Isolation, identification and fungicide screening of the main pathogens of ‘Encore’ fruit spot

Abstract:【Objective】‘Encore’hybrid mandarin, a hybrid offspring of wide-skinned citrus and sweet orange, was introduced from Japan in 1982 with sweet flavor, high quality, strong storage and transpor- tation resistance, long sales period, high economic value and broad growing prospects. In recent years, the severe fruit spot occurred in‘Encore’growing area of Qingshen county, Sichuan province. During the fruit hanging period, a large number of unchlorotic spots appeared on the fruit, and the disease spot was yellowish brown and chapped in the later stage of the disease, and the diseased spot on the mature fruit was more obvious, which seriously affected the fruit appearance so that the produce income de- creased sharply. Aiming at the fruit spot of‘Encore’, Sichuan Agricultural University has isolated the pahogens and identified that the spot is caused by Colletotrichum gloeosporioides, but the control of the disease with citrus anthracnose as the target was not effective. In order to identify the main pathogens of citrus fruit rot, in this paper, the identification of the pathogens and control of the disease were studied, so that the prevention and control plan could be formulated, which could provide theoretical basis and practical guidance for the production.【Methods】The diseased citrus fruits collected from the high inci- dence area of Qingshen county, Sichuan province were isolated by tissue separation method. The colo- ny, mycelium, sporulation structure and spore phenotype of pathogens were observed under a micro- scope. The shape and size of mycelium on the slide were observed and measured. 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 morphology and size of spores and sporogenic cells were observed and measured. The genomic DNA of pathogenic fungi was extract- ed and identified based on molecular biology (ITS and TUB bar code). The pathogens were inoculated on citrus fruits and leaves, and their pathogenicity was determined. After inoculating pathogen strains MK15 and MK13, the citrus fruits and leaves were kept moist on the wet absorbent cotton, and cultured in the incubator at 28 °C for 12 hours, lasting 3-15 days, and meanwhile statistical analysis was carried out for several times. The virulence of 11 fungicides was tested by mycelium growth rate method. There were three repeats for each treatment, and the untreated PDA medium was served as the control【. Re- sults】In the aspect of morphology, the colony of pathogenic strain MK15 was cultured on PDA plate, the colony was round with neat edge, and there were lines on the hyphae. During microscopic observation, it was found that the conidia were long fusiform erect or slightly curved, with 4 cells, and the co- nidiophores were long, light, non-branched without septum. In terms to molecular biology, MK15 andPestalotiopsis microspora were clustered into one branch, so it was clear that MK15 was P. microspora; while MK13 had round or oval colonies, neat edges, compact hyphae, well-developed aerial hyphae and dark gray color at the initial stage of growth. Microscopic examination showed that a large number of conidia were cylindrical, blunt round at both ends, single cell, transparent without septum, containing two oil balls, so MK13 was identified as Colletotrichum gloeosporioides. In the virulence test, MK13 and MK15 were strongly pathogenic to the fruits of‘Chunjian’,‘Wo ’and‘Encore’, and to the leaves of citrus,‘Wo’and‘Encore’, but the fruit and leaf symptoms of‘Encore’were severer than those of other varieties, so we speculated that the main reason for the occurrence of the disease in‘Encore’was that‘Encore’was highly susceptible. In addition, according to the fact that the pathogen was more viru- lent to fruits than to leaves, we speculated that this was also the reason why the pathogen caused Encore fruit spot rather than leaf spot. In the toxicity test of fungicides, prochloraz and pyrazolyl carbendazim had obvious antifungal effects on MK15 and MK13, in which the EC50 against MK15 was 0.001 4 μg·mL-1and 0.015 9 μg·mL-1, respectively; and the EC50 against MK13 was 0.002 2 μg·mL-1 and 0.016 μg·mL-1, respectively. It is especially worth noting that the EC50 of mancozeb to MK13 was 0.703 9 μg·mL-1, and the EC50 of mancozeb to MK15 was 52.893 μg · mL- 1, which indicated that mancozeb can effectively control the citrus anthracnose caused by C. gloeosporioides, but not the fruit spot caused by P. microspo- ra. According to the investigation, only mancozeb was used to soak the infected soils in the susceptible area. Therefore, we speculated that neglect of control was another reason for the prevalence of‘En- core’fruit spot.【Conclusion】P. microspora and C. gloeosporioides were the main pathogens of‘En- core’fruit spot disease, while prochloraz and pyrazolyl ester had obvious antifungal effect. It is also the first time to report that P. microspora can cause citrus fruit spot. This paper provides theoretical basis and practical guidance for early diagnosis and timely prevention and control of the disease.