| Lecanicillium lecani is an important entomopathogenic fungus in control of agricultural pests, suchas aphid, aleyrodid and thrips. However, few results of survival status of Lecanicillium lecani applied inthe field were reported. This dissertation studies aim to clarify the persistence dynamics ofLecanicillium lecani in soil after application and the essential demand characteristics of carbon andnitrogen nutrition. The main results are as follows.1. A selective medium adding YMT was optimized for isolation of Lecanicillium lecani from soil.Its constituent included oats20g/L, agar power18g/L, YMT0.3g/L and chloromycetin0.5g/L.Non-target microorganism could be completely inhibited in this selective medium, while the colonies ofLecanicillium lecani could be clearly identified. Also, the selective medium had high separationefficiency, and it is convenient to count.2. TaqMan RQ-PCR for Lecanicillium lecani was established. Firstly, the cell lysis and othertechnical parameters were improved and optimized, and then a method of extracting DNA ofLecanicillium lecani from soil was established. After DNA extraction of pure culture samples, artificialsoil samples and natural soil samples, the application effects were jointly evaluated by the concentrationmeasurement, agarose gel electrophoresis and PCR amplification. The results showed this method washigh sensitive to Lecanicillium lecani. Humic acid and other inhibitors in soil were wiped off, and theextracted DNA had high quality. Secondly, the TaqMan RQ-PCR fluorescence quantitative PCRdetection system and standard curve were developed based on the specific primers and fluorescenceprobes of Lecanicillium lecani. The standard curve is Y=-3.5065X+44.038(R~2=0.9978). The primersand probes are specificity; there were significant fluorescence signal for Lecanicillium lecani, and nofluorescence signal for the other species of entomogenous fungus.3. The persistence dynamics of Lecanicillium lecani in soil were clarified. The quantity of fungalCFU (colony forming unit) from the soil samples showed different trends in different time. After fourkinds of different concentration of Lecanicillium lecani applied, the quantity of CFU all decreasedrapidly to67.5%,41.6%,7.5%and2.2%of the initial CFU in the first30days, respectively. Also, thehigher the initial CFU, the greater the CFU fell. After90days, the quantity of CFU was in a stable range,and the order of magnitude remained at10~4. In the rest time of persistence, the quantity of CFUfluctuated in this scope, or decreased slowly, or remained the same level, or increased a little. In the fivedifferent depths of soil layers, the quantity of CFU decreased fast at first, and then declined slowly inthe first30days. Compared with the initial CFU, the CFU dropped to37.2%,28.4%,17.5%,13.8%and10.8%in the30thday, respectively. Also, the deeper from the surface, the more CFU dropped. In theearly stage of the persistence, the upper soil layer was more suitable for the growth of Lecanicilliumlecani; and in the later stage, the middle soil layer was more suitable. After90days, the quantity of CFUof five different depths of soil layers was fluctuated in a stable range, and the order of magnituderemained at10~4.4. The carbon and nitrogen nutrition demand characteristics of CA-14strain of Lecanicillium lecani were studied. The results showed that the conidia of this strain could germinate and grow quicklyand sporulate highly. The best ratio of C/N was20:1, and the best concentration of C was1mol/L.Among the sugar materials tested in this study, the best for growth and sporulation was polysaccharose,and the strain was weak to monosaccharide, such as D-ribose and L-sorbose. Among the nitrogensources, organic nitrogen was better for growth and sporulation than inorganic nitrogen, and this strainwas weak to ammonium salts. In the study of persistence dynamics of Lecanicillium lecani, the soil ofexperimental zone had high fertility. The organic matter in the soil could provide adequate nutrition forgrowth and sporulation of Lecanicillium lecani, and it was good for the activity and population density. |
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