Colonization Of Beauveria Bassiana And Metarhizium Anisopliae In Plant Rhizosphere And Its Inhibition On Some Soii-borne Phytopathogenic Fungi

Entomogenous fungi are an important regulator of insect populations and they spreadwidely in agro-ecosystems and play an important role for biological control of crop,orchard, forest and medical insects. Metarhizium anisopliae and Beauveria bassiana aretwo most important entomogenous fungi, which has a wide range of insect hosts and hasbeen widely used for fungal insecticides. In this paper, some isolates from maizerhizosphere soil collected from Guoyang, Xiaoxian and Mengcheng, north Anhui wereidentified as B. bassiana by both morphological method and rDNA ITS sequenceanalysis. They and some M. anisopliae isolates were tested for their sensitivity tocarbendazim, the colonization ability in plant rhizosphere, the inhibition on soil-borneplant pathogenic fungi, and the effects of different carbon and nitrogen sources on theirbiological characteristics and anti-fungal activity. The results were as follows:1. With selective medium, thirty-nine strains of Beauveria were isolated from maizerhizosphere soil, collected from Counties of Guoyang, Xiaoxian, and Mengcheng in thenorth of Anhui province. The strains were classified and identified as B. bassiana by bothmorphological and molecular （rDNA ITS sequence analysis） means. The colony diameterand conidia number of different strains were tested in vitro and showed significantdifference.2. The genetic diversity of thirty-seven B. bassiana strains isolated from maizerhizosphere was estimated using inter-simple sequence repeats （ISSR） markers. Elevenamong forty ISSR primers were chosen for their reproducibility and high polymorphism.Each primer resulted in7.5markers and totally83fragments were amplified, in which69（83.13%） were polymorphic. Genetic diversity analysis revealed a relatively high level ofintraspecific genetic diversity of B. bassiana. The percentage of polymorphic loci （PPL）was83.13﹪, Nei’s genetic diversity （H） was0.3169and Shannon’s information index （I）was0.4657. It suggested that the thirty-seven B. bassiana strains had highly geneticdiversity.3. The sensitivities of B. bassiana and M. anisopliae to carbendazim were studiedby mycelial growth inhibition rate method. The results showed that the sensitivities ofB. bassiana and M. anisopliae to carbendazim had some differences. Among the testedstrains of B. bassiana, the EC₅₀value of GY^-17was the highest, but that of GY-6wasthe lowest. Among the tested strains of M. anisopliae, the EC₅₀value of Ma55was thelowest, followed by Ma27, while the EC₅₀value （4.05） of Ma10was the highest. 4. The resistant level of the carbendazim-resistant strains of B. bassiana and M.anisopliae were studied, by mycelium and conidium oriented screening. The resultsshowed that the resistant level of BC-4was the highest among thecarbendazim-resistant strains of B. bassiana, with EC₅₀and the resistant level at258.77and242.11, respectively. The resistance level of BC-3was the lowest amongthe carbendazim-resistant strains of B. bassiana, with EC₅₀at18.63. The resistantlevel of MC-2was the highest among the carbendazim-resistant strains of M.anisopliae, with EC₅₀and resistant level was397.06and102.35, respectively. TheEC₅₀of MC-4was the smallest （149.54）. The mycelia growth rate and sporulationability of the carbendazim-resistant mutants were studied too. It showed that themycelium growth rate and sporulation capacity had some differences among differentcarbendazim-resistant mutants. The growth rate of resistant mutants was smaller thanthat of the control （parent strain）, but sporulation ability was stronger than that of thecontrol （susceptible strain）, and different strains had some differences. Among thecarbendazim-resistant strains, the sporulation of BC-4mutant and MC-2mutant wasthe highest.5. The colonization ability and the influence on rhizosphere microorganisms of thecarbendazim-resistant mutants BC-8and MC-2in the plant rhizosphere were studied bymeans of dipping in conidial suspension. BC-8and MC-2were detected from outerrhizosphere and root surface of maize and cotton, but not from inside the roots. Itsuggested that BC-8strain and MC-2strain had a certain influence on the number of plantrhizosphere microorganisms, especially at the beginning, but the effects decreased withtime.6. Under laboratory conditions, the inhibition of M. anisopliae and B. bassianaagainst Fusarium oxysporium f. sp. vasinfectum and other soil-borne plant pathogenicfungi, and its mechanism were studied in vitro by dual culture, cup-plate test andmicroscopic examination. The results of dual-culture test showed that among three M.anisopliae isolates and four B. bassiana isolates which all displayed obvious inhibition onthe tested soil-borne plant pathogenic fungi, Ma55and Bb2092resulted in the highestinhibition. On the PDA plates mixed with different concentrations of conidial suspensionof Ma55and Bb2092, the inhibition rate by the suspension of106conidia per milliliterswas the highest to all tested fungi. The inhibition on mycelia growth of the tested fungi bysterile fermented broth of M. anisopliae and B. bassiana shake culture was obvious, andthose to production and germination of their conidia （sclerotia） were also significant. No hyperparasitism of M. anisolpiae and B. bassiana on the hyphae of the tested fungi wasobserved microscopically. All these evidences suggested that the inhibitory mechanisms ofM. anisopliae and B. bassiana against the pathogen of soil-borne plant diseases weremainly competition to nutrition, space and antibiosis.7. The biological characteristics of B. bassiana and M. anisopliae were compared indifferent carbon sources and nitrogen sources by measuring the mycelium growth, drymycelium biomass and conidia yield. The results showed that B. bassiana and M.anisopliae could use many carbohydrate and nitrogen sources for the mycelia growth. Bymeans of mycelium growth rate, the inhibitory rates of metabolic liquid cultured withdifferent carbon sources and nitrogen sources were tested. The anti-fungal assay of thesecondary metabolites of B. bassiana showed that the metabolic liquid cultured withdifferent carbon sources and nitrogen sources had significant difference on the5testedstrains of Fusarium. Among the metabolic liquid, the inhibitory rates of those withglycerol and ammonium sulfate as carbon and nitrogen source were the highest and thosewith lactose and urea as carbon and nitrogen source were the lowest. The anti-fungal assayof the secondary metabolites of M. anisopliae revealed that secondary metabolitesproduced in the culture with sucrose as the carbon source and/or the peptone as thenitrogen sources was the highest.