| Pear,the third most important temperate fruit species after apple and grape,is the perennial woody plant belonging to the subfamily Pomoideae in the family Rosaceae.Pear industry,as one of the most important fruit industries in China,plays an important role in agricultural economy.During the growth,the storage and transportation of fruits after harvest,pear is vulnerable to pathogens of pear anthracnose,which poses serious threats to the increase of pear yield and the healthy development of pear industry in China.In recent years,there are two different types of symptoms existed in the major pear producing areas in the south of the Yangtze River Basin.TypeⅠis typical which could produce large disease spots and cause fruits rotten;TypeⅡproduces black spots and abnormal early defoliation which leads to the secondary flowering which resulting in the excessive nutrition consumption of trees and deterioration of fruit quality,and cause serious economic losses to the local pear industry finally.At present,the control of pear anthracnose mainly depends on chemical control methods in pear producing areas in China.However,there are some problems in chemical control,such as pesticide residues,unfriendliness to the environment and drug resistance of pathogens.Biological control has good environmental compatibility and it is not easy to induce drug resistance of pathogens,which is in line with the development concept of green plant protection.It can overcome many disadvantages of chemical pesticides and help to increase crop yield.Therefore,it is of great significance to explore the causes of two different field symptoms of pear anthracnose and screen out the biocontrol bacteria with high antagonistic effect on pear anthracnose for analyzing the pathogenesis of pear anthracnose and promoting the healthy and sustainable development of pear industry.In this study,both of two pathogenic strains NC40 and FJL causing different symptoms were confirmed to be Colletotrichum fructicola by the phylogenetic analysis.We conducted a series of comparative studies on the biological characteristics of strains NC40and FJL:（1）There was no significant difference in colony morphology and growth rate between the two strains.Their colonies were white and round on the PDA medium.（2）The conidia yield of NC40 was much higher than that of FJL.（3）In terms of sexual reproduction,the black granulated raised ascomata were produced on the OA medium after induction,which were buried or half buried in the medium.（4）There was no obvious difference in tolerance to H2O2and HSAF.（5）Conidia could germinate appressoria producing melanin precipitation on onion inner epidermis and hydrophobic slides,and there was no significant difference in appressorium morphology.（6）There were significant differences in the expansion ability of invasive hyphae between strains NC40 and FJL.The appressoria produced by NC40 could penetrate the inner epidermis of onion to form expanded invasive hyphae and colonize between adjacent cells.Most of the appressoria produced by FJL could not penetrate the inner epidermis of onion,even a few of the appressoria that penetrated the inner epidermis of onion could not colonize between adjacent cells,and the morphological characteristics of appressoria are abnormal.Biocontrol strains CL01 and CL02 were isolated from the rhizosphere soil of healthy pear trees in Jurong,Jiangsu Province.Based on biological characteristics,physiological and biochemical characteristics and molecular phylogenetic analysis,the strain CL01 was identified as Paenibacillus ottowii,the strain CL02 was identified as Paenibacillus terrae.We studied the biological characteristics and antifungal mechanism of strains CL01 and CL02,then obtained the following results:（1）CL01 and CL02 were in the logarithmic growth phase during 12-24 h,which was the best period to study their characters.The optimal inoculation amount of CL01 and CL02 was 0.5%and 1%,respectively.The optimal growth p H of CL01 was 6,and that of CL02 was 7.（2）Extracellular enzymes activity detection showed that both CL01 and CL02 could produce cellulase and protease,but not chitinase.（3）Through the determination of antimicrobial spectrum,we found that strains CL01 and CL02 both had good inhibitory effects on Colletotrichum fructicola and three important pathogens of pear,namely,Valsa Mali var.Pyri,Botryosphaeria dothidea and Alternaria alternata.Besides,CL01 was a biocontrol bacterium with broad-spectrum antifungal activity which had strong antagonistic effect on the mycelial growth of other common pathogens of crops,such as Rhizoctonia solani,Phytophthora infestans,Botrytis cinerea and Sclerotinia scleotiorum.（4）With C.fructicola NC40 as the target,it was confirmed that strains CL01 and CL02 had obvious inhibitory effect on the mycelial growth of C.fructicola,and the inhibition bandwidth was（8.2±0.4）mm and（6.6±0.5）mm,respectively.Under the scanning electron microscope,the mycelia of C.fructicola treated with CL01 were shrunk and sunken,and the integrity of the cell walls were destroyed.While CL02 had a strong adhesion ability and could adhere to the surrounding mycelia,resulting in mycelia shrinkage and deformity.（5）In the conidial germination inhibition experiment of C.fructicola,the conidial germination rate of CK and Escherichia coli control group reached more than 80%after co-culture for 8 h,and that of CL01 or CL02treatment group was not higher than 55%,respectively.（6）We extracted the antifungal active substances from CL01 and CL02 fermentation broth by n-butanol extraction,ethyl acetate extraction and ammonium sulfate saturated precipitation,then tested on PDA medium.The results showed that the antifungal active substances produced by CL01 and CL02 existed in the organic phase of n-butanol and ethyl acetate,respectively.Through indoor control effect test,we found that CL01 and CL02 fermentation broth,and their crude extracts could effectively inhibit the infection and expansion of C.fructicola on the fruits,and the inhibitory activity of their crude extracts was more significant. |
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