| The mealy bug Phenacoccus Fraxinus Tang is a piercing sucking pest parasitized on ash tree Fraxinus spp, which cause rampant harm in a number of provinces and cities in China, and seriously affects the growth of trees and city landscape. It possesses a variety of wax glands which secret powdery waxy mass during the growth process on its body surface. The powdery waxy become a natural protective barrier to the body. The control effect of chemical insecticide is not very ideal, and cause serious pollution of the environment. The biological control of Rraxinus was not reported. Lecanicillium lecanii and Beauveria brongniartii are widely used as two kinds of entomopathogenic fungi. The metabolites from entomopathogenic fungi play important roles in the pathogenesis of insects. Fungal metabolite compositions are complex, and the separation and purification of metabolites through several steps are tedious and difficult. High-speed counter-current chromatography (HSCCC) is a new separation technology, which has been widely applied to the separation of natural products. The use of HSCCC for separation and purification metabolites from entomogenous fungi were reported rarely.In the present study, we investigated the mortality rates of P. Fraxinus infected with L. lecanii 3.4505, and the infection process and histopathological changes off. fraxinus by optical microscope, scanning electron microscope and transmission electron microscope. At the same time, HSCCC was applied to the separation and purification of metabolites from L. lecanii 3.4505 and B. brongniartii 2382, and isolates were detected and analyzed by GC/MS. These researches provide some evidences for the fungal application as potential biological control agent.The results are as follows.1. The bioassay result of L.lecanii 3.4505 against 2 instar P. Fraxinus female and male nymphs showed:the mortality rates of 2nd instar P. Fraxinus female and male nymphs infected with L. lecanii 3.4505 were high. After 2d, the mortality rates of male and female nymphal were above 40%, after 5d, the mortality reached 90%. The fungus could finish infection against P. Fraxinus in a short period of time. This may be that 2nd instar nymphs were weak, and wax filaments could not prevent the hyphal infection of L. lecanii 3.45052. By using optical microscope, scanning electron microscope and transmission electron microscope technology, we systematically observed the conidia attachment and germination on the body, hyphal penetration into the wall and the pathological changes of internal organization of P. Fraxinus nymph infected with L.lecanii 3.4505. The study found:at Id post inoculation, the conidia of L. lecanii 3.4505 were observed on the intersegmental folds, the hollow place of the body surface, and body segment contacted with the host plant. These places became important invasion ways of L. lecanii against P. Fraxinus. These showed the conidia of L. lecanii could break through the action of hydrophobic and bacteriostasis of the wax powder. After conidia germinated, the end of germ tube expended and formed appressorium which tightly adhered to the cuticle. The germ-tube continued growing and developed into hypha which passed through the waxy secretion along the body surface. Some hyphae produced a specialized cone-shaped penetration pegs at the end of hyphae and searched suitable sites for penetrating. The conidia and hyphae could secrete massive mucus which could cause physical visible damage to the host cuticle in the growth process. At 2d, the structure of the body wall was mostly destroyed by the hyphae which have entered into the haemocoel. The hyphae grew rapidly using the nutrients in the haemocoel and formed a lot of blast spores. The various organelles in the haemocytes are destroyed, such as the endoplasmic reticulum hypertrophied and agglomerated into obvious fingerprint-like; the mitochondria swelled and deformated. The mitochondria of muscle cells were also severely damage. The myofibril was unclear in the irregular state and parts of myofilament were break and dissolved. The trachea, fat body, Malpighian tubules, combined nerve ganglion and other internal organs were subject to varying degrees of damage. The mycelium fully occupied the whole of haemocoel. The whole bodies were wrapped in a white mycelium, mycelium extending radically outwards.3. Secondary metabolites Phenol, xylene, dibutyl phthalate and diisobutyl phthalate from L. lecanii 3.4505 fermentation liquid which was extracted by ethyl acetate can separated by HSCCC with two-phase solvent system n-hexane composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5). The content of dibutyl phthalate was higher in these isolates, and the purity was 98% by area normalization method.4. Secondary metabolites 2-coumaran ketone,3-Methyl-benzoic acid methyl ester, 4-methyl-benzoic acid methyl ester and dibutyl phthalate from B. brongniartii 2382 fermentation liquid could separated by HSCCC with two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3.5:5:3.5:5). The purities of them were 81%, 89%,80% and 78% by GC detection, respectively.5. The results of the toxicity test showed that 3-methyl-benzoic acid methyl ester and 4-methyl-benzoic acid methyl ester presented different degree of toxic effect on Dendrolimus tabulaeformis.3-methyl-benzoic acid methyl ester caused the mortalities 32.95% in breeding,32.56% in epidermis contact and 86.75% in injection on the larvae of D. tabulaeformis, while 4-methyl-benzoic acid methyl ester caused the mortalities 36.36%,36.05% and 90.36%, respectively. |
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