Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus

Abstract

Melanose disease caused by Diaporthe citri is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure Diaporthe-like boundary species. Then, a subset of thirty-eight representatives were selected to perform the phylogenetic analysis with combined sequences of ITS, beta-tubulin gene (TUB), translation elongation factor 1-α gene (TEF), calmodulin gene (CAL), and histone-3 gene (HIS). As a result, these representative isolates were identified belonging to D. citri, D. citriasiana, D. discoidispora, D. eres, D. sojae, and D. unshiuensis. Among these species, the D. citri was the predominant species that could be isolated at highest rate from different melanose diseased tissues. The morphological characteristics of representative isolates of D. citri were investigated on different media. Finally, a molecular tool based on the novel species-specific primer pair TUBDcitri-F1/TUBD-R1, which was designed from TUB gene, was developed to detect D. citri efficiently. A polymerase chain reaction (PCR) amplicon of 217 bp could be specifically amplified with the developed molecular tool. The sensitivity of the novel species-specific detection was upon to 10 pg of D. citri genomic DNA in a reaction. Therefore, the D. citri could be unequivocally identified from closely related Diaporthe species by using this simple PCR approach.

Phylogenetic and Haplotype Network Analyses of Diaporthe eres Species in China Based on Sequences of Multiple Loci

“…In brief, the ITS sequence was amplified with the primer set of ITS1/ITS4 [ 49 ], EF1-α with EF1-728F/EF1-986R [ 50 ], TUB2 with Bt-2a/Bt-2b [ 51 ], CAL with CAL-228F/CAL-737R [ 50 ], and HIS with CYLH3F/H3-1b [ 51 , 52 ]. PCR amplification protocols of the five loci are the same as those described previously [ 53 ].…”

Morphology Characterization, Molecular Phylogeny, and Pathogenicity of Diaporthe passifloricola on Citrus reticulata cv. Nanfengmiju in Jiangxi Province, China

“…The sections were surface disinfested with 1% sodium hypochlorite solution (NaClO) for 1 min, rinsed three times with sterilized water, dried, and then incubated on PDA plates amended with 100 µg/mL streptomycin and 100 µg/mL ampicillin at 25 • C for 2 to 5 days. Hyphal tips growing from the pieces of the sample were transferred onto fresh PDA plates and incubated at 25 • C for 30 days as previous methods [7]. After sporulation, single-spore-isolation was performed as previously described [63].…”

“…The chamber was wrapped with plastic film to maintain 95% relative humidity and incubated at 25 • C in the dark for 7 days. In all the pathogenicity tests, the conidial suspension (10 6 conidia/mL) of D. citri strain NFHF-8-4 [7] and sterile distilled water were used as positive and negative controls, respectively. Symptoms on fruit were observed.…”

“…Recently, the cultivation area reached 2.5 million ha, and the production was about 38 million tons [2]. Melanose, one of the most common fungal diseases of citrus worldwide [3,4], generally occurs in many citrus-growing regions of China, such as Chongqing, Fujian, Guangdong, Guangxi, Hunan, Jiangxi, Shaanxi, Shanghai, Zhejiang, and so on [5][6][7]. All commercial citrus varieties are susceptible to melanose.…”

Molecular Phylogenetic Diversity and Biological Characterization of Diaporthe Species Associated with Leaf Spots of Camellia sinensis in Taiwan

“…Species of Diaporthe have been frequently reported as pathogens, endophytes, and saprobes of various types of hosts [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. These taxa have been reported globally and cause various diseases on economically important plants and crops such as dieback of forest trees [ 2 ], leaf spots on tea and Ixora spp.…”