Isolation, Identification, And Pathogenicity Of The Entomopathogenic Fungi From Pine Sawfly

Pine sawfly (Insecta:Hymenoptera:Tenthredinoidea:Diprionidae) is a group of the most important pests in the coniferous forest. The larvae feed on the needle causing the needle to turn brown and wilt. In the past, releasing smoke, artificial and aerial spraying of chemical pesticides were used to control pine sawfly. Although these methods were in effect rapidly decreasing the population density of pine sawfly, they resulted in a series of problems in pesticide residue, environmental pollution and pest resistance. Attentions have been paid to entomopathogenic fungi as biological agent in the control of pine sawfly. In this study, the naturally diseased pine sawfly larvae were collected in the pine forests at Yitong of Jilin Province, Yangquan, Yuxian, and Xiaxian of Shanxi Province, respectively. The puipose is to select some higher virulent pathogens for this pest biological control. The content are as follows,(1) The collection and isolation of the pathogenic fungi, their morphology and ultrastructure observation, DNA extraction, PCR product sequences analysis, and molecular identification.(2) The effect of different temperature and UV irradiation on the colony and spore production. The extracellular enzyme activity (extracellular protease, lipase and chitinase enzyme) of the strains.(3) The change trend of the protective enzymes and detoxicate enzymes of the pine sawfly infected by the strains.(4) The pathogenicity test of the three strains to the host insects in laboratory and the control effect in forest. Observation of the fungal infection on the surface of pine sawfly larvae under the scanning electron microscope.The results are showed as followed:The three strains of entomopathogenic fungi were obtained by isolating from the naturally diseased pine sawfly larvae. They were designated as FDB01、FYQ01、FXX01and were proved to be the entomopathogenic fungi of pine sawfly by inoculating.Strain FDB01was isolated from Acantholyda parki ShinOhara＆Byun collected from Yitong, Jilin Province. The observation of culture character and morphological characteristics of the strain FDBO1showed that colonies were circular, opaque, powdery, conidiophores represented some conidiogenous cells flask-like, with globose base and distinct neck and extended, denticulate rachis.Strain FYQO1was isolated from Acantholyda posticalis Matsumura collected from Yangquan, Shanxi Province. The colonies were circular, opaque. Conidiophores with unsmooth surface were fusiform and they formed into conidial chain.Strain FXXO1was isolated from Acantholyda posticalis posticalis Matsumura collected from Xiaxian, Shanxi Province. Its colonies were round, opaque. The center of the colonies were puce viewed from the back. Conidiophores containing oil droplets were big.The three strains were identified based on the morphological characteristics, ultrastructure and PCR product sequences analysis. The results showed that the strain FDBO1was the species of Beauveria bassiana in the genus Beauveria. The strain FYQO1was the species of Paecilomyces farinosus in the genus Paecilomyces. The strain FXXO1belonged to the species of Clonostachys rosea in the genus Clonostachys.2. The biological characteristics of the three strains showed that the maximal spore production of three strains all appeared at25℃during culture. In which, the strain FDB01was the greatest. Its spore production reached to40.375±0.976×107/cm2at25℃, which was7.34and16.15times respectively to the strain FYQO1and FXXO1. The colony growth rate of FXXO1was the fastest, but its spore production was the lowest in the three strains. The anti-ultraviolet capacity of the strain FDB0l was the strongest, that means the strain FDBO1had higher ecological adaptitude. The strain FDB01possessed highest activity of extracellular enzymes, and it demonstrated that the strain FDBO1had highest virulence for the pine sawflv larvae.3. The activities of superoxide dismutase (SOD), catalase(CAT), peroxidase(POD) of pine sawfly larvae infected by different strains were determined. The results showed that the activities of SOD, CAT, POD changed in degree when the insects were infected. The activities rose at the first phase then droped at the last phase in the infection. But the enzymatic activity peak of the pine sawfly larvae appeared at different time with the infection of the three strains. This maybe indicated different virulence of the strains.The activities of acetylcholinesterase (AchE), glutathione-S-transferase (GST) of pine sawfly larvae infected by different strains were determined, and the results showed that the change trend of AchE and GST were similar. Their activities were stimulated at first and then were inhibited.4. Mortality rates of the pine sawfly larvae in laboratory condition were higher than that in the forest condition. Diseased rates of the pine sawfly larvae in the forest of Xiaxian were higher than that of Yangquan. Diseased rates of the pine sawfly larvae infected by the strain FDB01reached above90%.The fungal infection on the surface of pine sawfly larvae was observed using the scanning electron microscope. It was found that mycelium mainly appeared in the head, prothorax, anal segment, and intersegmental fold.