Study On Histopathology Of Dendrolimus Tabulaeformis Infected By Beauveria Bassiana And The Fungal Preparation

The pine caterpillar, Dendrolimus tabulaeformis Tsai et Liu, (Lepidoptera:Lasiocampidae), is the most serious defoliator and outbreaks frequently in the pine forest in Northern china. Although a variety of chemical pesticides have been used for the pest control, a long-term control effect for the pine caterpillar has not been achieved, due to the environment pollution and the ecological unbalance issues brought by pesticide application. Nowadays, Beauveria spp., a typical group of entomopathogenic fungi as biological control agent in pests control have been worldwide used. Some insecticidal preparations produced with Beauveria spp. have been available, such as Mycotrol(?), BotaniGard(?), Naturals(?) and so on, which are effective to the pest species more than sixty. In China, however, matolity studies on the Beauveria spp. application to control of pine caterpillars have been focused on those species occurring in southern regions, such as Dendrolimus punctatus (Walker). The reports on those species occurring in northern China controled with the biological preparations of Beauveria spp. are scarce. In this paper, two northern specificity strains,1573 of Beauveria bassiana and 2382 of Beauveria tenella that were originally isolated from the natural disease larvae of D. tabulaeformis, were used to infect this pine caterpillar. The infection process and histopathologic changes of the insect were observed by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscope (TEM). The strain 2382 of Beauveria tenella was subsequently tested for the three-class culture and fermentation (solid-liquid-solid). Water ratio, spore yields and protease activities were investigated so as to determine the best culture way and harvest time for the fungal preparation production. We hope this research could provide some useful evidences to guide the biological pest control in northern china.1. Histopathological changes in D. tabulaeformis infected by B. bassianaThis study showed the strain 1573 of B. bassiana is an effective pathogen to the pine caterpillar, D. tabulaeformis. It caused a series of infections in the insect integument and inner tissues and led the host insect to die. The strain 1573 generally infected the pine caterpillar by penetrating the integument. At 24 h after inoculation, the attached conidia were found on the vertex and the regions around ocellis, antenna and mouthparts on the head, while on the thorax and abdomen, they adhered on verrucas, tufts, the acanthaes and intersegments. At 36 h after infection, the conidia germinated into hyphae on the cuticle, and then the hyphal tip differentiated into appressoria and penetration pegs to penetrate the cuticle. At 48 h, hyphae penetrated the integument depending on both the mechanical pressure force and degradation action from hyphal extracellular enzymes, that caused cuticular rupture and melanism, resulted in the deformation of the procuticle. About 72 h after infection, the hyphae had entered the body cavity and infected hemolymph, fat body, muscle, alimentary canal, silk gland and nerve tissue. By utilizing the nutrition of haemolymph and internal organs and tissues, the fungus massively proliferated. The insect body showed swelling and dark. At 96 h after infection, as hyphae occupied the haemocoel, the internal tissue structure of the pine caterpillar was totally destoryed and the insects died. At last, the hyphae broke through the cuticle and released new conidia on the cadaver surface.2. Histopathological changes in D. tabulaeformis infected by B. tenellaThe pathogenesis of the strain 2382 to the pine caterpillar is similar to the B. bassiana1573, but its infection site to the insect body was more wide and the infection speed also more quick than B. bassiana1573. It generally also invaded the insect coelom by penetrating the integument, yet besides tormogen, acanthae and intersegmental fold, the clear area and the accessory organs on body surface, such as spiracle, anus, were also the significant invading sites of the strain 2382. In addition, its invading ocurrence was earlier than tha strain 1573. At 48h after infection, trace amount of mycelia were observed on the body surface, At 60 h, a part of tissues and organs of the host insect was destroyed and the hyphae could been seen on the throax and abdomen of the insect. At 84 h, the whole body of the infected insects were covered by hyphae. The result indicated the strain 2382 is a higher toxic pathogen to the pine caterpillar than the strain1573.3. Study of fungal preparationThe strain 2382 of B. tenella was used for the production of the fungal preparation. After liquid fermentation, the zymotic fluid was inoculated to the solid medium that contained in plastic bag, wedge-shaped bottle and preserve jar, respectively. During culture, some important indicators associated with the preparation's quanlity (water content, spore numbers and the protease activity) were detected. The results showed that the alive spore numbers and protease activity in the solid prepareation cultured in the wedge-shaped bottle were the highest in the three cultural vessels. The largest level of the alive spore numbers (19.13×107spore/g) and the protease activities (107.71±3.77 U/mg) appeared at 20 d in the wedge-shaped bottle. Thus, the solid preparation cultured in the wedge-shaped bottle was the most useful for reference in the mass production of B. tenella, while the 20th day was the best time for the solid prepareation harveast.