Preliminary Studies On SCAR Molecular Maker And Biocontrol Fungus Of Thrips Hawaiiensis Morgan

Thrips hawaiiensis(Morgan)is one of the most common thrips in several provinces of south China,which may cause great economic loss to the commercial production of fruits,such as mango and banana,and horticulture flowers.It is quite difficult to conduct a trip control due to its little size,cryptic behavior and high reproductive capacity.In order to solve the problems of inspection,quarantine and biological control,a research project on the SCAR molecular marker and biocontrol Metarhizium of T.hawaiiensis was carried out in current work.The study results are summarized as follows.1.The L9(34)orthogonal design method was used in this experiment for the optimization of RAPD-PCR reaction system.The optimized reaction system was involved in a total volume of 20jgL containing 0.2μmmol/L primer,0.2mmol/L dNTP,1.5mmol/L Mg2+,1U Taq DNA polymerase,and 20ng DNA.SCAR analysis on 8 species of thrips was performed based on their PCR production of 9 random primers using the optimal reaction system.A unique band to T.hawaiiensis was amplified by primer OPB-06 and T.hawaiiensis molecular marker was obtained in the test.2.The virulence of thirteen Metarhizium strains were first evaluated.Five strains including Ma1,Ma2,Ma3,Ma6,and Ma8,were chosed while showing higher pathogenicity to T.hawaiiensis adult than any other strains used.Impregnation method with 2×108 spores/1L suspension was employed to deal with T.hawaiiensis,the result indicated that T.hawaiiensis were susceptible to these Metarhizium strains,the cumulative mortality of thrips treated with Ma1 and Ma8 after 7 days of inculation was 100%,and that done with Ma2,Ma3 and Ma6 after 9 days reached 100%.3.The characteristics of five Metarhizium isolates that are highly pathogenic against T.hawaiiensis were determined,including the spore viability,colonial growth rate,conidial yield,as well as the enzyme activity of the protease,lipase,and chitinase.The results showed that the order of spore viability is Ma1>Ma3>Ma8>Ma2>Ma6,with a range from(97.83±0.11)%to(94.70±0.09)%,and the differences have no significant difference(P>0.05).The colonial growth rate of Ma1 was higher than that of any other isolates.Ma1 and Ma6 separately produced the maximum and minimum spores,and the spore yields of these five isolates were(1.67 ± 0.47)x107 spores/mm2 to(2.24±0.35)×107 spores/mm2,without significance in the differences.The order of protease activity is Ma2>Ma6>Ma8>Ma1>Ma3,and the protease activity of Ma3 was significantly less than that of other four isolates.The order of lipase activity is Ma1>Ma2>Ma3>Ma8>Ma6,and their differences were not significant(P>0.05).The order of chitinase activity is Ma6>Ma1>Ma3>Ma8>Ma2,and the differences in the chitinase activity of Ma6 vs that of Ma3,Ma8 vs Ma2,have significant difference,as well as Ma1 vs Ma6.4.The infection of T.hawaiiensis by the Metarhizium isolate Ma was investigated using scanning electron microscope(SEM)and stereo microscope.The infection process of M.anisopliae to T.hawaiiensis is revealed as follows.Firstly,the conidia of M.anisopliae attached to the cuticle surface at different parts of the host.The conidia started germination and produced a short germ tube when the environmental conditions were favorable.As germ tube continued to grow,it could directly penetrate the cuticle,and it could form an appressorium followed by the penetration into the host cuticle.When the entomopathogenic fungus got successfully into the host body,it began to multiply until the host immune system collapse and finally led the host to death.M.anisopliae began to germinate after 8 hours inoculation on T,hawaiiensis adults and the penetration of germ tube into the host cuticle could be seen in 16 hours after inoculation.Large numbers of conidia appeared and caused serious damage to the cuticle within 48 hours postinoculation.By 72 hours postinoculation,the mycelia had already infected all parts of the host body,and the insect body was covered by lots of hyphae and spores.Through observation by stereo microscope,we found that a lot of white hyphae emerged on the insect body that had been kept in high-humidity environments for 1 day,and green spores began to appear in the second day.By the sixth day,the insect body had been surrounded by green spores.