Isolation Of Entomogenous Fungi And Screening Of Pathogenic Isolates For Biological Control Of Colorado Potato Beetle

Colorado potato beetle [CPB, Leptinotarsa decemlineata (Say)] is an important invasive alien species and quarantine pest and it is now distributed in northern Xinjiang Autonomous Region of China.The objectives of the present work were to isolate entomogenous fungi from diseased samples of CPB adults and to screen highly pathogenic isolates for future applications in biological congtrol of the CPB pest.Isolation of CPB pathogens:Diseased samples of CPB adults were collected from different venues of northern Xinjiang and 46 fungal isolates were obtained using the conventional tissue culture techniques in the laboratory.Initial morphological identification showed that these fungi belonged, respectively, to Beauveria, Verticillium, Fusarium, Penicillim, Trichoderma and Aspergillus genera.Pathogenicity tests on silkworms:2nd-instar larvae of Bombyx mori Linnaeus were inoculated with fungal suspension. The results showed that the 36 isolates were pathogenic to silkworms including 30 isolates of Beauveria sp.,3 isolates of Verticillium sp.,2 isolates of Fusarium sp. and 1 isolate of Trichoderma sp.The pathogenicity of Beauveria isolate MJ-07 was the strongest; the cumulative mortality rate reached 95.6% 11d after inocultion and the mid lethal time (LT50) was 7.5d; the inoculation with Verticillium MJ-20 cumulative mortality and LT50, respectively 61.5% and 9.1d; Fusarium MJ-27 and Trichoderma MJ-32 would be weak pathogenicity, after the 11d of the cumulative mortality rate was only 8.6-11.0%. The test results to determine the pathogenicity of CPB had a foundation for the areas concerned but also for future control of CPB in the application of fungus when taken to ensure the safety.Pathogenicity tests on CPB: A number of 36 fungal isolates were obtained from diseased adults of CPB, collected from Kashi, Akeshu and Artai of Xinjiang Autonomous Region of China, and they were morphologically identified as the species of Beauveria (30 isolates), Verticillium (3 isolates), Fusarium (2 isolates) and Trichoderma (1 isolate), respectively. Results from inoculation of CPB 2nd-instar larvae with spore suspension of 1.1×109 mL-1 showed that all the Verticillium, Fusarium and Trichoderma isolates had only relatively low pathogenicity to CPB. The cumulative lethal rates of these isolates after 9 days of inoculation (LR9d) were only 4.3%-11.3%. Most of the Beauveria isolates were shown much higher pathogenic than other tested fungi. Amongst Beaveria isolates,5 (MJ-01, MJ-03, MJ-07, MJ-08 and MJ-09) were highly pathogenic to CPB larvae and the death of larvae was first observed only 5 days after inoculation whilst the LR9d were 82.3%-93.3% and the mid lethal time (LT50)and mid lethal contrations(LC50) were 6.71-7.53d and 4.53x106-5.89x107 mL-1, respectively. The most virulent two isolates of Bearveria (MJ-07 and MJ-09) were further tested agianst CPB adult and 1st-4th instar larvae. MJ-07 was shown very pathogenic to 1st-,2nd instar larvae and adults with LR9d of 100%,93.3% and 98.0%, but much less virulent to 3rd- and 4th-instar larvae with LR9d of 70.0%, and 43.3%. The bodies of MJ-07 killed CPB became contracted and were completely wraped by the properously produced mycelia and conidiospores by the fungus. MJ-09 was demonstrated highy pathogenic to 1st- and 2nd instar larvae with LR9d of 100% and 90.0%, but significanly less virulent to 3rd-,4th-instar larvae and adults with LR9d of only 63.3%,33.0% and 13.3%. The bodies of MJ-09-killed CPB became dark-brown and were partially covered by the less properous mycelia and conidia of the fungus. It can be concluded that the Beauveria isolates MJ-07 and MJ-09 are pathogens of CPB with high virulence and may be potential candidates for using to develop mycopesticide against CPB.Greenhouse and field tests of MJ-07 and MJ-09 against CPB: The two most pathogenic isolates of Beauveria (MJ-07 and MJ-09) were applied to control CPB and field the results indicated that CPB was effectively controlled by spraying spore suspensions of both MJ-07 and MJ-09. In the greenhouse tests, the lethal rates of 1st and 2nd instar larvae reached more than 62% at 15d after treatment. In the field, CPB lethal rates of about 40% were recorded 15d after separate sprays of the two isolates on the 1st and 2nd-instar larvae. The control efficacy was significantly higher than that of a commonly used Beaveria bassiana pesticide (control,30%).Morphological and molecular identification of MJ-07 and MJ-09:On SDAY medium plates, the colonies were downy, whiteor yellowish wite. Aerial hyphae transparent, thin-walled, branches were thin and slender, width 1.8-2.8μm; conidia were produced on the conidiophores (sized 1-2μm in diameter). They were single-spored with thin walls, transparent, smooth, mostly spherical, sized 2-3 x 2-2.5μm (in diameter).The rDNA ITS sequences were obtained from the genomic DNAs of MJ-07 and MJ09 through polymerased chain reaction (PCR) with the general fungal primer pair ITS4 and ITS5. These sequences were registered in Genbank with accesion numbers of GU233851 and GU989641, respectively, and they shared maximum identities of 98%-100% with those from isolates of Cordyceps bassiana existed in Genbank.Therefore, MJ-07 and MJ-09 were identified as Cordyceps bassiana based on the about morphological and molecular descriptions and the asexual stage of the fungus was Beaveria bassiana.Cultural conditions of Beauveria isolates:For the two B.bassiana, MJ-07 and MJ-09, the best medium was SDAY. The most suitable temperature for its colony growth was 25℃.The optimum germination temperature of 25-28℃. The fungual isolates grew normally on media with glucose, sucrose, tryptone and yeast extract sources. Light treatment had positive effect on fungal sporulation. Conidial lethal conditions for 55℃treatment 10min. UV radiation had negative effect on spore germination and after treatment of 60min-UV irradiation for 24h there was no spore germination.The conidia germinated best on medium plate with 2% glucose+1% peptone medium.The five insecticides commonly used for CPB control were tested to show their effect on MJ-07 and MJ-09. Results showed that lambda-cyhalothrin and imidacloprid had minimum reverse effect on the mycelial growth and spore germination of the isolates while the other three insecticides (methomyl, emamectin benzoate and acetamiprid) significantly suppressed mycelial growth and spore germination of the two isolates.Entomogenous fungi as a class of valuable biological resources are widely distributed in natural fields or environment. With the rapid development of science and technology entomogenous fungi will be given greater attention in crop pest management. The two isolates of Beaveria bassiana had been shown potentially valuable in producing mycoinsecticides and in future CPB control.