| Apocheima cinerarius Erschoff is an explosive defoliator on poplar trees, the larvae feeding on the tree from late March to early May each year. The larvae may eat whole leaves and make trees die when the population density is much higher. The larvae were controlled by using spray insecticide on the tree in the past. According to the habit of mature larvae pupating over winter and summer for nine months, we did the research on biological control of application of entomopathogenic fungi in this thesis of Master. In this study, Apocheima cinerarius took place and were collected in poplar forests of Hongtong County of Shanxi Province. Pupae of three developmental stages were sampling in may, July and October respectively. Morphological characteristics and internal anatomy of pupae were observed by stereo microscope. Three strains of Beauveria bassiana the lab saved were selected as tested strains. The entomopathogenic fungi TST05 was isolated from Carposina niponensis; Walsingham overwintering larvae. FDBOlwas isolated from Acantholyda parki. SYN01 was bought in China General Microbiological Culture Collection Center, preservation number was CGMCC 3.4428. The host was Dendrolimus kikuchii Matsumura. The virulence of three Beauveria bassiana strains TST05, FDB01 and SYN01 infected on Apocheima cinerarius pupae was compared. At the same time, the entomopathogenic fungus from Apocheima cinerarius pupae was isolated.The content are as follows,(1) Morphologic observation and inner structures of Apocheima cinerarius pupae and comparison of mortalities of Beauveria bassiana TST05, FDB01 and SYN01 infected on them;(2) The infection process, extracellular enzymes activity and the cuticle components;(3) Isolation, purification, culture characteristics, morphological characteristics observation under electron microscopic, molecular identification of entomopathogenic fungi from Apocheima cinerarius pupae.Results:1. The results of external morphology and internal anatomy of Apocheima cinerarius pupae in three developmental stages showed that the morphological characteristics of pupae were about the same. The pupae had ommateum, antenna, mesostigma, wing bud, cremaster, etc. The cuticle of pupae in early and middle stages was yellowish-brown,while pupae in later stage was taupe. The internal structures of pupa varied in different stages. Pupae had not fully developed in early stage, only can see rectal caecum in the central. Pupae in middle stage had basically developed to adulthood. The midgut and Malpighian tube had differentiated. There were eggs in later stage female pupae.Infection experiment shows that three strains were able to infect pupae and dead pupae had white mycelium along with culturing. Although the curves which corrected mortality of each period of A.cinerarius pupae changes with time were not the same, the corrected mortality of TST05remained top tendency on 4d. The corrected mortalities of strain TST05 in three periods were the highest on 8d. The corrected mortalities reached 71.05%,70.73% and 61.18%, respectively. LT50 were 4.76d, 4.91d and 5.60d, the lowest and significant difference. The mortalities of TST05 on pupae in early stage and middle stage reached 70% on 8d and significant difference. LT50 were less than 5d. Conclusion:TST05 is definitely a strain with great potential for controlling the pest in soil. The early pupa stage in May and the rainy season in July or August are best time for controlling the pest.2. The pupae in later stage were infected by TST05 spore suspension (1×108 spore·mL-1). The infection process was observed by SEM. There were many nonuniform punctums on the abdomen of Apocheima cinerarius pupa. Conidium attached to punctums, abdominal spiracle and cuticle the abdominal spiracle and germinated to form hyphae. Hyphae went through the cuticle and extended on the backside of cuticle. At last, the hyphae infected the developing adult cuticle.Made pupae in the different development stages as the only carbon and nitrogen source, the activities of subtilisin-like protease, chitinase and lipase were determined. The results showed that the ability of TST05 and FDB01 strains in decomposing cuticular protein was stronger. The protease activity reached the maximum value 4.581±0.392 U/ml and 5.753±0.095 U/ml respectively. The ability of SYN01 in decomposing cuticular protein was weaker. The protease activity reached the maximum value 4.247±0.368 U/ml. The ability of three strains in decomposing cuticular chitin was the same when the pupae in early stage were the only carbon and nitrogen source. The chitinase activity reached the maximum value 0.206±0.005 U/ml, 0.193±0.014 U/ml and 0.200±0.012 U/ml respectively. The chitinase activity reached the maximum value 0.379±0.019 U/ml. The three strains of ability in decomposing cuticular chitin from strongest to weak were SYN01, TST05 and FDB01, when the pupae in middle and late stage were the only carbon source. The chitinase activity reached the maximum value 0.379±0.019 U/ml,0.346±0.036 U/ml and 0.347±0.044 U/ml respectively. The three strains of ability in decomposing cuticular lipid from strongest to weak were TST05, FDB01 and SYN01, whatever the only carbon and nitrogen source were the pupae in early, middle or late stage. The lipase activity reached the maximum value 1.475±0.314 U/ml,1.300±0.372 U/ml and 1.053±0.240 U/ml respectively.The protein, chitin and fat contained in the culticles of Apocheima cinerarius pupae in different stages were also determined. The protein contents were 0.055±0.002g,0.053±0.004g and 0.052±0.003g per gram of culticles corresponding to early stage, middle stage and later stage respectively, but the diversity was not remarkable. The chitin contents were 0.021±0.001g,0.023±0.004g and 0.026±0.002g respectively. The chitin contained in the culticles of pupae in late stage was higher than pupae in early stage and significant difference. Middle stage had no evident difference. The fat contents were 0.025±0.001g,0.024±0.001g and 0.022±0.003g respectively, but the diversity was not remarkable. The result of mortalities and the activities of extracellular enzymes showed that content of protein in cuticle was the most and the subtilisin-like protease played an important role in the process of pupae infection. The Chitinase were important in the infection process when the content of chitin increased obviously in later stage of pupae. Because there were so much fat in pupae, the lipase played an important role in the process of pupae infection after the hyphae went into the coelom. The ability of TST05 strains in decomposing protein, chitin and fat were stronger. TST05 were selected as higher virulent pathogen for Apocheima cinerarius pupae control.3.The strain was designated numbers as YHT01 which isolating from the naturally infected Apocheima cinerarius pupae. YHT01 was proved to be the pathogens of Apocheima cinerarius pupae by re-inoculation test on the host insect. The observation of culture character in PDA of the strain YHTO1 showed that colonies were ellipse, white and fluffy. The colour of colonies was orange viewed from the back. The coremium were like brooms and phialides on the conidiophores were whorled or solitary. The diameter of phialides was 0.4～1.1μm, while the length was 4.2-13.1μm. The conidium was small, oval to ellipsoid, smooth。 The strain was identified based on the morphological characteristics, ultrastructure and PCR. The results showed that the strain YHT01 was the species of paecilomyces farinosus in the genus paecilomyces. |
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