| Two-spotted spider mite,Tetranychus urticae,had been developed as a key pest in greenhouse and field crops worldwide at the present.Chemical pesticides were long-term used in the control of spider mite,some negative effect,such as development of pesticide resistance of pest mites,reducing of predators,increase of environmental pollution and health damage,have led to public concerns about environmental impact and safety of chemical applications and stimulated agricultural practitioners to change their pest management strategies.Microbial pesticides may be a solution to the issue of the excessive use of chemical pesticides and could play an important role in integrated pest management(IPM).Many insect pathogens have been investigated as potential agents for mite control,and insect/mite-related fungi were regarded as most potential agents for managing pests.In the present dissertation,the Yeast-like symbionts(YLS)of two-spotted spider mite and the araneogenous fungi being new resource for the mite control were studied in order to have basic data for the green control of two-spotted spider mite.The results were as follows:1.The YLS existed in two-spotted spider mite.The primer NS1-NS2 was selected for the follow-up work based on YLS primers which were used in the previous studies.According to the phylogenetic tree of YLS sequences obtained using the primer NS1-NS2,the sequences were clustered in a separate clade with a high bootstrap(100/99/98),and had closer relationship with the fungal pathogen Hypomyces chrysospermus.The phylogenetic position analysis revealed that the YLS from two-spotted spider mite were clustered with the entomoptahogenic fungi Paecilomyces tenuipes(Isaria tenuipes),Beauveria bassiana and B.brongniartii.All those fungi belong to Pezizomycotina,and also supported by phylogenetic network analysis.Two morphological types of YLS were observed in two-spotted spider mite,one was cylindrical to fusiform,and another was ellipsoidal to globose in shape.2.The YLS from different mite species were significantly different.The YLS of seven mite species,Oligonychus sp.,Gamasiphis sp.,Nothrus sp.,Cosmolaelaps sp.,Allothrombium sp.,Hygrobates sp.and T.urticae,belong to Pezizomycotina and have a closer relationship with reported YLS in insect.The base compositions were different among the YLS of seven mite species.The adenine was generally the highest base except for Nothrus sp.,from which the highest base was guanine.The content of G+C was also different among the YLS from the seven mite species.The highest and the lowest G+C were observed in YLS from Hygrobates sp.and T.urticae,and the G+C average values were 51.4% and 43.4%,respectively.There were closer relationship among the YLS of Gamasiphis sp.,Nothrus sp.and Allothrombium sp.The genetic distance within groups and among groups revealed that both of them were different.The highest genetic distance,314.333,was determined from YLS of T.urticae within the group.3.The YLS of two-spotted spider mite populations on different host plant species were significantly different.The YLS of nine two-spotted spider mite populations fed on nine plant species were different in base composition.The plants are Vigna unguiculata cylindrical,Ipomoea batatas,Raphanus sativus,Gossypium arboretum,Solanum tuberosum,Lycopersicon esculentum,Brassica chinensis,Vigna unguiculata and Cucurbita pepo.Generally,thymine content was higher in YLS of the mite populations except those of B.chinensis and G.arboreum,with highest adenine.The content of G+C was in the range of 45.1% to 45.5% in YLS of the mite populations on V.unguiculata cylindrical,S.tuberosum,R.sativus,V.unguiculata,B.chinensis and C.pepo.The highest G+C content was of the YLS of mites on L.esculentum,I.batatas and the lowest content was of that on G.arboreum,respectively.The highest genetic distance,reached on 110.833,within groups was detected from YLS of the mites on L.esculentum,I.batatas and G.arboreum.The highest genetic distance between groups,reached to 132.750,was determined from YLS of the mites on L.esculentum,I.batatas and G.arboreum.There were 22 haplotypes and divided into 5 clades,which suggested that there were at least 4 to 5 different YLS types in two-spotted spider mite.4.Five fungal new species with the first report of araneogenous ones in genera Beauveria and Clonastachys were identified.There were 11 strains isolated from 25 spider and insect specimen infected by fungi in Guiyang,Guizhou Province.Through molecular and morphologic identification,10 strains were identified as 5 new species and 1 new host isolate of a known species,i.e.: Isaria albocitrina sp.nov.,DX1,DX2,QLS1,QLS2,SN1;Lecanicillium araneogenum sp.nov.,GZU1032Lea;B.araneola sp.nov.,GZU0317bea;C.aranearum sp.nov.,QLS0625 clo and Metarhizium dendrolimatilis sp.nov.,IFR1006.I.farinosa,newly recorded from spider,DX3.Another strain,GZU12141,was Beauveria sp.It is worth mentioning that the strains of Beauveria and Clonostachys were firstly recorded as araneogenous fungi in the tow genera.5.The pathogenicity of araneogenous strains to two-spotted spider mite was proved.T.urticae were susceptible,in dose-dependent manner,to all 11 isolates used in the bioassay.Strains GZU0317 bea and GZU1032 Lea caused the maximum mortalities,95.2% and 95%,and the lowest mortality,80%,was observed with strain QLS1 at a concentration of 1 × 108 conidiaˇmL-1.The results of alcalase activity tests revealed that strain GZU12141 showed the highest activity,32.38 U/mL,and strain DX2 the lowest activity,5.29 U/mL.Therefore,the alcalase activity could be treated as screening indicator for selecting araneogenous fungus with high pathogenicity to T.urticae.6.The enzymes of araneogenous fungi associated with infection were verified.The activity of alcalase,lipase and chitinase of the fungal cultures were obviously changed during 7 d when the strain B.araneola GZU0317 bea was cultivated in the medium containing the powder of T.urticae.The highest activity of alcalase,lipase and chitinase appeared in 2 d,6 d and 7 d,respectively.The three enzymes were produced in the order of alcalase,lipase,and chitinase in the process of GZU0317 bea infecting two-spotted spider mite.7.The detoxification and protection enzymes of two-spotted spider mite were activated with infecting of araneogenous fungi.The enzyme system of T.urticae responded quickly to infecting of the two araneogenous fungus,GZU12141 and GZU0317 bea.The activity of three detoxification enzymes and protection enzymes except for SOD were significantly increased.The highest activity of CarE,Gst,CAT,SOD,POD and PPO,which induced by the strain GZU12141,occurred and reached to 1.34 U/mL,20.316 U/mL,14.916 U/mL,14.88 U/mL,0.015 U/mL,6.7U/mL in 5 d,4 d,2 d,2 d,4 d,3 d,respectively.The activity increases of the enzymes caused by the strain GZU0317 bea were similar to the strain GZU12141.The enzymes playing major role in protecting T.urticae from fungal infection were Gst of detoxification enzyme system and CAT of protection enzyme system.In general,the YLS is confirmed in the two-spotted spider mite,and may be different among mite species and even vary among populations of the same mite species feeding on different plants.The new species of Beauveria,Clonastachys and other genera are valuable new araneogenous fungus resources for pest mite control.The strains with highest pathogenicity to the two-spotted spider mite are detected with infecting proofs.The activity performances of infection-related enzymes in the fungi and defence-related enzymes in the mite are illustrated.The results have expanded entomopathogenic fungi resource with their potential use in the pest mite control,which could be contribution to the theoretical innovation in green control to the pest mite,and have practical significance in exploitation of fungal biopesticide against the pest. |
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