Plant Disease • 2017 • 101:1022-1028 • http://dx.doi.org/10.1094/PDIS-12-16-1824-RE Characterization, Pathogenicity, and Phylogenetic Analyses of Colletotrichum Species Associated with Brown Blight Disease on Camellia sinensis in China Yingjuan Chen, Wenjun Qiao, Liang Zeng, Dahang Shen, and Zhi Liu, Department of Tea Science, College of Food Science, Southwest University, Chongqing, 400715, China; Xiaoshi Wang, Agricultural Committee of Liangping County, Chongqing, 405200, China; and Huarong Tong, Department of Tea Science, College of Food Science, Southwest University, Chongqing, 400715, China Abstract Brown blight disease caused by Colletotrichum species is a common C. gloeosporioides. Phylogenetic analysis derived from individual and and serious foliar disease of tea (Camellia sinensis). Fungal isolates combined ITS and GAPDH sequences clearly clustered C. acutatum from several tea plantations causing typical brown blight symptoms and C. gloeosporioides into separate species. Pathogenicity tests vali- were identified as belonging to the Colletotrichum acutatum species dated that both species were causal agents of tea brown blight disease complex and the Colletotrichum gloeosporioides species complex and were highly pathogenic to tea leaves. However, the two groups based on morphological characteristics as well as DNA analysis of of C. gloeosporioides with low levels of variability within their ITS the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate and GAPDH regions differed in their virulence. This study reports for dehydrogenase (GAPDH). Colletotrichum acutatum, a new causal the first time the characterization of C. acutatum and C. gloeospor- agent associated with C. sinensis, showed high phenotypic and ioides causing brown blight disease on tea (Camellia sinensis (L.) O. genotypic diversity compared with the more commonly reported Kuntze) in China. Tea (Camellia sinensis (L.) O. Kuntze.) has been widely planted as now used to assist identification of species within this genus (Cai an important economic crop worldwide and is mainly cultivated for et al. 2009; Cannon et al. 2000). So far, molecular diagnostic tech- beverage production. Brown blight disease is one of the foliar niques have not been extensively used to diagnose diseases of tea. diseases of tea prevalent in China, Japan, Sri Lanka, and India The objective of this study was to characterize the species of Colleto- (Chakraborty et al. 2002; Guo et al. 2014). This disease is very destructive trichum from a number of isolates collected from the leaves of tea as- and has become highly limiting for tea cultivation and the tea indus- sociated with brown blight disease in Chongqing district of China, try. The pathogens causing brown blight are described as Colletotri- based on DNA sequence data, morphology, and pathogenicity. We chum species, which are among the most important plant pathogenic used a combined application of molecular tools with traditional meth- fungi worldwide. Colletotrichum gloeosporioides (syn. Glomerella ods including morphology and pathogenicity to study brown blight of cingulata (Schena et al. 2014; Silva-Rojas and Avila-Quezada´ 2011; tea caused by Colletotrichum. Accurate identification of plant patho- Sutton 1992), belonging to the C. gloeosporioides species com- gens, especially in the Colletotrichum complexes, is essential for effec- plex (Weir et al. 2012), is a common Colletotrichum species that tive disease management (Cai et al. 2009; Cannon et al. 2000). has been reported to occur on C. sinensis in India (Chakraborty et al. 2002) and China (Guo et al. 2014). The Colletotrichum acutatum Materials and Methods species complex (Damm et al. 2012) has been reported to cause dev- Fungal isolates and morphological characterization. Colletotri- astating crop losses in many agriculturally important hosts, including chum isolates from necrotic lesions of tea leaves showing brown strawberry (Fragaria ananassa) (Garrido et al. 2009; Sreenivasaprasad blight symptoms were collected from several main tea plantations and Talhinhas 2005; Ureña-Padilla et al. 2002), blueberry (Vac- in Chongqing district of China during the growing seasons of 2014 cinium corymbosum L.) (Xu et al. 2013), apple (Malus domestica and 2015 (Fig. 1). To characterize the pathogen, more than seventy L. Borkh.) (Mari et al. 2012), pepper (Capsicum spp.) (Liao et al. symptomatic leaves were collected for isolations. Tissue was re- 2012), celery (Apium graveolens) (Pollok et al. 2012), olive (Olea euro- moved from the margin of lesions, surface-sterilized in 0.1% HgCl2 paea L.) (Mart´ınandGarc´ıa-Figueres 1999), and hazelnut (Corylus avel- for 1 min and 70% ethanol for 30 s, rinsed three times in sterile dis- lana L.)(SezerandDolar2012). Only recently has C. acutatum tilled water, cultured on potato dextrose agar (PDA), and incubated in (teleomorph: Glomerella acutata) (Guerber and Correll 2001) been a chamber at 25°C with a 12-h photoperiod for 5 days. According to reported as causing brown blight of tea (Chen et al. 2016). Tradition- the similarity of cultural characteristics on PDA and symptoms on ally, Colletotrichum species were identified and characterized based leaves in the field (Fig. 1), 20 isolates were selected from more than on morphological characteristics, such as size and shape of conidia or 70 for further identification and analysis. Of these 20, cultural and co- existence of setae, and cultural characteristics such as colony color nidial morphology of 5- to 10-day-old isolates were observed with a and texture (Bailey and Jeger 1992; Smith and Black 1990). How- U-TV0.5XC-3 microscope (Olympus, Tokyo, Japan). For each iso- ever, morphological characteristics of Colletotrichum have been late, colony characteristics were noted, as well as the color of the col- found to be unreliable for identification of species and the teleomor- ony top and bottom; conidia (50 per isolate) were measured and their phic stage is rarely formed (Baroncelli et al. 2015; Cannon et al. shape noted. The 20 isolates were placed into three groups based on 2000; Schena et al. 2014). Molecular phylogenetic methods are colony characteristics (shape of conidia and colony color). Among these 20, four isolates showed similar cultural and morphological characteristics (Colletotrichum 1). The other 16 isolates were placed Corresponding authors: Y. J. Chen; E-mail: [email protected], and H. R. Tong; in two other groups, Colletotrichum 2 and Colletotrichum 3 (Fig. 2). E-mail: [email protected]. DNA extraction and PCR amplification. Total genomic DNA Accepted for publication 6 February 2017. was extracted from the mycelium of each isolate by a CTAB method according to the procedure of Than et al. (2008). All DNA extracts were stored at −20°C before use. The ITS and GAPDH regions were © 2017 The American Phytopathological Society amplified by PCR with the universal primers ITS4/ITS5 (ITS4: 1022 Plant Disease / Vol. 101 No. 6 5¢-TCCTCCGCTTATTGATATGC-3¢;ITS5:5¢-GGAAGTAAAA (1 × 106 or 1 × 108 spores/ml) onto the middle of the reverse side of GTCGTAACAAGG-3¢) (White et al. 1990) and GDF1/GDR1 the leaves. After inoculation, the twigs were placed in glass culture (GDF1: 5¢-GCCGTCAACGACCCCTTCATTGA-3¢;GDR1:5¢- dishes and maintained at 25°C in an incubator with constant relative hu- GGGTGGAGTCGTACTTGAGCATGT-3¢) (Templeton et al. 1992), midity of 90% and a 12-h photoperiod. The inoculated tea seedlings respectively. The amplification program was as follows: 95°C for were covered with plastic bags to maintain high relative humidity for 3 min; 32 cycles of denaturation at 95°C for 30 s, annealing at 55°C 2 days, and placed in the greenhouse at the same temperature described for 30 s, elongation at 72°C for 50 s; and a final extension at 72°C above. Leaves were monitored for the onset of symptoms for 25 days. for 10 min. The PCR products were analyzed by electrophoresis in Disease incidence (infected leaves) was assessed 5 to 15 days after in- 1% agarose gels and then single bands of the expected size were purified oculation by counting the number of leaves showing necrotic lesions, using the Gel Extraction Kit (Omega Bio-Tek, Norcross, GA) according compared with the total leaves inoculated. Virulence was evaluated to the manufacturer’s protocol. The purified products were sequenced by measuring the diameter of the necrotic leaf lesions 5 days after inoc- at Sangon Biotech (Shanghai, China). ulation for the wounded leaves and after 8 days for the nonwounded Phylogenetic analyses. The ITS and GAPDH sequences obtained leaves. The experiment was carried out three times. Differences in vir- in this study were compared with homologous sequences of several ulence caused by Colletotrichum species was determined by one-way other species of the genus Colletotrichum from GenBank. GenBank ANOVA and means were compared by Tukey’stest(P # 005) using accession numbers, molecular groups, and hosts are shown in IBM SPSS Statistics 20.0. All fungal isolates included in the pathogenic- Table 1. The sequences were analyzed using Molecular Evolutionary ity tests were reisolated from inoculated leaves to confirm their identity Genetic Analysis (MEGA) software version 6.0. Multiple sequence using both molecular and morphological approaches. alignments of each gene used Clustal W as implemented in MEGA v.6 and manually adjusted to allow maximum sequence similarity Results (Tamura et al. 2013). The evolutionary history was inferred by using Morphological and cultural characteristics. Colletotrichum 1 both the maximum-likelihood