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The Screening of Metarhizium Anisopliae Isolates and the Pathogenesis of the Highly Virulent Isolate to Anoplophora Glabripennis Larvae

Author: WangDa
Tutor: HuangDaZhuang
School: Agricultural University of Hebei
Course: Forest cultivation
Keywords: Metarhizium Anoplophora glabripennis Filter Biological characteristics Pathogenesis
CLC: S476
Type: PhD thesis
Year: 2009
Downloads: 181
Quote: 3
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Abstract

The application of Metarhizium anisopliae against Anoplophora glabripennis adults and larvae is an important way for the comprehensive management of Anoplophora glabripennis. In order to reveal Metarhizium optical Shoulder Star alternatus larvae pathogenic mechanism and control potential, clear green anisopliae and Anoplophora alternatus larvae each other for relationship, this article Tenebrio lure set method from the soil lure set and screening has a higher cause light shoulder star alternatus larvae disease force the green anisopliae MS01 strain, and the system studied its biological characteristics, optical glabripennis pathogenic nature and its impact factors (temperature, humidity, passage passage frequency), Metarhizium optical Shoulder Star alternatus larvae invasion and induced disease process, Anoplophora the beetle larvae infected by Metarhizium defense reaction pathological changes of Anoplophora glabripennis infected with M. anisopliae. The results are as follows: insects Tenebrio as bait trap from soil samples collected 8 Metarhizium isolated on Anoplophora glabripennis larvae bioassay. The results showed that different strains 50 d instar larvae of Anoplophora glabripennis virulence differences, the MS01 strain virulence of the highest corrected mortality and infection rates were 100% and 83.33%, lethal LT50 to 2.69 d. MS01 strains commercially available powder is formed by the rejuvenation of the parasites after 95 d age of Anoplophora glabripennis larvae biological measurement results show that, relative to commercially available strain MS01 rejuvenation strain virulence higher correction to the 8th day mortality rate of 100% infection rate to 90.4%, the LT50 to 5.25 days. MS01 strain on the 95 d age of Anoplophora glabripennis larvae median lethal concentration LC50 of 1.68 × 106 spores · mL-1. Therefore confirmed MS01 strains of Metarhizium Anoplophora glabripennis larvae with high virulence. Determined by biological research the MS01 strain vegetative growth and sporulation optimum medium PPDA. The strain was grown at 26 ° C under nutrition best, 28 ° C under the highest sporulation, spore germination speed the fastest germination rate was the highest. The strain vegetative growth and sporulation optimum relative humidity range of 95% to 100% relative humidity and spore germination rate, germination rate was positively related to the highest relative humidity of 100%. Anoplophora glabripennis adult vaccination is low, medium, and high concentrations of M. anisopliae strain MS01, fecundity, egg hatchability and hatched larvae survival were significantly reduced, and the inoculum spawning and incubation survival rate of larvae was negatively Relevance. MS01 strain Anoplophora glabripennis larvae pathogenic role of the optimum temperature is 26 ℃, and the optimum humidity of 100%. Through the the PPDA medium passage 2 strain virulence no significant impact will result in reduction of strain virulence passaged 4 times; improve by Anoplophora glabripennis larvae subculture will be different degrees of virulence of the strains. Scanning electron microscopy and transmission electron microscopy showed that the main parts of the Metarhizium infection Anoplophora glabripennis larvae film abdominal section, attached to a large number of conidia, faster germination, germination rate and a higher rate of invasive; followed by the area near the valve. Two conducive Metarhizium invade the weak link. Inoculation after 12 h, conidia Anoplophora glabripennis larvae surface began to germinate and the emergence of the bud-like protrusions, followed by a germ tube and attached to the structure, after 16 h of inoculation, the host epidermis observed a small amount of mycelium segment to 24 h mycelium has been widely invasive germ tube penetrate rely on mechanical pressure and the joint action of the enzyme in the invasion process. 36 h to 48 h conidia can penetrate the body wall into the blood chamber. Hyphae and mycelium segment blood lymph circulation invade the organs and tissues. From 48 h to 72 h, with the rapid proliferation of hyphae and mycelium segments in the intestinal wall cells, intestinal tissue is gradually destroyed, the shedding of the intestinal microvilli of intestinal cells to form vacuoles peritrophic decomposition disappear, the final collapse of the intestinal tissue disintegration. When the larvae is inoculated by different passage strains of Metarhizium the blood lymph phenol oxidation enzyme activity, although the ups and downs of change but all appear a peak value that host by Metarhizium invasion stimulation, the immune each other for so phenoloxidase activity increased to the peak of the phenol oxidase activity gradually increased with the strain adaptive rendering fluctuating decline. On larval blood lymph inoculated with different strains of phenol oxidation activity of the differences significant resistance analysis showed that, with respect to the host pass-generation strain, medium pass on behalf of the strains were inoculated light shoulder Star beetle larvae cause the blood lymph phenol oxidation enzyme activity higher. Alternatus larvae of Anoplophora blood cell invasion of Metarhizium has certain defensive ability, blood cells involved in the defense response is mainly tablets blood cells and plasma blood cells in the defense reaction peak period (48 h ~ 72 h), the total number of blood cells sharply rising grain blood cells and the proportion of pulp blood cells change, while blood cell adhesion, aggregation, phagocytosis, the package is a series of defensive reaction, the formation of nodules or cysts block. However, due to the rapid proliferation of mycelium segment in the blood, the blood cell defense mechanisms Metarhizium only a temporary obstacles and inhibition, and ultimately the collapse of. Anoplophora glabripennis adult vaccination low, medium and high three concentrations of M. anisopliae strain MS01, their food intake was significantly lower inoculum concentration was negatively correlated with food consumption. The Anoplophora beetle larvae infected with Metarhizium their food consumption and body weight were lower than the control group; the blood lymph protein concentration, although fluctuations than the intense but overall showed a declining trend; Anoplophora beetle larvae blood lymphoid Chinese Communists to detect 17 kinds of free amino acids, green infection anisopliae in different periods after the first decreased and then increased trend of free amino acids, different kinds of amino acids in inconsistent changes. The Metarhizium the infection caused by Anoplophora glabripennis larvae protective enzymes and detoxification enzyme activity occurs with varying degrees of change. In early contamination superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity rapidly increase in contamination post-SOD and POD activity decreased in varying degrees, and the CAT activity rises to a higher level. Carboxylesterase (CarE) activity performance first increased and then decreased gradually; overall performance of the activity of glutathione-S-transferase (GSTs) of acetylcholinesterase (AchE) to activate → inhibit → activation → inhibition; dynamic can be summarized activated to curb → → inhibit → Activation → inhibition. Clear histopathological changes and Metarhizium Anoplophora beetle larvae infection in the process of development of the host body. Metarhizium invasive, the host epidermis and skin cell layer separation, into the host blood chamber, with the mycelium blooms in the body cavity, the various organs and tissues of infested obvious lesions, such as fat body loose muscle tissue cracks, trachea tissue disintegration and failure of, Markov tube deformation, gastrointestinal disintegration.

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