| Carposina sasakii (Matsmura) is an important fruit borer pest. The entomogenous fungi for biological control can be applid in the soil to infect C. sasakii overwintering larvae. Applying entomogenous fungi can sustainable control on the population of the pest because the fungi can survival in a long time in the soil and the infected larvae can be a new source of infection. In the thesis, one higher virulent strain TST05of entomopathogenic fungus was obtained by isolating from the naturally disease larvae of Carposina sasakii. Based on morphological characteristics and ITS gene sequence analysis, the taxonomy position of the strain was identified. The pathogenicity and virulence of strain TST05against C. sasakii larvae was studied. The histopathological characteristics and physiological and biochemical reaction of C. sasakii larvae infected by the strain were investigated. A series of experiment were conducted on the strain in biological characteristics, fungal powder preparation, application in the field condition, the survival ability in natural environment, and the compatibility with common chemicals pesticides. The results may provide a base in theory and application for biological control on C. sasakii.The contents are as follows:1. The strain TST05was isolated from the natural cadaver in winter-cocoons of Carposina sasakii. It was proved that the strain TST05was a pathogen of C. sasakii larvae by re-inoculation test on the host insect. Cultured on potato dextrose agar (PDA) media, the colony of the strain was white and villous in early cultivation and produced thick conidia in yellow and powdery-like in later cultivation. The upper of conidiogenous cell extended into a "Z"-bending conidiogenous axis. Conidia located on the small pectina which were born on the axis. Many conidiogenous cells densely clustered on vegetal mycelia. And many spherical conidiogenous cells group were observed under microscope. Conidia were oval or near spherical. Based on the morphological identification and alignment analysis of rDNA ITS region, the strain TST05was identified to be Beauveria bassiana.2. The virulence of strain TST05to C. sasakii larvae was tested. The results showed that by innoculation with107-109conidia/mL of strain TST05, adjusted accumulative mortality rate of the larvae was68.57%97.50%during9d after treatment. The larvae began to die at2d after treatment, and the peak time of death appeared in4-7d. The death peak appeared earlier and quicker with conidia concentration increasing in treatment. The median lethal time (LT50) decreased from6.57d to4.31d with conidia concentration changing from1x109to1x107conidia/mL in the treatment. The median lethal concentration (LC50) was2.16x109conidia/mL at4d but it was4.36x106conidia/mL at6d. Strain TST05displayed a higher virulence to C. sasakii larvae and a rapid action in killing host. The strain can be used as a new strain for biology control on C. sasakii and was preserved in China General Microbiological Culture Collection, No.4526.3. The conidial adhesion and germination on C. sasakii larvae surface, the integument penetration of hyphae on the larvae, the internal tissues infection, defense response of the larvae, and the host pathological changes were investigated by using light, scanning electron microscopy and transmission electron microscopy. The result showed that the head capsule of the larva was so strong and smooth that very few conidia attached to there. But there were many conidia in the basal area around the mouthparts and antennae. On the thorax and abdomen of the larvae, dense acanthae were distributed over the cuticle and in which few long seta scattered. Conidia adhered mainly to the area around the acanthae. Some conidia were observed in the peritreme socket of the spiracles and on the smooth seta.After conidia germinated, the fungus invaded the larvae mainly by penetrating the integument. The conidia germinated during a24-36h period after inoculation, and the germ tubes might directly penetrated the cuticle or produced a infection peg at the hyphal tips to penetrate into the cuticle. The fungal infection destroyed the regular protein-chitin parallel lamellar structure in the procuticle. The defensive response of the larvae to the fungal attack was indicated by some dark spots appeared on the cuticle and some melanization appeared in the hemocoel. After overcoming the host’s defense system, the fungus grew and reproduced many blastospores in the hemocoel, then infected the internal tissues and organs. Infection symptoms were observed in the fat body, alimentary canal, the Malpighian tubules and the muscle tissue. The fungus attacked and colonized the sericin layer and the liquid fibroin in the silk gland lumen. Finally, the fungus emerged through the cuticle of the dead insect and released conidiophores that could act as new pathogens to infect other larvae. The results clearly revealed the process of strain TST05infected C. sasakii larvae.4. The physiological and biochemical reaction of C. sasakii larvae were studied when they were infected by the strain TST05. The results showed that after infected, the content of trehalose and protein in the larvae decreased obviously, the trend of changes of glucose content was increased at first and then decreased, the activity of trehalase rose to1.91times as that of control groups at5d after inoculation.During the initial infection stage, the activities of PO, GSTs, CarE, SOD, POD and GSH-Px that play a role in immune and protection for the host, increased quickly, while during the later stage, their activities decreased at different degrees. The change of GSTs and GSH-Px activities were biggest in those enzymes. Their activities were2.84times and2.95times respectively as those in control groups at3d after inoculation. The activities of SOD and POD were1.61times and1.53at3d, the activeties of PO and CarE was1.48times and1.50times as that of control groups at5d, respectively. The results indicated that the infection of strain TST05affected nutrient metabolism and induced the physiological defense reaction of C. sasakii larvae.The activity of CAT in infected larvae always lower than that in control larvae, and the lowest activity is56.78%as control activity. It indicted that the infection of the strain TST05can inhibited and destroyed CAT. As the same time, the increase of the AChE activity in infection period indicted the infection or the toxin of the strain can effect the nerve conduction of larvae.5. The effect of culture media, temperature, and humidity on the mycelium growth, conidia yield, and conidia germination of the strain TST05were investigated. The results showed that the strain could grow well in the four culture media, PDA, PPDA, SDAY and SMAY. The fungal colonies were sick and dense, and the conidia yield exceeded3.95x107conidia/mL in these culture media.The TST05strain was adapt to a wide range of temperature and humidity. The conidia could germinate, and the mycelia could grow and sporulate in15-30℃and RH30%-100%. With the temperature closing to25℃and the humidity increasing, the rate of conidia germination and mycelium growth and the conidia yield were all significantly increased. The conidia yield was1.32x107conidia/mL under15℃, RH100%,1.37x107conidia/mL under25℃, RH30%, and6.19x107conidia/mL under25℃, RH100%. The conidia germination rate was52.28%under15℃, RH100%, and90%or more under25℃, RH>80%. The results indicted that the strain TST05was easy to culture and adapt to low temperature and humidity in north. The strain could become a biological agent to control C. sasakii.6. The powder preparation of strain TST05was preparated by cultured in wedge-shaped bottle and plastic bag with glass cavity tube in the both ends. The results showed that the strain grew vigorously in the two culture vessels. The white dense mycelia overgrow in the solid media. The conidia yield and the protease activity of powder preparation were12.50x109/g and770.34U/mg pr respectively at20d in the plastic bag, which were1.42times and1.54times respectively as that in wedge-shaped bottle. The results indicted the good ventilation effect is more conducive to the growth and sporulation of strain TST05.7. The control efficiency of powder preparation to C. sasakii mature larvae in an orchard was determined. In September, when the mature larvae of C. sasakii bored out jujube and dropped in the soil for overwintering, the fungal powder preparation was mixed with soil in the proportion of1:200and then was applied in the upper soil layer under the tree. The larvae were laid on the surface of the soil and naturally bored into the soil. The mortality rate of powder preparation to C. sasakii larvae was determined. The adjusted mortality rate of C. sasakii larvae reached75.29%by the fungus infecting in the soil.8. The survival ability of the strain TST05in soil was tested. The fungal powder preparation was scattered into soil by using the method as above mentioned. The CFUs (colony forming unit) of the strain TST05that survived in soil were determined. The results showed that the CFUs of the strain were1.09x104CFU/g after4months, while the value at the start was108.67x104CFU/g. But in the following months, the CFUs were relatively stable. The conidia yield of the strain isolated from the mixed soil can reach106conidia/mL after6and8months. The results indicated that the strain TST05possessed higher survive ability in the field condition which was propitious to sustainable control on the population of C. sasakii.9. The compatibility of the strain TST05was studied with seven common chemicals pesticides including beta cypermethrin, cypermethrin-emamectin benzoate, phoxim, cyhalothrin, abamectin, Avi monosultap and tebufenozide. The effect of the chemicals pesticides on mycelium growth, conidia germination and conidia yield of the strain were tested.The results showed that the inhibition rate of the mycelium growth was27.93%-38.74%under the conventional concentrations of the pesticides. In the group treated with tebufenozide, the conidia germination rate was33.19%, and the conidia yield was only0.05×106conidial/mL, while in other groups treated with other6pesticides, the conidia germination rate ranged from50.34%to70.62%, and the conidia yield was2.51-3.58x106conidial/mL. The inhibition action of the pesticides to the strain decreased with the concentration decreasing. When the pesticides were diluted5times of the conventional concentrations, the conidia germination rate was up to72.08%-84.92%, and the conidia yield was3.01-9.61x106conidia/ml. The results showed that the compatibility of the strain TST05with the six pesticides was relatively good. And the compatibility was increased with the concentration of pesticides decreasing. |
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