Virulence And Histopathology Of Lecanicillium Lecanii To Scale Insects

Scale insects are the most important pests in agricultural corps, forests, fruit trees and ornamental plants. They live on plant juice with their sucking mouthpart piercing into leaves of branches, which cause harm to the host. At the same time, the scale insects possess wax glands, which can secrete wax substances that cover on the surface of insect body to protect the insect and make chemical control difficult. Application of insect pathogenic fungi as biological control agent is an important way to achieve sustainable control of scale insects. Lecanicillium lecanii (Zimmermann.) Gams & Zare is probably the most important entomopathogenic fungus of the scale insects in nature reported in the world. In this paper, the virulence and histopathology of the scale insects by Lecanicillium lecanii was investigated, which can provide theoretical and practical basis for further field applications of the pathogenic fungi on scale insects. The main contents including, (1) effect of the strains No.V3.4504 of L. lecanii successive transfer cultured on seven different substrates on its colony growth characteristics, extracellular protease and chitinase enzyme activities and the virulence against scale insects; (2) extracellular lipase activity trend of L. lecanii and growth characteristics cultured on the substrates prepared with Rhodococcus sariuoni and Ceroplastes japonicus integument respectively, and fat contents with the integument of two species of scale insect; (3) histopathological changes of Didesmococcus koreanus infected by L. lecanii, The results of these studies are showed as follows.1. The strain No.V3.4504 of L. lecanii were successive transfer cultured on seven different substrates respectively. The colony characteristics were observed and the growth rate, spore quantity, activity of extracellular protease and chitinase of the fungus, and mortality of the scale insects by L. lecanii were determined. The results revealed that, the fungus cultured on PDA medium (A) showed most fast in colony growth, the minimal in sporulation, straight decline of extracellular protease and chitinase activity with generation increasing, and the minimal mortality of the scale insects. In medium (B), no significant effect was observed promoting the virulence of the fungus by adding peptone to PDA. In the media D (added cadavers of R. sariuoni), E (added cadavers of C. japonicus) and F (added cadavers of R. sariuoni and C. japonicus), although the fungus appeared lower in the colony growth rate, the spore quantity of the two strains were higher upward 8.83×106-9.13×106 spores/cm2. Extracellular protease and chitinase activities of the two strains reached 2.13-2.16 U/g and 1.01-1.03 U/g respectively, and the mortalities of the two species of the scale insects were 55%-58% and 39%-42% respectively. The fungus exhibited the highest extracellular enzymatic activities among the seven media after cultivated on the two species of scale insects for three successive generation, viz. its protease and chitinase were 2.92-3.08 U/g and 1.42-1.45 U/g respectively and the mortalities of the two scale insects were 71.30% and 58.89% respectively. A linear correlation was found between extracellular protease and chitinase activities of the fungus and the mortalities of the scale insects.2. The culture medium prepared with cuticle materials of R. sariuoni and C. japonicus, were respectively inoculated with conidial suspension of L. lecanii No.V3.4504 with a concentration of 7×107 spores/mL. The growth characteristic of the fungus cultured on the integument substrates were observed under dissecting microscope. Fat contents of two scale insects with cuticle materials and the activity of the extracellular lipase of L. lecanii on cuticle medium were determined. The results showed that mycelium flourished on the substrate prepared with cuticle materials of R. sariuoni, by contraries, mycelium on the substrate of C. japonicus was thin. The lipase activity of the fungus cultured on the substrates prepared with R. sariuoni integument reached the maximum values (0.117 U/g) in the sixth days; comparatively, the lipase activity of the fungus cultured on the C. japonicus substrate reached the maximum value (0.442 U/g) in the seventh, which was late one day. The lipase activity of L. lecanii on cuticle medium of C. japonicus was far higher than that R. sariuoni's, which was about three times than that on cuticle mediums of R. sariuoni; Fat contents of C. japonicus was about two times than that on cuticle of R. sariuonsi's, that means the lipase activity of the fungus was positively correlative the fat contents of the cuticle.3. The D. Koreanus adult females were inoculated with conidial suspension of L. lecanii, No.V3.4504 with the concentration of 7×107 spores/mL. The infection process and invasion mode of L. lecanii, and the histopathogenic changes of D. Koreanus by the fungus were observed by light and electron microscopy. It was observed that L. lecanii generally infected the scale insect by penetrating the integument, especially around the base of mouthparts and legs, over the vulva, stigma, the body margin and the anal area were susceptible places. The infection process of L. lecanii to the scale insects were shown as follows:the conidia adhered to the surface of the scale insect within 24 h after inoculation; then the conidia germinated into germ tube and further developed into hyphae under appropriate humidity and temperature conditions; 48 h after inoculation, the tips of the hyphae specialized into penetration pegs to invade the integument. After penetrated the cuticle, the hyphae developed along the integument or branched laterally to invade the epidermis of the integument. In the epidermis, the hyphae invaded into gland cells were observed, then the integument was penetrated. On the surface of the integument, the anal area was covered by a mass of mycelia at the same time; 72 h after inoculation, the hyphae nearly entirely invaded into the insect body. On the ventral surface of the insect, amounts of of mycelia developed, while on the dorsal surface, the hyphae broke through the waxy layer. The whole insect body was covered by a thick layer of mycelia. After penetrating the integument, the hyphae invaded the hemocoele. In the hemocoele, the fungus invaded the hemocytes in blastospores. The blastospores grew rapidly and made vacuole appear in the hemocytes. Besides invading the hemocytes, the fungus also can invade the internal tissues and organs. The muscle fiber, gut, filter chamber, suboesophageal ganglion and tracheae were observed invaded by the hyphae. The hyphae made the muscle fiber become loose, furthermore, muscle fiber and basement membrane separation. The alimentary canal was destroyed with the characteristic of many vacuole appeared. Finally, the insect body was filled with mycelia and new produced conidiophores. The internal tissues and organs were decomposed. The scale insect was dead with its body shrink and became dark and shriveled.