| Mulberry is an important economic crop,which the roots,stems,leaves and fruits are of great economic value.Because mulberry leaf is the best natural food for silkworm(Bombyx mori),it has brought huge income for sericulture industry.However,the occurrence of mulberry diseases has seriously restricted the development of the sericulture industry.A new mulberry disease named mulberry shot-hole disease was reported in recent years,and it was noticed to significantly reduce both yield and the quality of the mulberry leaves,as well as affect growth of mulberry tree.The plant shot-hole disease is mainly caused by pathogenic microorganisms,including fungus,bacterial,and virus.The typical symptom of this disease is the shot hole occurred on diseased leaf which was shedding in the end.Besides,the branches and fruits also could be affected by the disease.Although mulberry shot-hole disease has been reported in 2008,the authors only described the situation and the symptom of the disease,but not determined the pathogen.Therefore,the pathogen of mulberry shot-hole disease is not clear so far.In August of 2015,a suspected shot hole disease on mulberry leaf was broke out in Santai county,Sichuan province,which was accompanied with a large numbers of tiny-pests.The complex ecological phenomenon makes the pathogen of mulberry shot-hole disease more difficult to be identified.We have recorded the environmental condition of the mulberry garden in which the shot hole disease outbreaks in Santai County,and we have also collected the disease leaves for further analysis.The microorganism on disease leaves were isolated and identified.The tiny-pests which distributed on the disease leaf surface were also identified and counted.According to the Koch's postulates,the suspected pathogens of the isolates were inoculated to healthy mulberry leaves to verify the pathogenicity via pathogenicity rat calculation and symptom comparison.After that,the pathogen was re-isolated from diseased leaf,and both 16 S rDNA or ITS rDNA sequence and morphological characteristics were compared with the original strain to fulfill Koch's postulates.The hyper-virulent strain which caused the highest rate of shot hole on mulberry leaves was selected to investigate the biological characteristics.The factors that affect mycelial growth were investigated,such as temperature,pH value,light conditions and minimum lethal temperature.The main contents and results of this research are as follows: 1.Investigation of microorganisms and tiny pests on mulberry leaves with shot-hole diseaseAccording to the normal method of isolating plant pathogenic microorganisms,five bacteria strains and six fungal strains were obtained from the mulberry leaves with shot-hole disease.All the isolates were identified combine with phylogenetic analysis by using 16 S rDNA or ITS rDNA sequences and morphological characteristics.The isolated bacterial strains were identified and named as Pantoea agglomerans MB01,Pantoea ananatis MB02,Pseudomonas oryzihabitans MB03,Pseudomonas argentinensis MB04 and Acinetobacter calcoaceticus MB05,respectively.The fungal strains were identified and named as: Nigrospora sp.MF01,Nigrospore oryzae MF02,Nigrospora sphaerica MF03,Phanerochaete sordid MF04,Phomopsis sp.MF05 and Epicoccum nigrum MF06,respectively.In addition,there is a large numbers of tiny-pests distribute on the disease mulberry leaf surface which were recorded and initially identified as Pseudodendrothrips mori,Pealius mori,Spider mite and chrysomeloidea sp.,respectively.2.Pathogenicity tests of suspected pathogensAccording to references,2 bacteria strains(Pantoea agglomerans MB01 and Pantoea ananatis MB02)and 3 fungal strains(Nigrospora sphaerica MF03,Phomopsis sp.MF05 and Epicoccum nigrum MF06)were determined as suspected pathogens of shot-hole disease.On the basis of the Koch's postulates,the suspected pathogens were inoculated to the healthy mulberry leaves to verify the pathogenicity.After two-week continuous observation,we found that the bacterial isolates could not cause shot hole disease of mulberry leaf.To verify the pathogenicity of fungal strains,mulberry leaves were pretreated as wound and unwound.In unwounded treatment all three fungal strains could infect mulberry leaves and cause very low probability of the lesion,but could not produce shot hole symptom.In wounded treatment,all three fungal strains could infect mulberry leaves and cause very high probability of the lesion,and all the strains could produce typical shot hole symptom.Among these strains,the most virulent strain was Nigrospora sphaerica MF03 which caused a very high shot hole rate after inoculated to mulberry leaves whether in summer or autumn.The other two fungal strains Phomopsis sp.MF05 and Epicoccum nigrum MF06 which were inoculated to mulberry leaves could also cause a very high rate of shot hole and lesion on mulberry leaves in summer,but cannot cause shot hole symptom and only cause a very low probability of lesion in the autumn.The leaves with typical shot hole were collected to re-isolate the dominant strains for further re-identification.The ITS rDNA sequences and morphological characteristics of the re-isolated strains were compared with the original inoculated strains,and results were matched each other that fulfill Koch's postulates.3.The biological characteristics of the pathogenic fungi Nigrospora sphaerica MF03According to the results of pathogenicity test,the hyper-virulent fungal strain Nigrospora sphaerica MF03 was selected to investigate the biological characteristics which include the growth cycle of the strain and the factors affecting growth,such as temperature,pH value,light conditions and minimum lethal temperature.The sexual reproduction cycle of strain Nigrospora sphaerica MF03 was not be observed in laboratory,and the strain were propagated by spores and hyphae.The tolerance ranges of mycelium to growth temperature and pH value are very wide.When the culture temperature was higher than 35? or the pH value was lower than 2.0,the mycelium could not grow at all.Besides,we found that the mycelial growth did not need light,and the minimum lethal temperature was around 50 ?... |
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