Identification of pathogen causing Guanxi honey pomelo anthracnose in Pinghe county of Fujian province

Abstract：【Objective】Honey pomelo is one type of perennial woody tree, which is widely planted in Pinghe county of Fujian province. With the development of the honey pomelo industry, more and more species were produced and their planting area increased year by year, and the diseases associated with honey pomelo increased gradually. Anthracnose is one of the main diseases in honey pomelo production. The disease causes up to 60% of damages to leaves and branches in production fields. Three species have been reported as causal agents of citrus anthracnose: Colletotrichum gloeosporioides, C. acutatum and C. truncatum.In this study, we identified the Colletotrichum spp. causing honey pomelo anthracnose, and aimed to determine whether the same or different species occur in honey pomelo nurseries and production fields in Pinghe, and supply a theory as the basis for future scientific control of this disease.【Methods】Investigated the prevalent situation of the Guanxi honey pomelo anthracnose in the fields from 2014 to 2015. The strains were collected from Banzai town, Guoqiang township, Anhou town and Xiazhai town in Pinghe county of Fujian province. We identified the pathogen on the unhealthy honey pomelo through morphology and recorded the spore. One centimeter disks of the diseased honey pomelo leaf and branch were collected from the field and were cut and surface-sterilized with 75% ethanol and 1‰ Hg Cl2. The disks were placed on a potato sucrose agar (PSA) medium in order to establish cultures at a temperature of 25 ℃ for 7 d. Through separating the pured pathogen single spores, to confirm the Colletotrichum spp. isolates as the causative pathogen, healthy honey pomelo fruit were sprayed with a conidial suspension (approximately 1.0 × 10⁶CFU·m L^-1) of the isolates and incubated at 25 ℃ and 90% relative humidity with plastic and incurring natural cycles of 12 h light and 12 h darkness. Four days after inoculation, lesions started to form on thehoney pomelo fruit which were sprayed with the putative Colletotrichum spp. isolates and the symptoms were quite similar with that of the other fields. We then compared the isolated spores from the tie-back fruit with the spores isolated from the diseased fruit collected from the field. We extracted the genomic DNA from the pathogen, which was identified through r DNA-ITS sequence analysis. We sequenced using the NCBI database, to obtain the results of the Blast alignment.【Results】Isolates of Colletotrichum spp. obtained from leaf, branch and fruit from different districts showing typical anthracnose symptoms were selected for morphological identification. Based on taxonomic criteria of morphological characteristics (colony characters, conidial and appressoria morphology) , the conidia morphology of the pathogen were similar to that of C. gloeosporioides. Conidiophore is colorless, single-spore, oblong or cylindrical, the size of the spores are (9.6-17.2) μm× (3.3-5.2) μm and the average is 14.0 μm×4.8 μm. However, morphological criteria can be unreliable and non-predictive, because of highly variable morphological characteristics which possibly failed to distinguish C. gloeosporioides and others from honey pomelo plants. The DNA of strain GX (3) was used as a template, and one r DNA-ITS fragment of 600 bp was amplified. The obtained sequences with the highest homology belonged to C. gloeosporioides. Their homology reach up to 100%.These studies suggested that the pathogen causing the Guanxi honey pomelo in Pinghe county of Fujian province was C. gloeosporioides. As for honey pomelo, it is still to be determined whether there are any other types of Colletotrichum spp. for subsequent research. In addition, we will study the occurrence rules, biological characteristics of pathogen and laboratory toxicity and field trials, seeking the best effective method for prevention and cure, which provides the basis for ensuring the healthy development of the Guanxi honey pomelo industry.【Conclusion】Based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA (ITS) , the pathogen of Guanxi honey pomelo in Pinghe county of Fujian province was identified as C. gloeosporioides. C. gloeosporioides can be isolated from leaves, branches and fruits, C. acutatum and C. truncatum cannot be isolated, as to whether it can be isolated from floral organ will require further research.