Indentification And Pathogenicity Analysis Of A Fungal Strain Newly Isolated From The Diseased White Mulberry Fruits

Mulberry is a common fruit in our lives, which are delicious sweet and sour sauce. In addition, mulberry also is very rich in nutrients and deep in loved by the people. Mulberrt is a kind of collective fruit and easy to be infected in the process of maturing. The white mulberry fruits disease is one of common disease. Under normal circumstance, the white mulberry fruits’ surface is showing white in whole or in part and the internal of drupelets have a black or dark gray nugget. Now some of pathogen fungi leading to the white mulberry fruits disease have been reported, such as Sclerotinia sclerotiorum, Ciboria shiraiana, and Phoma morioola. In our study, we got a strain which is a suspected pathogen of the white mulberry fruits disease from the white mulberry fruits, and the strain number was SC1104. It was identified by the morphology observation and the molecular biology methods and the results displayed that this strain belongs to Imperfect Fungi, Coelomycetes, Sphaeropsidales and Phomopsis genus [Phomopsis (Sacc.) Bubak]. The strain was named as Phomopsis sp. SC1104. Phomopsis is a common pathogenic fungal which has a wide host range and is mainly infecting angiosperms and gymnosperms. Phomopsis commonly infects in certain parts of plants, such as leaves, branches and fruits. In China, it has been reported that Phomopsis could infest fruits and agricultural products, for example, blueberries, chestnut, eggplants, bean and so on. But now, there is no report that Phomopsis could infect the mulberry fruits that lead to the white mulberry fruit disease.According to Koch’s Postulates, we did a pathogenicity experiment in order that confirming the strain SC1104 is the pathogen which could cause the white mulberry disease in this study. We inoculated the mulberry fruits in different periods of growth with Phomopsis sp. SC1104. Then we observed the symptoms of mulberry fruits and test the pathpgenicity. Then, we chose Carbendazim, Chlorothalonil and Thiophanate Methyl to do the suppressive experiments of Phomopsis sp. SC1104 growth. The purpose of these tests is in favor of providing new ideas and methods for preventing and curing the white mulberry disease caused by Phomopsis.The research contents and results are as follows:1. The diseased mulberry fruits were collected from the mulberry orchard in Nanbu County, Suining City, Sichuan Province. These mulberry fruits’surface is white, and the internal of drupelets have a black or dark gray nugget. After surface disinfection, we got a suspicious strain SCI 105 which was cultivated in fungal screening culture medium. The colonies of strain SC1104 is white in the PDA mediums and the strain could generate the black and irregular shaped sporodochidium which are very hard. Microscopic observation showed that the strain could produce two types of conidia. The a type conidium is fusiform and the size of a type conidium is approximately 4.2-7.8× 2.0～2.8μm. The interior of a type conidium contains 2-6 small oil balls, and the small oil balls are unevenly distributed and have different sizes. The (3 type conidium is linear and some of this type conidium is hooked at one end. The β type conidium has no small oil balls and could not germinate in the condition of laboratory culture. And the size is approximately 16～30×0.5～1μm. The ITS rDNA、18S rDNA and 26S rDNA gene of the strain SC1104 were amplified and sequenced. Then it was compared by BLAST comparison in NCBI and did the phylogenetic analysis. The results showed the strain belonged to Phomopsis and was named as Phomopsis sp. SC1104.2. According to Koch’s Postulates, we did field trials in order to confirm the pathogen of strain SC1104 for the mulberry fruits. We prepared the conidia suspension whose concentration was 2-5 X 106, and inoculated into the medium susceptible mulberry trees. And the negative control was water. After 20 days, the mulberry fruits were claret-colored in the control group, but white in the testing group. And there was a black nugget inside the drupelet. We isolated one strain from the white mulberry fruits which were inoculated by the conidia suspension was the same as the strain SC1104 in morphology. However, in the control group the mulberry fruits were not infected and we did not found this strain in the normal mulberry fruits. Finally, we identified the strain by molecular biology method and amplified the ITS rDNA、18S rDNA and 26S rDNA gene to get the sequence. Then it was compared with the strain SC1104, and the result was that the similarity of these two strains was 100%. The result illustrates the strain is pathogenic for the mulberry fruits bringing about the white mulberry disease likely.3. We select Carbendazim, Chlorothalonil and Thiophanate Methyl which are general antifungals to inhibit the growth of strain SC1104, and investigate the relative inhibition rates of mycelia growth and the spore germination rates. The results showed that Thiophanate Methyl inhibit the mycelia growth was the best that its EC50 was 19.3 ng/mL. Diluted in 200 times to 600 times, a type conidium would be ruptured. And diluted in 800 times to 1,400 times, the spore germination rates were 33.79%-65.12%. The EC50 of Chlorothalonil was 3.7722μg/mL, and the inhibiting effect was weak in the testing concentration. And in the inhibiting spore germination test, the spore germination rates were 11.71%-60.62% diluted in 200 times to 1,400 times and the effect was wonderful. The EC50 of Carbendazim was 2.2646μg/mL, and the relative inhibition rates of mycelia growth was only 29.06% in highest testing concentration. Meanwhile, the effect of inhibiting spore germination was also weak, and the spore germination rate was 83.01% diluted in 400 times.