The Entomopathogenic Fungus Simplicillium Lanosoniveum Strain TYL001 Isolated From Pseudaulacaspis Pentagona

Pseudaulacaspis pentagona(Targioni-Tozzetti)is a worldwide insect pest on the flower plants and trees.This insect happens 3 generations a year and passes the winter with the fertilized female adult in the north of China.Female adults oviposit under shell in the late April.The first instar nymphs crawl out the shell in May and spread on the trunks and twigs of host plants.As soon as fixed at an appropriate position,the insects begin to suck plant juice with piercing-sucking mouthparts.As feeding,the insects begin to secrete wax by wax glands and gradually form scale or shell.The wax shell is a protective layer for the insect.It is difficult for the chemical reagent to permeate into the wax shell,so that the chemical control is not efficient.In present study,P.pentagona on green ash trees in Taiyuan,Shanxi province is studied.One strain of entomopathogenic fungus is isolated from P.pentagona and a series of pathological researches are conducted.This study result will provide a significant basis for biological control.Research content:1.The development and microscopic morphology of Pseudaulacaspis pentagona in each instar are observed.The slide specimens of each instar are prepared.2.The characteristic observation of naturally diseased P.pentagona,insect carrions collection,isolation and purification of entomopathogenic fungi,infection test,micro and ultra-morphological characteristics observation of the strain TYL001 and species identification,gene sequencing(ITS,the small rRNA subunit and the NADH dehydrogenase subunits 3 of mitochondrial gene),the construction of phylogenetic tree and molecular identification for the strain TYL001.3.The female adult of P.pentagona is inoculated with conidial suspension of the strain TYL001 in five concentrations(1.0×10~6conidia/mL,5.0×10~6conidia/mL,1.0×10~7conidia/mL,5.0×10~7codinia/mL,1.0×10~8 conidia/mL),and the infection rates and IT50 are studied.Entomopathogenic fungus infecting process to P.pentagona and pathological features were observed with optical microscopy,scanning electron and transmission election microscopy techniques.The liquid medium with the integument of P.entagona female adult as the sole carbon for the fungus is prepared,and three extracellular enzymes(lipase,Prl and chitinase)activities of the strain TYL001 infecting the integument of P.pentagona are studied.Research results:1.The shell of P.pentagona female adult is rounded or oval,offwhite,the back uplift with obvious helicoid,and closely attaches to tree bast.The female adult is wide pear shape or short egg shape,the color from pale yellow to orangish.The body is about 1 mm in length.The antennae,legs and eye are degenerated.The body surface has various glands and setae.When the female adult oviposits,perivulvar glands secrete wax to protect eggs from adhesion each other.After oviposition,the bodies shorten to the head,dry and die.The eggs are elliptic,pink and orangish.The first instar nymph is orangish,flat oval.The eye,antennae,legs and tail filaments can be clearly observed by light microscope.The antenna has 6 segments,two glands pores present middle of two eyes,and two tail filaments are located at the end of abdomen.The antennae,legs,eyes and tail filaments of the second instar nymph are degenerated,the morphology is similar to the female adult,but,perivulvar glands are not formed.2.Some populations of P.pentagona are collected and the naturally diseased individuals are found.The body colors are different with the naturally infected degrees,the color is pale,tan and brown in the early,medium,and last stage of pathopoiesia,respectively.Hyphae cover the body surface.The insect body has obvious body fluid in the early and middle stage,but the body becomes dry in the last stage.The spores are directly picked from the naturally diseased P.pentagona by using inoculation loops,and are inoculated on the plate culture medium.After several isolations and purifications,one strain TYL001 of entomopathogenic fungus with white hyphae is got.The surface of body appeared the same symptoms as the naturally diseased scales by re-inoculation test by conidial suspension of the strain TYL001 in the concentration 1.0×10~8conidia/mL.It shows that TYL001 is the entomopathogenic fungus that could lead P.pentagona to disease and death.The morphological characteristics of the strain TYL001:The sporophores of the strain TYL001 are borm on the hyphae,and are mostly single,some in pairs,and very few in whorls.When the conidia newly grow,balls of conidia are born on the sporephoers,but after mature,conidia are in single.On the basis of morphological characteristics,the strain is identified to be belonged to the family Cordycipitaceae.The gene sequencing and molecular identification of TYL001:The comparative results of three sequences in NCBI Blast on the web are as follows,the similarity rate of ITS and Simplicillium sp.(sequence number:FJ948132)is 99%,the small rRNA subunit(rns)and Simplicillium lanosoniveum(sequence number:EF513006)are similar to the rate of 97%,and the NADH dehydrogenase subunits 3(nad3)and Simplicillium lanosoniveum(sequence number:EF512887)are similar to the rate of 92%.By combining the morphological characteristics with molecular identification results,the strain TYL001 is determined as the species Simplicillium lanosoniveum.3.After inoculated by conidial suspension in the five concentrations(1.0×10~6conidia/mL,5.0×10~6conidia/mL,1.0×10~7conidia/mL,5.0×10~7 codinia/mL,1.0×10~8 conidia/mL),the infection rates of P.pentagona are 80%,83.33%,86.67%,90%,and 93.33%,respectively.At the same time,the median infection times(IT50)are 10~1.1 h,95.0 h,94.2 h,85.5 h and 82.5 h,respectively.Infection process and pathology:After infected by conidial suspension of TYL001(1.0×10~8 conidia/mL),the mouthparts,dorsal tubes,vulva,perivulvar glands,anus and pygidial intersegmental folds of P.pentagona are easily attached by conidia.The conidia germinate to germ tubes and hyphae,they are able to intrude into the insect body through organs that interlinked with the outside,such as mouthparts,dorsal tubes,vulva,perivulvar glands,and anus.Also,the hyphae could penetrate integument with the mechanical pressure and extracellular enzymes by themselves.After entering into the insect hemocoel,the hyphae infect hemocytes,muscle tissue,etc.and largely grow and breed.When the nutrition in the insect hemocoel is used up,the hyphae break through integument,and spread to outside,and the insect body is covered by hyphae.The strain TYL001 is cultured in the liquid medium with the insect integument for 6 days.The activities of three extracellular enzymes of the strain TYL001 show a trend of rising up at first then draping down.The activity peaks of lipase,Prl and chitinase appeared at the second day,the third day and the fourth day,respectively.