci in greenhouse agroecosystems in China.2. Sublethal effects of B.bassiana on subsequent generations of B.tabaciThe objective of this study is to compare life tables of subsequent generations of B. tabaci arising from parental generations exposed in the third nymphal stages to B.bassiana and controls, and to investigate the sublethal effects of fungus on life table parameters of B. tabaci offsprings. Results showed that: there were significant differences in pre-adult developmental time, total preoviposition period, 1st-4th instars survival and life table parameters between fungus-treated whiteflies and control. These results indicated that offsprings of fungus-treated B. tabaci compared to control increased their population slower, had higher mortality. However, fungal treatment didn’t impose significant effect on fecundity of B. tabaci.3. Comparative growth kinetics and virulence of three isolates of B.bassiana in B.tabaciIn order to examine the relationship between the fungal growth kinetics during infection and its virulence against the insect host, the virulence of three B.bassiana isolates, GZGY-1-3, SCWJ-2 and WLMQ-20 was evaluated using the 4th-instar nymphs of B.tabaci as target insect host at the concentration of 1×108spores/m L(high dose) and 1×106spores/m L(low dose). Fungal growth in insect was detected using quantitative real-time PCR(q RT-PCR), and fungal infection process was observed using fluorescence microscopy method. Bioassays showed that isolate GZGY-1-3 took the shortest time to kill the whiteflies. Whiteflies were killed more quickly by the high dose of spores than by the low dose of spores for each isolate. The results of q RT-PCR revealed that more fungal biomasses were detected in the whiteflies inoculated by the high dose of spores than those inoculated by the low dose for each isolate. After inoculation, a similar pattern of fungal growth on/in the insect was detected for each isolate and at different dose. However, there were quantitative differences of fungal cells among the isolates that with similar growth vigour. Insects infected with high virulent isolates or treated with the high spore dose could produce high fungal biomasses. The infection process of B.bassiana on B. tabaci observed by using fluorescence microscopic technology confirmed the results of quantitative PCR. These results suggest that variation of virulence among isolates of B.bassiana might be determined by quantitative differences of fungal growth kinetics to some extent.4. Transcriptome analysis of B.bassiana infecting B.tabaci nymphIn this study, high throughput sequencing technology was employed in the transcriptome analysis of B.tabaci 4th nymphs infected and uninfected(control) groups. Differentially expressed genes(DEGs) and their functions, classifications and signaling pathways were analyzed using bioinformatics tools. This research assembled 232,554 non-redundant Unigenes. The average length of all Unigenes were 674,24 bp. Among the 1166 DEGs(p<0.05), 474 were up-regulated genes and 692 were down-regulated genes; The Unigenes were enriched in 156 biological processes, 89 cellular components and 154 molecular functionsby GO analysis. KEGG pathway analysis indicated that 1145 DEGs were mapped to 309 distinctive pathways, and enriched in 76 pathways. The results provide a bioinformatics basis for applying entomopathogenic fungi to control whitefly at the molecular level.5. Identification of immune-related genes and q RT-PCR assayOn basis of sequence similarity to genes which have been known to involve in immune response, 182 immune-related genes have been identified in the transcriptome data. They include 46 pattern recognition proteins; 36 genes involved in extracellular signal modulation and amplification; 30 genes for signal transduction; and 70 immune responsive effectors. The results of q RT-PCR assay revealed that the expression patterns of these genes varied throughout the whitefly development and during the infection process of B.bassiana. |
