Identification Of Pathogen Causing Mango Dew Blotch Disease And Screening Of Its Antagonistic Microorganisms

In recent years, mango dew blotch disease occurs in Hainan, Guangdong, Guangxi,Yunnan, and other major mango producing areas of China. The symptom appears as a irregular dark, clouded stain on peel in the early period. The whole mango is covered by blots in the later stage of the disease, which seriously affects the appearance of the mango fruits and causes great lose of commodity value. This paper presents the screening and identification of pathogen causing mango dew blotch disease and screening of its antagonistic microorganisms. The Cladosporium cladosporioides was thought to be the major pathogen. The antagonistic Yeast Y-l and antagonistic Bacillus TE-7 were screened out, which had great practical signification in both research and production. The influence on fruit quality of mango and the antibacterial mechanism were discussed. The results were summarized as follows:(1) Scanning electron microscopes showed that a large amount of mycelium attached to the diseased peel, which colonized in the wounds and lenticels of mango. And the number of hyphae increased with the severity of disease.The pathogen was isolated from the diseased mango by tissue isolating method, and was identified as Cladosporium cladosporioides according to the pathogenicity, morphology and ITS sequence analysis.The pathogenicity of strain J1 reached 86.6%, which was determined by Koch’s postulates.(2) A total of 56 strains were isolated from the rhizosphere soil of mango trees in Changjiang Hainan province, and 9 antagonistic strains of Cladosporium sp. were screened by the method of plate confrontation. The strains JT-16, Y-1 and TE-7 had the most antagonistic effects to Cladosporium cladosporioides, and results showed that the inhibition zone diameters were 15.17 mm, 14.93 mm and 21.56 mm. Based on morphological, physiological and biochemical characteristics, and molecular biological methods, JT-16, Y-1 and TE-7 were identified as Burkholderia gladioli, Debaryomyces hansenii, and Bacillus atrophaeus.(3) After soaked with different concentrations of antagonistic strain suspension for 10 minutes, treated mangoes had better maintaining of the fruit chroma a* values, hardness,titratable acid, ascorbic acid and soluble solids content compared with the control.(4) The possibility of the antagonistic strain induced resistance was investigated in mango fruits. The results showed that Y-1 and TE-7 could enhance the activities of the defense related enzymes including polyphenol oxidase, peroxidase, phenylalnine ammonialyase, chitinase and β-1,3-glucanase in mango fruits. At the same time,Cladosporium cladosporioides had positive effects on the induction effect.(5) The volatile compounds produced by Y-1 and TE-7 could delay the germination of the spores and lead to the mycelium swelling, folding, and losing the ability of normal growth. But this kind of volatile antibacterial substance can inhibit the pathogen in a short time. The study found that the fermentation filtrate of strain TE-7 had strong antagonistieffects on the pathogenic. The antibacterial substance reached the largest accumulation after the strain TE-7 cultured at 30℃ with 200 r/min in NB medium for 60 hours.(6) The crude active substance was extracted from the Bacillus atrophaeus TE-7 culture by 6 mol/L HC1 precipitation. The crude extract had a strong inhibitory effect on the C.cladosporioides. The crude extract was heat-stable, which still maintained antibacterial activity after 100 ℃ treatment for 30 minutes. And the crude extract maintained a higher antibacterial activity in acidic and alkaline conditions. The antibacterial substance was also stable to ultraviolet rays. And it has good solubility in organic solvents such as methanol, ethanol, acetone and acetonitrile.(7) The morphological abnormalities were observed in the spores and hyphae of C.cladosporioides after treatmented 12 h by the crude extract of TE-7. The spores appeared swelling, deformation, plasma membrane folding and contraction, cell membrane degradation, loss of germination ability. And The hyphae appeared swelling or shrinking,twisting and folding,adhesion,disorder, and loss of normal morphology.(8) The freeze-dried crude extract from the fermentation broth of TE-7 was analyzed by FT-IR. And the results showed that the main component of crude extract was lipopeptide. The lipopeptide substrates in crude extract were separated by reversed-phase high performance liquid chromatography (HPLC). The results showed there were surfactin and iturin A in crude extract according to the same peaks of standard surfactin and inturin A.