Plant Disease • 2017 • 101:1022-1028 • http://dx.doi.org/10.1094/PDIS-12-16-1824-RE

Characterization, Pathogenicity, and Phylogenetic Analyses of 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 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 (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 /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 and neighbor-joining methods based isolates after 5 to 10 days on PDA were circular, raised, and produced on the Kimura 2-parameter model (Kimura 1980; Saitou and Nei white-to-gray fluffy aerial mycelium containing salmon-colored 1987). All positions containing gaps and missing data were elimi- nated. Clade stability was assessed using a bootstrap analysis with 1000 replicates (Saitou and Nei 1987; Tamura et al. 2013). Pathogenicity of Colletotrichum isolates. All four C. acutatum and sixteen C. gloeosporioides isolates were cultured on PDA at 25°C as described above. After 7 days, conidia were harvested by add- ing 5 ml of sterilized distilled water containing Tween 80 at 0.01% (v/v) onto the culture, which were then gently dislodged using a sterilized curved glass rod. The conidial suspension was filtered through two layers of muslin cloth and adjusted to two concentrations of 1×106 and 1×108 spores/ml in sterile water. Pathogenicity assays were per- formed both on leaves of detached tea twigs and two-year-old potted tea seedlings of cv. Fudingdabai. Tea leaves were surface-sterilized with 75% ethanol and rinsed twice with distilled water. Both tea twigs and tea seedlings were randomly divided into four groups, which were inocu- lated with Colletotrichum 1(C. acutatum), Colletotrichum 2and3 (C. gloeosporioides), and sterile water suspension (as a control). For each isolate, 20 leaves on detached tea twigs were inoculated with and without wounding, and 15 leaves on tea seedlings were inoculated only after wounding (Kanchana-udomkan et al. 2004; Lin et al. 2002; Than et al. 2008). Wound inoculation involved pin-pricking the middle of the leaves (2 sites per leaf along the longitudinal axis) and then plac- Fig. 2. Morphological and cultural traits of Colletotrichum spp. isolated from tea. A, ing a 5-ml conidial suspension (1 × 106 spores/ml) over the wound. Upper and B, reverse sides of cultures on PDA grown at 25°C for 7 days. C, Nonwound inoculation involved placing an 8-ml conidial suspension Conidia (scale bar = 20 mm).

Fig. 1. Brown blight on tea caused by Colletotrichum sp. under natural conditions.

Plant Disease /June 2017 1023 conidial masses; the underside of the colony was pink (Fig. 2a and b). C. sinensis (Guo et al. 2014), but were similar to the earlier re- Conidia were aseptate, guttulate, primarily fusiform and measured 12 ports for C. acutatum on yellow dryad (Dryas drummondii L.) to 16 × 3.5 to 6.5 mm(n = 50). These characteristics were not con- and strawberry (Gunnell and Gubler 1992; Michel et al. 2011). Ac- sistent with the published descriptions of C. gloeosporioides on cordingly, Colletotrichum 1 isolates were identified as C. acutatum.

Table 1. Isolates of Colletotrichum cultured from tea in this study, and reference isolates used for sequence comparisons and phylogenetic analysis GenBank accession no. Isolate/strain Identified asz plant Geographical origin ITS GAPDH CS01 C. acutatum C. sinensis China KU145148 KU728152 CS02 C. acutatum C. sinensis China KY342374 KY342367 CS03 C. acutatum C. sinensis China KY342375 KY342368 CS04 C. acutatum C. sinensis China KY342376 KY342369 CS05 C. gloeosporioides C. sinensis China KU145149 KX426530 CS10 C. gloeosporioides C. sinensis China KU145150 KX426531 CS17 C. gloeosporioides C. sinensis China KU145151 KX426532 CS23 C. gloeosporioides C. sinensis China KU145152 KX426533 CS32 C. gloeosporioides C. sinensis China KU145153 KX426534 CS69 C. gloeosporioides C. sinensis China KU145154 KX426535 CS08 C. gloeosporioides C. sinensis China KY342377 KY342370 CS09 C. gloeosporioides C. sinensis China KY342378 KY342371 CS11 C. gloeosporioides C. sinensis China KY342379 KY342372 CS12 C. gloeosporioides C. sinensis China KY342380 KY342373 CS06 G. cingulata f.sp.camelliae C. sinensis China KU234560 KX426536 CS07 G. cingulata f.sp.camelliae C. sinensis China KU234561 KX426537 CS16 G.cingulata f.sp.camelliae C. sinensis China KU234562 KX426538 CS19 G.cingulata f.sp.camelliae C. sinensis China KU234563 KX426539 CS35-1-1 G.cingulata f.sp.camelliae C. sinensis China KU234564 KU728153 CS64-1 G.cingulata f.sp.camelliae C. sinensis China KU234565 KU728154 BB#7 G. acutata Vaccinium corymbosum USA JQ040069 JQ040079 XTG07 C. acutatum Citrus Brazil KF515693 KF963617 XTG05 C. acutatum Citrus Brazil KF515692 KF975660 LF603 C. fioriniae Citrus China KJ955175 KJ954876 TR-123 C. fioriniae Citrus Brazil KF944356 KF944354 FJFHG C. gloeosporioides f. sp. camelliae Citrus Brazil KP635415 KP635415 FJZHD C. gloeosporioides f. sp. camelliae Citrus Brazil KP635401 KP635414 ICMP10643 G. cingulata f.sp. camelliae C. sinensis India JX010224 JX009908 LGMF811 C. gloeosporioides citrus leaves Brazil KM278588 KM278649 LGMF810 C. gloeosporioides citrus leaves Brazil KM278587 KM278648 LGMF808 C. gloeosporioides citrus leaves Brazil KM278585 KM278646 CQ1A9 C. camelliae C. sinensis China KT319703 KT319694 CQ1A8 C. camelliae C. sinensis China KT319702 KT319693 HEN1A1 C. camelliae C. sinensis China KT319717 KT319687 C1259.2 C. siamense Pistacia vera Australia JX010270 JX010002 C1276.5 C. siamense Dioscorea rotundata Nigeria JX010245 JX009942 CBS:129944 C. lupini Cinnamomum verum Portugal JQ948178 JQ948508 IMI:375715 C. lupini Australia JQ948161 JQ948491 CBS:124969 C. karstii Quercus salicifolia Panama JQ005179 JQ005266 CBS:127591 C. karstii Sapium integerrimium Australia JQ005186 JQ005273 CBS:129815 C. karstii Solanum betaceum Colombia JQ005187 JQ005274 CBS:128547 C. boninense Camellia sp. New Zealand JQ005159 JQ005246 CBS:123756 C. boninense Crinum asiaticum var. sinicum Japan JQ005154 JQ005241 CBS:123755 C. boninense Crinum asiaticum var. sinicum Japan JQ005153 JQ005240 CBS:114801 C. destructivum Crupina vulgaris Greece KM105219 KM105574 CBS:119187 C. destructivum Crupina vulgaris Greece KM105220 KM105575 CBS:128509 C. destructivum Medicago sativa Canada KM105214 KM105569 CBS:133704 C. fuscum Digitalis dubia Netherlands KM105176 KM105526 CBS:133702 C. fuscum Digitalis lutea Netherlands KM105178 KM105528 CBS:133701 C. fuscum Digitalis lutea Germany KM105174 KM105524 ICMP 17928 C. kahawae Coffea sp. Kenya JX010235 JX010037 C1266.2 C. kahawae Coffea arabica Malawi JX010233 JX009970 C1254.10 C. clidemiae Vitis sp. USA JX010274 JX009909 CBS 129826 C. annellatum Hevea brasiliensis Colombia JQ005222 JQ005309 CBS 129828 C. oncidii Oncidium sp. Germany JQ005169 JQ005256 CBS 175.67 C. phyllanthi Phyllanthus acidus India JQ005221 JQ005308 CBS 128544 C. torulosum Solanum melongena New Zealand JQ005164 JQ005251 CBS 129814 C. tamarilloi Solanum betaceum Colombia JQ948184 JQ948514 CBS 129811 C. tamarilloi Solanum betaceum Colombia JQ948185 JQ948515 CBS 330.75 C. costaricense Coffea arabica Costa Rica JQ948180 JQ948510 CBS 211.78 C. costaricense Coffea sp. Costa Rica JQ948181 JQ948511 z Colletotrichum acutatum = Glomerella acutata; Colletotrichum gloeosporioides = Glomerella cingulata.

1024 Plant Disease / Vol. 101 No. 6 Colletotrichum 2 isolates on PDA produced white-to-gray fluffy aer- of the conidia (12.5 to 17.3 × 3.9 to 5.8 mm) that was reported by Guo ial hyphae (Fig. 2a) and the underside of the colonies were darkly et al. (2014). In addition, nearly all isolates of Colletotrichum 3 could pigmented (Fig. 2b). The conidia were primarily cylindrical and be easily distinguished from Colletotrichum 2 by their dense white 12.6 to 18.5 × 4.3 to 7.5 mm (Fig. 2c), distinctly larger than those hyphae on PDA, the two groups showed similar shape and size of of Colletotrichum 1. These characteristics were similar to published conidia, which suggested that the two groups may belong to the same descriptions of C. gloeosporioides on C. sinensis except for the size species.

Fig. 3. Molecular phylogenetic obtained through maximum-likelihood method based on concatenated sequences of the ITS and GAPDH genes from collected isolates and published sequences. The percentage of replicate in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown above the branches and only values above 50% are shown. Groups with a bootstrap below 75% should be considered hypothetical. The bold indicate isolates obtained in the present study.

Plant Disease /June 2017 1025 Molecular characterization. The ITS and GAPDH sequences Phylogenetic analyses. The individual and combined ITS and were used to correctly separate and group isolates belonging to dif- GAPDH sequences of our 20 isolates compared with 41 sequences ferent species of Colletotrichum. The newly obtained sequences in of Colletotrichum species reported on different hosts from GenBank this study were submitted to GenBank, with the accession numbers (Table 1) allowed us to allocate the isolates into the molecular groups indicated in Table 1. GenBank BLAST searches confirmed that the of Colletotrichum species. Phylogenetic analyses performed by both amplification products of ITS and GAPDH regions of Colletotri- neighbor-joining and maximum-likelihood methods produced simi- chum 1 showed 99 to 100% sequence similarity to the sequences lar topologies, and only one tree constructed by maximum-likelihood of C. acutatum. BLAST searches for Colletotrichum 2 and Colleto- method is shown here (Fig. 3). In the maximum-likelihood tree (isolates trichum 3 isolates showed the highest similarity with C. gloeospor- in bold were collected by the authors), all the Colletotrichum isolates ioides and G.cingulata f. sp. camelliae, respectively, which were were separated into four distinct groups. The sequences of Colletotri- reported to cause anthracnose on different hosts worldwide (Hartill chum 1 (CS01-CS04) were clustered within C. acutatum species com- and Everett 2002; Munaut et al. 2001; Xiao et al. 2004). Multiple se- plex with a strong bootstrap support of 100%. Although the 10 quence alignments of ITS and GAPDH regions to detect differences isolates of Colletotrichum 2 and the six isolates of Colletotrichum 3were among the species of Colletotrichum revealed that 10 Colletotrichum clustered in different subgroups with the bootstrap value at 91% and 2 and six Colletotrichum 3 isolates shared higher sequence similarity 80%, respectively, both the subgroups belonged to C. gloeosporioides within each group but slightly lower sequence similarity between the species complex. Based on molecular analysis and morphological two groups. Furthermore, a much lower similarity between the iso- characteristics, we conclude that Colletotrichum 1 isolates are C. lates of Colletotrichum 1 and Colletotrichum 2 (or Colletotrichum acutatum and Colletotrichum 2andColletotrichum 3isolatesare 3) was found by nucleotide sequence identities. C. gloeosporioides.

Table 2. Pathogenicity of Colletotrichum isolates on cv. Fudingdabai 5 days after wound inoculation and 8 days after nonwound inoculation Infected leaves (%)w Lesion diameter (mm)x Leaves on detached Leaves on seedlings Leaves on detached Leaves on twigs (n = 20) (n = 15) twigs seedlings Species complex Isolate Woundedy Nonwoundedz Woundedy Woundedy Nonwoundedz Woundedy C. acutatum Colletotrichum 1 CS01 50.0±5.0 11.7±2.9 53.3±6.7 12.5±1.4bc 3.5±0.3d 7.9±0.3bcd CS02 55.0±5.0 10.0±0.0 48.9±7.7 12.7±1.3bc 3.5±0.1d 7.0±0.8d CS03 56.7±7.6 13.3±2.9 60.0±0.0 11.4±1.4bc 3.6±0.3cd 7.5±1.1cd CS04 51.7±2.9 13.3±2.9 51.1±10.1 12.8±1.9bc 3.1±0.3d 7.9±0.9bcd C. gloeosporioides Colletotrichum 2 CS05 56.7±5.8 8.3±2.9 53.3±13.4 12.2±1.4bc 3.4±0.4d 7.8±0.7bcd CS10 53.3±5.8 5.0±5.0 57.8±3.9 12.5±1.1bc 3.5±0.5d 6.9±0.9d CS17 56.7±2.9 10.0±0.0 60.0±11.5 12.1±1.0bc 3.6±0.3cd 7.4±0.7cd CS23 51.7±2.9 10.0±5.0 55.6±7.7 12.6±0.8bc 3.8±0.5bcd 7.6±1.3cd CS32 55.0±5.0 11.7±2.9 44.4±7.7 12.6±1.1bc 3.4±0.4d 7.6±0.5cd CS69 60.0±5.0 13.3±2.9 48.9±11.5 13.7±1.3bc 3.8±0.3bcd 7.4±0.8cd CS08 48.3±10.4 6.7±2.9 46.7±11.5 9.8±1.1c 2.3±0.3d 8.1±0.8bcd CS09 41.7±7.6 5.0±0.0 51.1±7.7 10.2±1.9bc 3.0±1.0d 6.9±0.9d CS11 46.7±7.6 8.3±5.8 53.3±6.7 11.2±0.7bc 3.0±0.4d 7.8±1.1bcd CS12 58.3±7.6 6.7±2.9 53.3±11.5 11.2±0.9bc 3.2±0.6d 7.2±0.9cd Colletotrichum 3 CS06 61.7±7.6 8.3±2.9 60.0±6.7 16.5±1.1a 5.7±1.1a 9.9±1.8abcd CS07 60.0±5.0 11.7±5.8 53.4±6.7 17.9±0.8a 5.6±0.6a 10.9±1.2ab CS16 63.3±2.9 11.7±2.9 55.6±7.7 17.2±1.1a 5.4±1.0abc 10.3±1.7abc CS19 70.0±5.0 13.3±2.9 62.2±3.9 17.6±1.4a 5.3±0.9abc 11.3±1.8a CS35-1-1 63.3±2.9 16.7±2.9 53.3±6.7 18.1±1.0a 5.3±0.6abc 11.2±1.2a CS64-1 71.7±2.9 18.3±2.9 53.3±11.5 18.0±0.8a 5.9±0.7a 10.8±0.9ab w Incidence of disease by percent following inoculation of wounded and unwounded tea leaves. Data represents the mean of 3 replications, each with 20 and 15 leaves on detached twigs and seedlings respectively. x Lesion diameter following inoculation of wounded and unwounded tea leaves. Data represents the mean of 3 replications, each with 20 and 15 leaves on detached twigs and seedlings respectively. Columns with the same letter do not differ significantly per Tukey’s test (P # 0.05). y Wounded by pinprick, inoculated with 5-ml suspension (106 conidia/ml). z Unwounded, inoculated with 8-ml suspension (108 conidia/ml).

Fig. 4. Typical symptoms observed in pathogenicity tests on leaves of detached twigs of cv. Fudingdabai caused by B, C. acutatum, and C and D, C. gloeosporioides 5 days after wound inoculation. A, CK, leaves inoculated with sterile water.

1026 Plant Disease / Vol. 101 No. 6 Pathogenicity tests. With wound inoculation, lesions appeared reduced. Tea leaves have a thick cuticle and this may explain 3 days after inoculation for 41 to 71% of all inoculated leaves on why Colletotrichum does not easily infect nonwounded leaves detached twigs (Table 2). Five days after wound inoculation, dis- (Pring et al. 1995). The differences in pathogenicity within the tinct circular necrotic spots developed (Fig. 4) which gradually C. gloeosporioides group reported here are most likely the result expanded to large, dark brown lesions; defoliation occurred of variation in pathogenicity of the since the host was of within 10 to 15 days. These symptoms were similar to those that one cultivar and environmental conditions were uniform through- occurred under natural conditions in the field. All isolates were out the inoculation trials. Although the two groups of C. gloeospor- highly pathogenic to tea leaves (Fig. 4b-d) but Colletotrichum 3 ioides shared a low level of variability within their ITS and GAPDH isolates were the most virulent; there were no significant differ- regions, they differed highly in their virulence, which revealed a ences (P # 0.05) in virulence between Colletotrichum 1andCol- correlation between the gene sequence diversity of the two groups letotrichum 2 isolates (Table 2). When nonwounded plants were and their pathogenicity. To develop a more complete picture of the inoculated with 1 × 106 conidia/ml, few symptoms developed Colletotrichum population structure causing brown blight of tea, a (data not show); however, when inoculated with 1 × 108 wider geographic range of isolates should be gathered and more conidia/ml, up to 18.3% of the nonwounded leaves developed le- representatives of the C. acutatum complex in particular should sions. Lesions on nonwounded leaves averaged 4.3 mm in diam- be examined. Further investigations of the molecular genetics of eter while lesions that developed from wounds averaged 14.4 mm Colletotrichum species on tea and other plants may help elucidate in diameter (Table 2). Control leaves in all cases remained non- mechanisms of pathogenicity and open new avenues for developing symptomatic (Fig. 4a). On tea seedlings, wounded leaves devel- management strategies. oped lesions later and were smaller than lesions on wounded detached leaves (Table 2). For each of the inoculated treatments, Acknowledgments isolates were recultured and their identity confirmed by morphol- This work was supported by the Natural Science Foundation Project of ogy and ITS sequences. CQ CSTC (cstc2015jcyjA80019), Chongqing Science and Technology Funds (cstc2013jcsfC80002), and Fundamental Research Funds for the Central Universi- Discussion ties (SWU 113043 and XDJK2015C039) for financial support. The Colletotrichum species responsible for brown blight are diffi- cult to determine with morphological characteristics alone (Cannon Literature Cited et al. 2000). In this study, both molecular and morphological tech- Bailey, J. A., and Jeger, M. J. 1992. Colletotrichum: Biology, Pathology, and niques, together with pathogenicity, were used to identify and distin- Control. CAB International, Wallingford, UK. guish the species of Colletotrichum causing brown blight. The Baroncelli, R., Sarrocco, S., Zapparata, A., Tavarini, S., Angelini, L. G., and Vannacci, Colletotrichum G. 2015. Characterization and epidemiology of Colletotrichum acutatum sensu lato of most taxa within was previously based (C. chrysanthemi) causing Carthamus tinctorius anthracnose. Plant Pathol. 64: primarily upon morphological variation in conidial traits and colony 375-384. characteristics (Bailey and Jeger 1992). 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