| As a classic fungal biocontrol agent, Beauveria bassiana has been developed into formulations for application to control of numerous crop or forest insect pests. Enviromental temperature is one of the factors that known to affect the ambient shelf-life of the formulations or field performance after application. To search for more poteintial candidates for formulations adaptable to pest control in southern China during hot summer and to study the stress biology of Beauveria bassiana, this study was performed to evaluate conidial thermotolerance quantitatively of 12 B. bassiana strains derived from different host and geographic origins, and sequencing the full length sequences of the gene encoding amino acid sequence of a 15.0-kDa FAE protein, which designated CWP15. Detailed results are summarized as follows.Conidial thermotolerance evaluation. The assay protocol to evaluate quantitatively conidial thermotolerance was developed by exposing fungal conidia to the thermal stress of 48°C(in water bath) for certain periods of time. The ratio of conidial viability(measured as germination rate) after a given period of the thermal stress to that of blank control (unstressed conidia) was defined as survival index I_s. When the conidia of 12 B. bassiana with different host and geographic origins were separately exposed to 48°C for 30min, the survival indices of all over the time of exposure(t) fit very well to the logistic equation I_s= 1/[1+exp(a+bt)](r~2≧ 0.97, P < 0.01). The process of the survival indices of all over the time of exposure(t) was separated into three parts, which were the slow declined part, the sharp declined part and the gentle declined part. For all the isolates, the survival indices was declined along with the prolongation of the exposed time, and the declined speed of the survival indices of all over the time of exposure(t) differed significantly among the strains tested. The fitted equation was used to estimate median lethal time, LT50, of its conidia under the thermal stress. This estimate was a charateristic index for the conidial thermotolerance of each isolate. As a result, the LT50S of twenty B. bassiana isolates were averaged as 12.6(6.7～21.2)min whereas four strains with the LT50S were below 10min and most of the strains with the LT50S were between 10～20 min. FAE protein analyse. The conidia FAE proteins was hypothesized that they were involve into the conidia thermotolerance. SDS-PAGE profiles of the FAE proteins were consistent for each fungal species, but there were a few differences among the strains of B. bassiana. The FAE proteins with molecular weights of 15.0 and 17.5kDa were constitutively expressed in the conidia of B. bassiana. However, for some strains of B. bassiana the protein with molecular weight of 12.0 were not expressed.Sequencing and analysis of the FAE protein CWP15. The FAE protein with molecular weights of 15.0 was designated as CWP15. It was a new thermotolerance-relating protein found by our own lab. A common, conserved sequence of amino acids (HNDRVVGAWDQDVK IV) was found from the CWP15 sequences of the twenty B. bassiana. Based on divergence in amino acid sequences of the CWP15, the two groups CWP15a and CWP15b were classified. The comparison of CWP15 amino acid sequences of three strains of B. bassiana from Yunnan Province suggests that they were in two different group. This suggests there were much diversity among the strains of B. bassiana even they were with the same geographic origins. This suggests a possible use of CWP15 as a new molecular marker for the phylogeny of B. bassiana.In summary, variation in conidial thermotolerance was large among B. bassiana strains. The declined speed of the survival indices of all over the time of exposure(t) differed significantly among the strains of B. bassiana. The results provide new insights into stress biology of B. bassiana and suggest a promising approach to improving ecological fitness or field persisitence of fungal formulations.
|
PDF Full Text Request