Pathogen identification and fungicide screening of ‘Orah’ fruit spot disease

Abstract: 【Objective】‘Orah’is a kind of wide-skinned citrus introduced from Israel. It has the advan-tages of beautiful skin, late ripening, high sugar content, early bearing and high yield. And then it has high economic value and broad planting prospect. In recent years, a large area of fruit spot disease has occurred in‘Orah’planting area of Kaizhou, Chongqing. When it happened seriously, it damaged the newly sprouted branches and young fruits, resulting in the death of new sprouted branches, yellowing of fruits and serious fruit drop. The disease weakened the seedling vigor and reduced fruit yield, which brought serious economic losses to local fruit growers. In order to identify the main pathogen of citrus fruit spot disease, the present experiment was carried out to provide theoretical basis and practical guid- ance for local fruit growers.【Methods】The diseased citrus fruits collected from the high incidence area of Kaizhou county, Chongqing were isolated by tissue separation method. The colony, mycelium, sporu- lation structure and spore phenotype of pathogen were observed under a microscope. The shape and size of mycelium on the slide were observed and measured under the microscope. Under the micro- scope, the hyphae were colorless and transparent. We observed the change of mycelium color during the growth of pathogenic strains cultured on PDA medium for 20 days. The genomic DNA of pathogenic fungi was extracted and identified based on molecular biology (ITS, TUB2, GPDH and LSU bar code).Furthermore, the pathogens were inoculated on citrus fruits, leaves and branches, and their pathogenici-ty was determined. After inoculating pathogen strain WC2, the citrus fruits and leaves were kept moist on the wet absorbent cotton, and cultured in the incubator at 28 ℃ for 12 hours, lasting 3-6 days, during  which statistical analysis was carried out for several times. The virulence of 18 fungicides was tested by mycelium growth rate method. There were three repeats for each treatment, and the untreated PDA me-dium was used as the control.【Results】In the aspect of morphology, the colony of pathogenic strain WC2 was cultured on PDA plate, the colony of WC2 was round with neat edge and developed aerial hy-phae on PDA plate; the colony was white at the initial stage and gray at the later stage. Through micro-scopic observation, it was found that the conidia were spindle-shaped, single-celled, colorless and trans-parent. In terms of molecular biology, WC2 and Neofusicoccum parvum are clustered into one branch, so it is clear that WC2 is N. parvum. In the pathogenicity test, obvious spots were found in all of four variet-ies. With the increase of the disease onset time, the disease spots increased gradually. The maximum di-ameter of the disease spots could reach 40 mm after 4 days of culture. The surface of leaves and branch-es was gray brown, and the surface of young fruits was black and slightly sunken. When the humidity was kept above 70%, there were white hyphae in the diseased area. The fruits and branches were hard and firm without rotten and soft phenomenon. The pulp was also black due to the invasion of pathogenic fungi, without obvious decay and special smell. WC2 had stronger virulence to Eureka lemon, tarocco and‘Orah’except Citrus reticulatea‘Dayagan’, and WC2 had stronger pathogenicity to branches than to leaves and fruits. On the basis of the pathogenicity test in vitro plant tissue, the experiment in pot was carried out. Eight days after inoculation with pathogenic fungi, the plants began to suffer disease symp-toms. Most of the buds on the damaged branches would be damaged. The infected buds can’t germinate normally, or they would wither just after germination, and the newly sprouted young leaves would be-come brown from the petiole. Then the disease spot spread along the vein, leading to the new sprouting leaves falling off, the branches were in the shape of bare stalks, and finally the whole branches died. The disease weakened the seedling vigor of‘Orah’and reduced the fruit yield caused by a large number of fruit drop, which brought serious economic losses to local fruit growers. In the toxicity test of fungi-cides, difenoconazole, trifloxystrobin-tebuconazole, tebuconazole-kresoxim-methyl and boscali had ob-vious antifungicidal effect on WC2 strain, with EC50 of 0.24, 0.72, 1.04 and 5.28 µg·mL^-1, respectively.However, pyraclostrobin, mancozeb, prochloraz, thiophanate-methyl, benziothiazolinone, captan, thia-methoxam, diniconazole, copper (Ⅰ) oxide, bromothalonil, oxine-copper, buprofezin, copper chloride oxide, azoxystrobin, pyrazole-metiram had almost no inhibitory effect on WC2. These fungicides should be avoided in the field application to prevent and control the disease so as to reduce the economic expen-diture.【Conclusion】The disease caused by Neofusicoccum parvum was pathogenic to many citrus vari-eties, mainly to the branches. Among them, difenoconazole, trifloxystrobin- tebuconazole, tebucon-azole- kresoxim-methyl and boscali had obvious antifungicidal effect on WC2 strain, which was recom-mended for field control of the disease. This paper provides theoretical basis and practical guidance for early diagnosis and timely prevention and control of the disease.