Identification Of Three Strains Of Beauveria Bassiana And Their Pathogenicity To Gonocephalum Mongolicum

Gonocephalum mongolicum is an underground pest that causes severe damage to tobacco plants in Liangshan,Sichuan Province in recent years.So far,control of this insect pest mainly relies on chemical insecticides.However,extensively application of chemical pesticdes is unfriendly with the environment and does not help the improvement of tobacco quality.The use of entomopathogenic fungi for biological control of G.mongolicum meets the demand for green pest control.In this study,three strains of entomopathogenic fungi were isolated from naturally infected G.mongolicum collected in the field.Their taxonomic classification was determined,and their biological characteristics and pathogenicity to G.mongolicum were analyzed.Furthermore,a combination of fungi and insecticides against G.mongolicum was determined in a simulated field environment.Our findings provide a basis for developing biological control technology of G.mongolicum.The results of this study are as follows:1.Isolation and identification of three strains of entomopathogenic fungiThree strains of entomopathogenic fungi were isolated from naturally infected G.mongolicum collected from a tobacco field in Liangshan,Sichuan Province.The three fungal strains were cultured separately on SDAY medium,and the colony morphology,conidial yield and conidial germination rate were determined.The fungal morphology was observed using light microscopy and scanning electron microscopy.By combination with the results of 18S rRNA,β-tubulin sequencing and phylogenetic analysis,all of the three fungal strains were identified as Beauveria bassiana and designated Bb18,Bb19,and Bb20.2.Pathogenicity of three fungal strains on G.mongolicumThe pathogenicity of Bb18,Bb19,and Bb20 was measured against G.mongolicum adults in a suspension at a concentration of 1×10⁸conidia/mL.The results showed that all three fungal strains were pathogenic to the test insects,among which Bb18 was the most pathogenic.At 20 days after the treatment,the corrected mortality rate of Bb18 was83.33%,which was significantly higher than those of other two strains,and the LT₅₀was11.05 d,which was significantly shorter than those of other two strains.Bb20 was less pathogenic,with a corrected mortality rate of 42.19%.The conidia suspensions of Bb18at different concentrations（1×10⁴,1×10⁵,1×10⁶,1×10⁷,and 1×10⁸conidia/mL）were prepared and used to infect G.mongolicum adults.The lethal intermediate concentration(LC₅₀)of the fungus was 1.16×10⁶conidia/mL.3.Symptoms of G.mongolicum infected with Bb18The G.mongolicum adults were treated with Bb18 suspension at a concentration of1×10⁸conidia/mL.After 2 d of treatment,the adults were less vigorous and slow in feeding and crawling.Death of the test insects was observed after 3 d.At 5 d after the treatment,white mycelium appeared on the head,abdominal prothoracic dorsal plate and abdominal end of the insects.After that,a large number of mycelium appeared from the head,prothoracic dorsal plate and intersegmental folds until the whole insects were wrapped,and then the mycelium gradually changed from white to light yellow and produced a large number of conidia.4.Compatibility of Bb18 with matrineMatrine（500-fold dilution）had significant inhibitory effect on the growth of Bb18colonies,while other concentration of matrine（1000-,2000-,3000-,and 4000-fold dilution）did not show significant inhibitory effect on the growth of Bb18 colonies.Among them,the inhibition of Bb18 colonies was only 3.85%after a 15 days treatment with a 1000-fold dilution of matrine.5.Virulence and simulated control efficacy of Bb18 in combination with matrine against G.mongolicumThe bioassay results showed that the corrected mortality of G.mongolicum adults treated with Bb18（1×10⁸spores/mL）or matrine（1000-fold dilution）alone was 70.57%and 62.32%,respectively,and the LT₅₀was 11.05 d and 9.73 d,respectively.While the corrected mortality significantly increased to 90.56%and LT₅₀decreased to 6.13 d when using Bb18 with matrine.Results of the simulated trails showed that the corrected efficacy of Bb18 or matrine alone was 44.83%and 54.02%,respectively,while the corrected efficacy of Bb18 in combination with matrine increased to 76.24%.In addition,the infection rate was 64.51%when using Bb18 alone,while the rate increased to76.25%when Bb18 combined with matrine.These results indicated that matrine was able to improve the infection of Bb18 on G.mongolicum.