Identification of pathogenic fungi causing mulberry fruit sclerotiniose and their resistance to four fungicides in Zhejiang

Abstract:【Objective】Fruit sclerotiniose is an important fungal disease on mulberry, and it has been re- ported that more than one pathogenic fungus is responsible for it. There were some reports on the patho- gen of mulberry sclerotinia, but the identification results of the pathogen of mulberry sclerotinia were different in different areas and no information in detail about pathogen characterization was available. As the main fruit mulberry producing areas in China, no systematic identification of the pathogen of mulberry sclerotinia in Zhejiang province has been carried out until now. The objectives of this study were to find out the pathogen species in mulberry fields in Zhejiang province, and to reveal the resis- tance status of pathogen population to the DMI fungicides (i.e., prochoraz, difenoconazole) and the di- carboximides fungicides (i.e., dimethachlon, procymidone). Our work will provide scientific basis for the reasonable prevention and control of mulberry fruit sclerotiniose.【Methods】Diseased mulberry fruits were collected from different geographical regions of Zhejiang province and pathogens were iso- lated by cultivating the split sclerotium. After disinfection with 75% alcohol for 1 min and 3% sodium hypochlorite solution for 3 min, the sclerotium was rinsed in sterilized water for 3 times, lasting 30 seach time. The sterilized sclerotium was drained on the sterile filter paper, and the white part was longi- tudinally cut with sterile scalpel to reveal the white part. The white part was inverted on the PDA plate and put into the dark condition of 25 °C incubator for nourish and observation. A small amount of myce- lium was taken on the inclined surface of PDA. The isolated strains were systematically classified in combination of morphological with molecular characteristics. After purification, systematic classifica- tion was carried out based on morphological characteristics (i.e, growth colony, sporulation structures, conidia) and the molecular identification through amplifying the internal transcribed spacer (ITS) of ri- bosome using the universal primer pair ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’). The resistance to dimethachlon, procymidone, prochoraz and di- fenoconazole was determined by the method of differential dosages. The isolates that could not grow on potato glucose agar plates (PDA) amended with 5 mg · L- 1 fungicide were sensitive(S), and the isolates that cloud grow on 5 mg·L-1 but cloud not grow on 50 mg·L-1 were defined as low resistant (LR) , and those that cloud grow at 50 mg·L-1 were determined as high resistant (HR)【. Results】Our results indicat- ed that Scleromitrula shiraiana (I), Sclerotinia minor (II), Diaporthe cotoneastri (III) could caused sclerotiniose on mulberry fruits. After cultivation for 7 days, the colony of Scleromitrula shiraiana was gray villous with developed aerial mycelia, and then turned pale green with the matrix mycelia when the isolate was cultivated for 7 days or more. The colony edge of Scleromitrula shiraiana was not in or- der, and the small sclerotinia formed along the edge of the colony. The conidium of Scleromitrula shi- raiana was in ovoid shape with one end slightly acute, occupying (2.216-4.232) μm× (1.746-2.563) μm. The colony of Sclerotinia minor grew vigorously with gray surface and aerial mycelia, and when the mycelium grew all over the medium, the mycelia became thin and clinging to the medium. WhenSclerotinia minor isolates were cultivated for 14 days, black clumpy sclerotium and gray conidial mound were observed with a naked eye, and the size of conidium was (2.238-2.665) μm × (2.351- 2.678) μm. D. cotoneastri was cultured in PDA for 4 days, the surface of the colony turned grayish white to brown, and aerated hyphae gathered, forming a raised and creeping outward to grow close to the medium. White sclerotia were observed on the 7th day after culture. After culture to the 14th day, it was observed that black massive pycnidia produced and distributed in PDA medium. The conidium was ovoid and its size was (4.078-4.996) μm × (1.514-2.745) μm. The fungicide sensitivity tests showed that class I pathogenic fungus was sensitive to prochoraz, dimethachlon and difenoconazole, and the re- sistance frequency to procymidone was 42.3%, all showing a low level of resistance. The low level re- sistance frequency of class II pathogenic fungus to dimethachlon, difenoconazole and procymidone was 14.2% , 28.57% and 28.57% , respectively. Class III pathogens were sensitive to prochoraz, dimeth- achlon, and difenoconazole, and the frequency of high level resistance to procymidone was 100%【. Con- clusion】There was diversity in the pathogen of mulberry fruit sclerotiniose in Zhejiang province, China:Scleromitrula shiraiana (72.2%, 26/36), Sclerotinia minor (19.4%, 7/36) and D. cotoneastri (8.3%, 3/ 36). A total of 36 isolated pathogens were sensitive to prochoraz, and their frequency of resistance to procymidone was up to 44.4%. In 36 isolates of mulberry fruit sclerotiniose, the low level resistance fre- quency to difenoconazole and dimethachlon was 5.6% and 2.8%, respectively. Our results indicated that prochoraz and mixtures containing prochoraz could be selected for the control of mulberry fruit scleroti- nosis.