Fungal Diversity DOI 10.1007/s13225-014-0310-9
Diverse species of Colletotrichum associated with grapevine anthracnose in China Ji-Ye Yan & M. M. R. S. Jayawardena & Ishani D. Goonasekara & Yong Wang & Wei Zhang & Mei Liu & Jin-Bao Huang & Zhong-Yue Wang & Jing-Jing Shang & You-Liang Peng & Ali Bahkali & Kevin D. Hyde & Xing-Hong Li
Received: 8 October 2014 / Accepted: 14 October 2014 # School of Science 2014
Abstract Grapevine anthracnose is an important disease, responsible for mild to severe yield losses in grape production, and is also an important post harvest disease. The disease was studied in vineyards in six provinces in China, with 34 isolates obtained from diseased grapes. Multi-gene (ACT, ITS, GAPDH, TUB2 and CHS) analysis coupled with morphology showed that Colletotrichum aenigma, C. hebeiense sp. nov. and C. viniferum were associated with grapevine anthracnose in China. Colletotrichum aenigma is reported for the first time as associated with grapevine anthracnose. Colletotrichum hebeiense is a new species introduced here. Pathogenicity testing showed that all species can infect grapes, causing anthracnose however, virulence of species and isolates showed great variation. Phylogenetic analysis showed that J.
C. viniferum is a cryptic species and its taxonomy needs to be resolved in the future. Keywords Colletotrichum aenigma . Disease symptoms . Virulence . Grape ripe rot . Multi-gene
Introduction Grapes are one of the most important fruits cultivated worldwide, both for wine production and for fruit consumption (Sung et al. 2008). There has been a rapid increase in areas that grow grapes throughout China. In 2012, the total area with grape vine production was 421,000 hm2, with a yield of 517,600 tons (China Agriculture Research System Statistical Data 2012). During the final stages of grape ripening, most of the grape growing regions in China receive high rainfall and relatively warm summer conditions which results in yield losses due to diseases and insects (Lu 2005). Among the grape disease, grape anthracnose or grape ripe rot is one of the most serious diseases in viticulture as it infects mature fruits and always reduces the crop value (Kummuang et al. 1996; Whitelaw-Weckert et al. 2007). Grape anthracnose may also be caused by Elsinoe ampelina, but symptoms differ in that Colletotrichum causes sunken water soaked necrotic lesions, while Elsinoe causes sunken lesions with grey centers and dark reddish-brown to violet-black margins (Ellis and Erincik 2008). The berries are susceptible to grape ripe rot from a small green size to the ripen stages, but do not show any symptoms until ripening (Whitelaw-Weckert et al. 2007). The genus Colletotrichum includes important phytopathogens of many economically important plants cultivated around the world (Hyde et al. 2009a, b; Cannon et al. 2012). Symptoms are broad-ranging from anthracnose to stem end rots, diebacks and seedling blights (Sutton 1992). Fruits
Fungal Diversity
especially are often infected by Colletotrichum species during pre- and post-harvest seasons (Sutton 1992). The first significant monograph on Colletotrichum was that of (von Arx 1957) which set a new era in Colletotrichum taxonomy. This monograph was mainly based on morphological characters, with little or no emphasis placed on pathology which led to the diminution in accepted species from around 750 to 11 (Damm et al. 2012; von Arx 1957). Hyde et al. (2009a) listed the Colletotrichum names in current use by accepting 66 species and with additional names considered as doubtful. At present many studies are focused on epitypification, identifying and understanding the morphological and molecular variations of Colletotrichum taxa worldwide (Weir et al. 2012; Damm et al. 2012; Cannon et al. 2012; Peng et al. 2012; Doyle et al. 2013; Gunjan et al. 2013; Hyde et al. 2014). The taxonomy and nomenclature of Colletotrichum has been confusing and it is difficult to identify Colletotrichum species due to instability of its morphological characters, depended upon experimental methods and conditions (Hyde et al. 2009b, 2010; Cannon et al. 2012). Many Colletotrichum strains have being successfully identified and epitypified by morphological and multilocus phylogeny which suggested molecular phylogeny could lead to better understanding and identification of this genus (Damm et al. 2012). This will help to identify phytopathogenic species which will result in controlling plant diseases. The objectives of the present study are as follows: (1) to evaluate the importance of grape anthracnose disease in China, (2) to identify the species of Colletotrichum causing
grape anthracnose by morphological and molecular approaches, (3) to determine the pathogenicity of the different Colletotrichum species and isolated strains and (4) to clarify the variation and distribution of the Colletotrichum species associated with grape anthracnose in China.
Materials and methods Vineyard survey and fungus isolation The vineyard survey was conducted between 2008 and 2012, in vineyards of Beijing, Hebei, Henan and Shandong provinces. Five plots were assessed (20 clusters were assessed in each plot) in every vineyard. Cultivar, location, time, fruit numbers in each cluster, and diseased fruit numbers per cluster were recorded. The fruit disease cluster ratio formula was calculated as follows: diseased fruit ratio (%)=(diseased fruit numbers in each cluster/ fruit numbers in each cluster)× 100 %. The disease ratio formula of clusters were calculated as follows: diseased cluster ratio (%)= (diseased cluster numbers/ cluster numbers) × 100 %. After assessing the vineyards the average diseased ratio was calculated (Table 1). Isolates were collected from different provinces (Hebei, Jiangsu, Peking, Shandong, Shanxi and Xinjiang) of China. Diseased grapevine samples were collected from vines showing characteristic ripe rot symptoms. Samples were collected from the most prevalent table and wine grape cultivars grown in the six provinces (Fig. 1) including Cabernet Franc,
Table 1 Vineyard Survey of Grapevine Anthracnose Diseased cluster and diseased fruit ratio Cultivar
Survey Time Aug. 1- Aug. 6
Aug. 14- Aug. 18
Aug. 23-Aug. 25
Diseased cluster ratio
Diseased fruit ratio
Diseased cluster ratio
Diseased fruit ratio
Diseased cluster ratio
Diseased fruit ratio
Viognier Riesling Cabernet Franc Cabernet -Sauvignon Merlot Chardonnay
13 6 0 0 0 7.68
18.2 4 0 0 0 14.33
83 – 1.6 3.2 18.2 –
61.14 – 16.52 20.3 34.94 –
– – 6.6 19.4 31.8 –
– – 17.98 39.19 35.1 –
Shiraz Sauvignon Blanc
37.67 0 0 0 0 0 0 0
14.33 0 0 0 0 0 0 0
50.4 55 21 53 34 20 0.8 0
16.52 25.29 48.83 32.44 16.91 26.59 34.94 0
– – 14.2 16 10.4 10.4 1.2 18
– – 15.71 42.16 23.53 28.52 5.67 49.56
Roussanne Petit Manseng Petit Verdot Gamay
Fungal Diversity Fig. 1 Symptoms of the ripe rot disease found in vineyards a Bunch of grapes affected by Colletotrichum viniferum b to c Grape fruits with salmon pink and orange coloured spore masses of C. viniferum d to e Severe infection of C. viniferum ripe rot in the vineyard
Cabernet Gernischet, Cabernet Sauvignon, Red Globe Seyval and Syrah. The isolates were obtained from infected fruits or leaves with fungal sporulation, by single-spore isolation methods as described in Chomnunti et al. (2014). Samples were surface-sterilized in 1 % sodium hypochlorite for 1 min,
70 % ethanol for 1 min and then rinsed three times with sterilized water before culturing on potato dextrose agar (PDA) at 28 °C. Single germinating conidia were transferred to fresh PDA (Potato Dextrose Agar) plates after 7–10 days and kept at 28 °C to obtain pure isolates (Chomnunti et al.
Fungal Diversity
2014). The pure isolates were cultured on PDA plates with sterilized filter paper pieces. Then the cultures on the filter paper pieces were dried on sterilized filter paper and stored at −20 °C (Table 2). Morphological characterization Morphological and cultural characterization followed the methods described in Cai et al. (2009). Mycelial discs (5 mm diam.) were taken from the growing edge of 5-dayold cultures and transferred to PDA plates and incubated in the dark at 28 °C. Three replicate cultures of each isolate were observed. Colony diameter was measured daily for 7 days, growth rate (mm/day) was calculated and the colour, conidial masses and zonation were recorded (Peng et al. 2012; Weir et al. 2012; Cai et al. 2009). Shape and colour of conidia were observed and the length and width of 50 conidia from two representative isolates were measured under a microscope (Nikon, NIS-Elements F3.0) Table 3. Appressoria were produced using slide culture technique (Weir et al. 2012). A small square of agar was inoculated on one side with conidia immediately covered with a sterile cover slip. After 14 days the cover slip was removed and placed in a drop of lactic acid on a glass slide. Isolates chosen for conidial characterization were also used to determine the effects of temperature and nutrients on colony growth and fruiting body formation. For the determination of temperature effect, a 5-mm-diameter colony plug from the margin of a five-day-old culture was placed in the centre of a 90-mm-diameter PDA Petri dish. Three replicates of each isolate were incubated separately at 5, 10, 15, 20, 25, 30, 35
and 40 °C. Colony diameters were measured after 24, 48, 72 and 96 h, and the resulting data were converted into radial growth. The experiment was conducted three times. For the determination of nutrient effect, a five-mm-diameter colony plug from the margin of a five-day-old culture was placed in the centre of a 50-mm-diameter PDA Petri dish. Three replicates of each isolate were incubated at 28 °C on media containing different nutrients including WA (water agar, 20 g/l), OA (oatmeal agar, 15 g/l), DA (dextrose agar, 20 g/ l) and PDA until sporulation was observed. The sporulation time was recorded. The experiment was conducted three times. Molecular characterization Isolates were transferred to 90-mm-diameter PDA Petri dishes and incubated at 28 °C for 7–10 days. Total genomic DNA was extracted by the modified protocol of Damm et al. (2009). DNA samples were detected by electrophoresis and Ethidium Bromide (EB) staining and used as templates for PCR amplification. Partial actin (ACT), Glyceraldehyde-3-phospate dehydrogenase (GADPH), Partial chitin synthase (CHS), βTubulin (TUB2), and the complete rDNA-ITS (ITS) were amplified with specific primers by PCR reaction (Table 6). The PCR were performed in an Applied Biosystems Veriti Thermal Cycler in a total volume of 25 μl. The PCR mixtures contained TaKaRa Ex-Taq DNA polymerase 0.3 μl, 12.5 μl of 2×PCR buffer, 2.5 μl of dNTPs, 1 μl of genomic DNA, 1 μl of each primer and 12.2 μl of ddH2O. The PCR conditions for ITS were 4 min at 95 °C, then 35 cycles of 95 °C for 30s, 57 °C for 30s, 72 °C for 45 s, and
Table 2 Synopsis of morphological data of Colletotrichum species on grape Species and isolate number
Conidia
Appresoria Length (μm)
Growth Rate (cm/ day)
Length (μm)
Width (μm)
Width (μm)
JZB330028 JZB330024 JZB330034 JZB330025 JZB330007 JZB330001 JZB330002
12.53(±0.98) 14.84(±3.32) – 14.79(±1.44) 13.44(±1.16) 13.12(±1.78) 12.21(±1.23)
4.24(±0.49) 4.98(±0.36) – 5.39(±0.61) 4.89(±0.5) 6.18(±0.98) 4.43(±0.42)
8.22(±1.42) 12.24(±2.48) 8.9 (±1.14) 10.88(±2.09) 9.64(±2.14) – 7.59(±1.31)
5.75(±0.81) 6.79(±0.73) 6.43 (±1.05) 6.31(±1.13) 5.94(±1.17) – 6.43(±0.68)
0.722(±0.020) 0.689(±0.015) 0.651(±0.027) 0.722(±0.041) 0.727(±0.014) 0.504 (±0.028) 0.355(±0.056)
JZB330003 JZB330005 JZB330011 JZB330017 JZB330023 JZB330033 JZB330037
13.56 (±1.12) 15.27(±2.4) 14.68(±2.05) 17.43(±2.94) 14.07 (±1.66) 12.96 (±0.77) 14.9 (±0.99)
4.24 (±0.43) 4.17(±0.39) 6.1(±0.93) 4.80(±0.48) 6.15(±0.72) 4.27 (±0.39) 4.8 (±0.47)
8.1 (±1.61) 6.66(±1.69) 8.32(±1.37) 8(±1.76) 7.84 (±0.121) 11.81(±2.51) 11.46(±4.23)
5.44 (±0.82) 5.99(±0.69) 4.9(±0.49) 4.76(±0.69) 6.58 (±0.92) 4.97 (±0.34) 5.67(±1.25)
0.542 (±0.106) 0.635 (±0.038) 0.625(±0.041) 0.596 (±0.077) 0.652 (±0.114) 0.421 (±0.097) 0.370 (±0.441)
Fungal Diversity Table 3 Growth rate (cm diam.) of isolates cultured on PDA at different temperatures (7 days old) in detail
No
Isolate
1 2 3 4 5
JZB330001 JZB330002 JZB330003 JZB330004 JZB330005
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
10 °C
15 °C
20 °C
25 °C
30 °C
0 0 0 0 3.6±0.4
14.0±0.5 16.9±0.4 30.3±0.4 9.3±0.6 31.5±0.2
33.8±0.4 31.1±0.2 41.5±0.9 21.1±0.3 51.9±0.9
55.5±0.3 36.3±1.0 59.7±1.1 37.6±1.2 70.0±0.4
65.5±0.6 39.0±0.6 61.0±0.9 42.0±0.7 64.6±0.9
8.0±0.3 3.3±0.3 5.8±0.5 1.9±0.5 6.9±0.7
JZB330007 JZB330008 JZB330009 JZB330011 JZB330012 JZB330013 JZB330014 JZB330015 JZB330016 JZB330017 JZB330018 JZB330019 JZB330021 JZB330022 JZB330023 JZB330025 JZB330026 JZB330027
15.8±0.4 2.2±0.2 4.9±0.5 3.9±0.7 0 2.3±0.4 0 0 2.4±0.5 2.8±0.4 0 1.9±0.3 0 4.6±0.6 11.2±0.2 14.7±0.3 2.5±0.5 14.3±0.1
49.6±0.5 21.0±0.5 38.8±0.9 21.7±0.7 10.8±0.5 25.3±0.5 35.5±0.5 12.8±1.1 30.3±0.4 43.6±0.3 19.9±0.5 19.8±0.4 11.6±0.2 37.1±0.6 43.1±0.3 51.8±0.6 22.1±0.8 40.8±0.5
69.9±0.5 37.0±0.7 50.2±0.6 49.5±0.7 26.7±0.2 44.0±0.6 43.8±0.5 25.6±0.6 41.5±0.9 48.0±0.8 28.8±0.7 34.1±0.3 13.9±0.7 51.1±0.8 49.0±0.9 65.4±0.6 35.8±0.4 49.3±0.9
80.0±0.1 52.7±0.2 67.1±0.3 68.8±0.4 44.3±0.7 58.3±0.9 62.8±1.0 41.9±0.7 59.7±1.1 65.6±0.6 45.2±0.4 41.7±0.5 32.3±0.9 66.5±0.7 71.8±1.1 79.5±0.4 45.2±0.3 65.0±0.9
78.8±0.2 53.8±0.4 63.4±0.2 58.5±0.5 33.2±1.1 56.5±0.3 66.6±0.6 43.4±1.1 61.9±0.4 52.6±0.7 50.6±0.7 47.8±0.5 42.8±0.7 69.4±0.6 73.1±0.2 78.4±0.3 43.8±0.4 61.9±0.4
8.7±0.7 1.9±0.2 14.0±0.9 7.2±0.7 2.4±0.1 4.1±0.2 12.3±0.4 5.8±0.6 5.8±0.5 6.7±0.5 10.2±0.5 74.4±0.4 17.6±0.9 6.6±0.2 15.7±0.5 55.1±0.2 12.0±0.2 3.2±0.3
JZB330028 JZB330029 JZB330030 JZB330031 JZB330032 JZB330033 JZB330034 JZB330035 JZB330036 JZB330037 JZB330024
14.1±0.5 4.0±0.2 9.1±0.1 2.3±0.4 0 0 8.3±0.6 6.7±0.7 1.8±0.2 0 4.8±0.9
47.1±0.6 33.0±0.9 42.2±0.6 25.3±1.1 12.1±0.3 19.5±0.6 32.7±0.8 29.3±0.7 19.8±0.4 19.4±0.6 47.2±0.9
64.4±0.9 47.9±0.3 54.9±0.6 39.0±0.3 19.2±0.4 29.0±0.5 51.6±0.3 50.1±0.4 33.9±0.2 25.5±0.7 55.4±0.7
79.5±0.2 61.4±0.2 75.1±0.7 57.6±0.6 31.9±0.7 46.4±1.0 71.7±0.3 67.5±0.5 41.7±0.5 34.3±1.1 75.6±0.9
78.9±0.3 48.4±0.5 78.9±0.3 56.9±0.8 33.2±1.2 35.1±0.2 51.8±0.9 52.8±0.7 47.8±0.5 40.8±0.4 66.7±0.8
17.3±1.2 5.7±0.2 25.0±0.4 3.3±0.6 1.0±0.2 6.1±0.5 2.0±0.7 14.8±0.2 7.4±0.4 1.4±0.2 7.0±0.6
then 1 min at 72 °C. The annealing temperatures differed for the other genes, with the optimum for each; ACT: 56 °C, TUB2: 56 °C, CHS: 59 °C and GADPH: 59 °C. Some isolates required altered temperatures and occasionally gave multiple bands, which were exercised separately from an electrophoresis gel and purified. PCR products were examined with electrophoresis, to be stained with on 1.2 % agarose electrophoresis gels and were purified according to the manufacturer’s instructions of a Qiangen purification kit (Qiagen, USA) and for ligation PMD – 18 T vector (Takara Company, Dalian, China) was used. DNA sequencing of the genes were conducted by Sunbiotech Company, Beijing, China.
35 °C
Phylogenetics analysis DNAStar and SeqMan were used to obtain consensus sequences from sequences generated from forward and reverse primers. Combination of multi-locus dataset of three gene regions was aligned using Clustal X1.81 (Thompson et al. 1997). The sequences were further aligned using default settings of MAFFT v.7 (Katoh and Toh 2008; http://mafft.cbrc. jp/alignment/server/) and manually adjusted using BioEdit (Hall 1999) where necessary. A maximum parsimonious analysis (MP) was performed using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded and gaps were treated as missing data. Trees were inferred using the heuristic search option
Fungal Diversity Table 4 Collection data of Colletotrichum species from six provinces
Species
Specimen no.
Host
Location
C. hebeiense
JZB330028 JZB330024 JZB330035 JZB330007 JZB330027
V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Gernischet V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Chardonnay
Qinhuangdao, Hebei, China Shandong, China Shanxi, China Fangshan, Beijing, China Qinhuangdao, Hebei, China
JZB330025 JZB330034 JZB330001 JZB330002 JZB330003 JZB330004 JZB330005 JZB330008 JZB330009 JZB330011 JZB330012 JZB330013 JZB330014 JZB330015 JZB330016 JZB330017 JZB330018 JZB330019
V .vinifera cv. Cabernet Franc V .vinifera cv. Cabernet Gernischet V. vinifera cv. Thompson Seedless V .vinifera cv. Red Globe V .vinifera cv. Syrah V .vinifera cv. Chardonnay V .vinifera cv. Cabernet Franc V .vinifera cv. Centennial Seedless V. vinifera cv. Autumn black V .vinifera cv. Cabernet Gernischet V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Syrah V .vinifera cv. Cabernet Gernischet V .vinifera cv. Cabernet Gernischet V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon
Qinhuangdao, Hebei, China Shanxi, China Changping, Beijing, China Changping, Beijing, China Fangshan, Beijing, China Fangshan, Beijing, China Fangshan, Beijing, China Daxing, Beijing, China Daxing, Beijing, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China
JZB330021 JZB330022 JZB330023 JZB330026 JZB330029 JZB330030 JZB330031 JZB330032
V .vinifera cv. Seyval V .vinifera cv. Seyval V .vinifera cv. Seyval V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon V .vinifera cv. Cabernet Sauvignon V. labruscana cv. Summer Black V. labruscana cv. Summer Black
Yantai, Shandong, China Yantai, Shandong, China Yantai, Shandong, China Qinhuangdao, Hebei, China Qinhuangdao, Hebei, China Qinhuangdao, Hebei, China Changzhou, Jiangsu, China
JZB330033 JZB330036 JZB330037
V. labruscana cv. Summer Black V. labruscana cv. Summer Black V. labruscana cv. Summer Black
C. aenigma
C. viniferum
with Tree Bisection Reconnection (TBR) branch swapping and 1,000 random sequence additions. Maxtrees were set up to 5,000, branches of zero length were collapsed and all multiple parsimonious trees were saved. Tree Length (TL), Consistency Index (CI), Retention Index (RI), Rescaled Consistency index (RC), and Homoplasy index (HI) were calculated for trees generated under different optimality criteria. The robustness of the most parsimonious trees was evaluated by 1,000 bootstrap replications resulting from maximum parsimony analysis (Hillis and Bull 1993). The Kishino-Hasegawa tests (Kishino and Hasegawa 1989) were performed in order to determine whether the trees inferred under different optimality criteria were significantly different.
Changzhou, Jiangsu, China Changzhou, Jiangsu, China Xinjiang, China Xinjiang, China
In addition, Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayers v. 3.1.2 (Ronquist and Huelsenbeck 2003). MrModeltest v. 2.3 (Nylander 2004) was used to carry out statistical selection of best-fit models of nucleotide substitution. Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100th generation. The first 2,000 trees, representing the burn-in phase of the analyses, were discarded and the remaining 8,000 trees used for calculating posterior probabilities (PP) in the majority rule consensus tree. Phylogenetic trees were drawn using Treeview (Page 1996). The alignments and trees are deposited in TreeBASE (www.treebase.org/treebase/ index.html). The fungal strains that were used for this study are listed in the Table 5.
Fungal Diversity Table 5 Strains of Colletotrichum spp. studied, with collection details and GenBank accessions Species
Strain
GenBank accession Numbers ITS
GAPDH
ACT
CHS
TUB2
C.aenigma C. aenigma C. aenigma C. aenigma C. aenigma C. aenigma C. aeschynomenes C. alatae C. alienum C. aotearoa C. asianum C. boninense C. clidemiae C. cordylinicola
ICMP 18608 JZB330025 JZB330007 JZB330034 JZB330027 JZB330035 ICMP 17673 ICMP 12071 ICMP12071 ICMP 18537 ICMP 18580 CBS 123755 ICMP 18658 ICMP 18579
JX010244 KF156860 KF156845 KF156869 KF156862 KF156870 JX010176 JX010190 JX010251 JX010205 FJ972612 JQ005153 JX010265 JX010226
JX010044 KF377492 KF377475 – KF377494 KF377502 JX009930 JX009990 JX010028 JX010005 JX010053 JQ005327 JX009989 JX009975
JX009443 KF377529 KF377512 KF377538 KF377531 KF377539 JX009483 JX009471 JX009572 JX009564 JX009584 JQ005501 JX009537 HM470235
JX009774 KF289005 KF288988 KF289013 KF289007 KF289014 JX009799 JX009837 JX009882 JX009853 JX009867 JQ005414 JX009877 JX009864
JX010389 KF288964 – – KF288974 KF288980 JX010392 JX010383 JX010411 JX010420 JX010406 JQ005588 JX010438 JX010440
C. dianesei C. endophytica C. fructicola C. fructivorum C. grevilleae C. gloeosporioides C. hebeiense C. hebeiense C. horri C. kahawae C. murrayae C. musae C. nupharicola C. proteae C. psidii C. queenslandicum C. rhexiae C. salsolae
CMM4083 LC0324 ICMP 18581 Coll 414 CBS 132879 CBS 112999 JZB330028 JZB330024 ICMP 10492 ICMP 17816 GZAAS5.09506 ICMP 19119 CBS 470.96 CBS 132882 CBS 145.29 ICMP 1778 Coll 1026 ICMP 19051
KC329779 KC633854 JX010165 JX145145 KC297078 JQ005152 KF156863 KF156873 GQ329690 JX010231 JQ247633 JX010146 JX010187 KC297079 JX010219 JX010276 JX145128 JX010242
KC329779 KC832854 JX010033 – KC297010 JQ005239 KF377495 KF377505 GQ329681 JX010012 JQ247609 JX010050 JX009972 KC297009 JX009967 JX009934 – JX009916
KC517298 KF306258 FJ907426 – KC296941 JQ005500 KF377532 KF377542 JX009438 JX009452 JQ247657 JX009433 JX009437 KC296940 JX009515 JX009447 – JX009562
– – JX009866 – KC297056 JQ005326 KF289008 – JX009752 JX009813 – JX009896 JX009835 KC296986 JX009901 JX009899 – JX009863
KC517254 – JX010405 JX145196 KC297102 JQ005587 KF288975 – JX010450 JX010444 JQ247644 HQ596280 JX010398 KC297101 JX010443 JX010414 JX145179 JX010403
C. siamense C. syzygicola C. temperatum C. theobromicola C. ti C. tropicale C.viniferum
ICMP 18578 DNCL021 Coll 883 ICMP 18649 ICMP 4832 ICMP 18653
JX010171 KF242094 JX145159 JX010294 JX010269 JX010264
JX009924 KF242156 – JX010006 JX009952 JX010007
FJ907423 KF157801 – JX009444 JX009520 JX009489
JX009865 – – JX009869 JX009898 JX009870
JX010404 KF254880 JX145211 JX010447 JX010442 JX010407
GZAAS5.08601 GZAAS5.08608 GZAAS5.08616 GZAAS5.08622 JZB330001 JZB330002 JZB330003 JZB330004 JZB330005
JN412804 JN412802 JN412807 JN412806 KF156840 KF156841 KF156842 KF156843 KF156844
JN412798 JN412800 JN412799 JN412796 KF377469 KF377470 KF377471 KF377472 KF377473
JN412795 JN412793 JN412790 JN412791 KF377506 KF377507 KF377508 KF377509 KF377510
– – – – KF288982 KF288983 KF288984 KF288985 KF288986
JN412813 JN412811 JN412809 JN412812 KF288965 – KF288966 – –
C.viniferum C.viniferum C.viniferum C.viniferum C. viniferum C. viniferum C. viniferum C. viniferum
Fungal Diversity Table 5 (continued) Species
Strain
GenBank accession Numbers ITS
GAPDH
ACT
CHS
TUB2
C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum C. viniferum
JZB330008 JZB330009 JZB330011 JZB330012 JZB330013 JZB330014 JZB330015 JZB330016 JZB330017 JZB330018 JZB330019 JZB330021 JZB330022 JZB330023 JZB330026 JZB330029 JZB330030 JZB330031
KF156846 KF156847 KF156848 KF156849 KF156850 KF156851 KF156852 KJ162927 KF156854 KF156855 KF156856 KF156857 KF156858 KF156859 KF156861 KF156864 KF156865 KF156866
KF377476 KF377477 KF377479 KF377480 KF377481 KF377482 KF377483 KF377484 KF377485 KF377486 KF377487 KF377489 KF377490 KF377491 KF377493 KF377496 KF377497 KF377499
KF377513 KF377514 KF377516 KF377517 KF377518 KF377519 KF377520 KF377521 KF377522 KF377523 KF377524 KF377526 KF377527 KF377528 KF377530 KF377533 KF377534 KF377535
KF288989 KF288990 KF288992 KF288993 KF288994 KF288995 KF288996 KF288997 KF288998 KF288999 KF289000 KF289002 KF289003 KF289004 KF289006 KF289009 KF289010 –
KF288962 KF288967 KF288968 – KF288969 KF288970 KF288971 KF288972 KF288963 – – – – KF288973 – KF288976 KF288977 –
C. viniferum C. viniferum C. viniferum C. viniferum C. xanthorrhoeae
JZB330032 JZB330033 JZB330036 JZB330037 ICMP 17903
KF156867 KF156868 KF156871 KF156872 JX010261
– – KF377503 KF377504 JX009927
KF377536 KF377537 KF377540 KF377541 JX009478
KF289011 KF289012 – – JX009823
KF288978 KF288979 – KF288981 JX010448
Pathogenicity testing Single spore isolates were grown on PDA for 7 days at 28 °C. Spores were harvested by adding 10 ml of sterilized distilled water onto the culture, which was then gently swirled to dislodge the conidia. The concentration was adjusted to a 106 conidia/ml using a haemocytometer and used as the standard inoculum for pathogenicity testing. The conidial
suspension was filtered through two layers of muslin cloth (Than et al. 2008). The inoculation method followed Than et al. (2008). Healthy grape fruits that were uniform in size and lacking visible disease symptoms on the outside were washed with tap water and then disinfected in 1 % Sodium hypochlorite for 5– 7 min. Disinfected fruits were washed three times with distilled, sterilized water and then dried with sterilized filter
Table 6 Primers used in this study Gene
Product name
ACT
Actin
Primer
Sequence
ACT-512F ATG TGC AAG GCC GGT TTC GC ACT-783R TAC GAG TCC TTC TGG CCC AT GAPDH Glyceraldehyde-3-phosphate dehydrogenase GDF GCC GTC AAC GAC CCC TTC ATT GA CHS-1
Chitin synthase
TUB2
β-Tubulin 2
ITS
Internal transcribed spacer
Reference Carbone & Kohn (1999) Carbone & Kohn (1999) Templeton et al. (1992)
GDR
GGG TGG AGT CGT ACT TGA GCA TGT
Templeton et al. (1992)
CHS-79F CHS-345R T1 T2 ITS-1F ITS-4
TGG GGC AAG GAT GCT TGG AAG AAG TGG AAG AAC CAT CTG TGA GAG TTG AAC ATG CGT GAG ATT GTA AGT TAG TGA CCC TTG GCC CAGT TG CTT GGT CAT TTA GAG GAA GTA A TCC TCC GCT TAT TGA TAT GC
Carbone & Kohn (1999) Carbone & Kohn (1999) O’Donnell & Cigelnik (1997) O’Donnell & Cigelnik 1997 Gardes & Bruns (1993) White et al. (1990)
Fungal Diversity
paper. Superficial wounds in the epidermis were carried out with a sterile scalpel. For inoculation with the fungus, 4-mmdiameter disks of PDA were removed from the edge of an actively growing culture and placed mycelium-side down on the wound. Fruits inoculated with plugs of sterile PDA were used as a negative control. Fruits that were surface sterilized were pricked with a sterilized insect needle and inoculated with 10 μl of the conidial suspension (106 conidia / ml). Control fruits were inoculated with 10 μl of sterile water. Five fruits from each plant were inoculated with the selected strain. The method of non-wound inoculation involved placing 6 μl of conidial suspension or the mycelium plug on the fruits without wounding. The test was carried out thrice to confirm the results. Fruits were kept individually in a 12 cm diam. Petri dish with a swab of cotton wall containing distilled water to maintain humidity, in plastic containers and incubated at 25–28 °C. Lesion diameters were measured 7 days after inoculation. The fungus was re-isolated from lesions that developed and incubated at room temperature and the resultant cultures were tested with morphological characters with Koch’s postulates. This was carried out in order to confirm the pathogenicity of the species isolated. All infected fruits were sterilized and disposed after autoclaving.
Results Field survey and collection of Colletotrichum species Grapevine anthracnose was common in all vineyards and in all provinces surveyed. The most severely infected vineyards were in Beijing, Hebei and Shandong. Anthracnose of wine grapes was more serious than that of table grapes, for example, diseased cluster ratios of wine grapes were 55–75 % in the BoLongBao Vineyard of Beijing (Fig. 1e), while diseased cluster ratios for table grapes were approximately around 5 %. The diseased cluster ratios for different grape cultivars grown in BoLongBao in 2010 are listed in supplementary Table 1. Thirty-four strains of Colletotrichum were isolated from Vitis vinifera and V. labruscana from 18 vineyards in the provinces (Table 4). Phylogenetic tree and analysis Phylogenetic trees were constructed using combined ACT, GADPH, CHS, TUB2 and ITS gene data set consists of 73 Colletotrichum strains with Colletotrichum boninense (MAFF 305972) as the outgroup taxa. The combined gene alignment comprised of 72 taxa and 1,786 characters including gaps. Parsimony analysis indicated that 1,237 characters were constant, 303 variable characters parsimony-uninformative and 246 characters parsimony-informative. The parsimony
analysis of the data matrix yielded single parsimonious tree (Fig. 2) (TL=960, CI=0.715, RI=0.792, RC=0.566, HI= 0.285). Of our 34 isolates 5 clustered together with ex-type culture of Colletotrichum aenigma B.S. Weir & P.R. Johnst. (Strains JZB330007, JZB330025, JZB330027, JZB330034), twenty-seven isolates clustered together with the type strain of C. viniferum. Strains JZB330028 and JZB330024 formed an independent branch with 90 bootstrap value and 0.98 Bayesian posterior probabilities (PP) support and is shown to be a sister taxon of C. aenigma. Pathogenicity testing The pathogenicity of the Colletotrichum strains was tested on fruits of V. vinifera to confirm Koch’s postulates. The three species recorded in this study showed different virulence. Almost all isolates tested were virulent on the grapevine cultivar Red Globe. Colletotrichum hebeiense isolates JZB330028 and JZB330024 were mildly virulent to Red Globe. C. aenigma isolates had varying virulence to Red Globe. C. aenigma isolate JZB330034 from Shanxi Province was the most virulent. Colletotrichum viniferum showed significant variation in virulence. Isolate JZB330022 from Shandong Province was the most virulent strain in all test isolates. Colletotrichum viniferum isolates JZB330021, JZB330023, JZB330009, JZB330013, JZB330016, JZB330018, JZB330031, and JZB330036 showed high virulence towards Red Globe, while isolates JZB330004, JZB330010, JZB330005 showed weak virulence towards Red Globe (Fig. 4). The C. viniferum isolates in particular had high variation in virulence. This suggests that the virulence of inter and intra species differs. Taxonomy Colletotrichum aenigmaB.S. Weir & P.R. Johnst. Five strains of this species were isolated from fruits causing irregular spots, two from V .vinifera cv. Cabernet Gernischet, one from V .vinifera cv. Cabernet Sauvignon, one from V .vinifera cv. Chardonnay and one from V .vinifera cv. Cabernet Franc. This is the first report of C. aenigma associate with causing grape ripe rot disease. C. hebeiense XH Li, Y Wang, KD Hyde. MMRS Jayawardena, JY Yan, sp. nov. Holotype: MFU14-0627 MycoBank: MB804580 Facesoffunginumber: FOF000307 Fig. 3 Etymology: hebeiense, referring to the Province in which the taxon was identified. Pathogen on fruits. Colonies grown from single conidia on PDA white becoming grey, reverse yellow and becoming black, reaching a maximum of 80 mm diam. in 7 days at
Fungal Diversity Fig. 2 Maximum Parsimonious tree obtained from a heuristic search of the combined ITS, GADPH, CHS, ACT and TUB2 sequence alignment. Bootstrap support values≥50 % and Bayesian posterior probability values≥0.5 are shown at the nodes. C. boninense is used as outgroup. Ex-type and Ex-epitype strains are bolded
28 °C, growth rate 5.4–8.5 mm/day (x =7.9, n=4). Aerial mycelium greyish white, dense, cottony. Asexual developed on PDA. Vegetative hyphae 1.96 μm in diam, hyaline, smooth walled, septate, branched. Conidiomata and setae absent. Conidiogenous cells hyaline, smooth-walled, cylindrical, 5.5–25.8×1.4–5 μm (x =15.45×2.64 μm, n=10), opening 0.5–2 μm diam, collarette<0.4 μm long, periclinal thickening visible to conspicuous. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical to clavate, apex round, base round, contents granular and mostly present at the polar ends leaving a space at the middle, 11.6–15.3×4.47–6.88 μm (x =
13.5×5.41 μm, n=10), L/W ratio=2.6. Appressoria 6.49– 12.98×5.98–10.1 μm (x =8.66×6.39 μm, n=10) pale to medium brown, mostly clavate to subglobose in outline, smooth-walled to undulate in margin, usually single, L/W ratio=1.2. Known Distribution: Shandong, Hebei M a t e r i a l ex a m i n e d: C H I N A , H e b ei P r o vi n ce , Qinhuangdao City, on fruits of Vitis vinifera cv. Cabernet Sauvignon, September 2009, X.H. Li (JZB330028) (MFU14-0627, holotype); ex-type living culture MFLUCC 13–0726=CGMCC3.17464.
Fungal Diversity
Fig. 3 Colletotrichum hebeiense a Conidiogenous cell b Immature conidia c Mature conidia d Germinating spore e to g. Appressoria h Upper view of colony (7 days old) i. Reverse view of colony (7 days old) Scale bars: a-g=5 μm
Additional specimen examined: CHINA, Shandong Province, on fruits of Vitis vinifera cv. Cabernet sauvignon, September 2009, X.H. Li (JZB330024) (MFU14-0628, isotype) ex-isotype living culture MFLUCC 13–0727 = CGMCC3.17465) Notes: Conidia of C. hebeiense are cylindrical to clavate with broadly rounded ends. In C. aenigma the gattulates of conidia are equally spread, while in C. hebeiense the granular contents are mostly present at the polar ends leaving a space in the middle. Appressoria of C. hebeiense are clavate to sub globose in shape while the appressoria of C. aenigma are subglobose, sometimes with few broad ends. Surface of the agar of the C. hebeiense colony shows a greyish black colour towards centre, while in C. aenigma it is pale orange towards
the center. This taxon differs from C. aenigma by eleven base pairs of GADPH and 1 base pair of β-tubulin. Colletotrichum viniferum L.J. Peng, L. Cai, K.D. Hyde & Zi Y. Ying Twenty-six strains of this species were isolated from fruits causing irregular spots, one from V. vinifera cv. Thompson Seedless, one from V .vinifera cv. Red Globe, two from V .vinifera cv. Syrah, one from V .vinifera cv. Chardonnay, one from V .vinifera cv. Centennial Seedless, one from V. vinifera cv. Autumn black, two from V .vinifera cv. Cabernet Gernischet, eight from V .vinifera cv. Cabernet Sauvignon, three from V .vinifera cv. Seyval, five from V. labruscana cv. Summer Black and one from V .vinifera cv. Cabernet Franc. Peng et al. (2012) introduced this new species from grapes.
Fig. 4 Pathogenicity test results: − JZB330022 showed the highest virulence of all isolates, JZB330028 and JZB330024 shows mid virulence, JZB330034 shows the highest virulence among C. aenigma strains,
JZB330004 and JZB330005 strains of C. viniferum showed weak virulence while JZB330013, JZB330016, JZB330018, JZB330021, JZB330023, JZB330031 and JZB330036 show highest virulence
Fungal Diversity
Phylogenetic analysis showed our strains to be conspecific, but some strains of the viniferum group displayed obvious phylogenetic divergence, although their morphology was similar.
Discussion Colletotrichum bunch rot disease that occurs on mature fruits as they ripen was first recorded in USA (Southworth 1891). Colletotrichum gloeosporioides (Penz) was previously considered to be the sole causal organism of the disease (Whitelaw-Weckert et al. 2007). Colletotrichum acutatum J. H. Simmons was later reported as a pathogen causing grape ripe rot in Australia, Japan, Korea and USA (Sung et al. 2008). Colletotrichum crassipes (Speg.) Arx (Hyde et al. 2009b), C o l l e t o t r i c h u m v i t i s I s t v. ( S a c c a r d o 1 9 1 3 ) a n d Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Peng et al. 2012) were also listed as causing ripe rot in grapes. The primary symptoms of grape ripe rot are rotting of the ripe fruit in the vineyard at harvest. Initially the affected grapes will develop circular sunken water soaked brown spots on the skins that subsequently enlarge to the entire berry. Rotting fruits are characteristically covered with salmonpink colored conidial masses (Daykin and Milholland 1984). (Greer et al. 2011) reported that grapes infected with C. acutatum displayed a distinctive mass of orange spores, with fruits shrivelling and compounds results in a bad taste. Winkler et al. (1974) reported that the diseased flesh of berries infected by ripe rot becomes reddish-brown or rose-coloured, and the surface is sunken. Lesions enlarge in concentric zones until they cover the whole berry, which becomes a mass of sticky, salmon coloured conidia, which spread to other parts of the vine by rain splash (Shiraishi et al. 2007) (Fig. 1). The grapevine disease ratio of wine grapes is more severe than that of table grape (unpublished data). Since C. gloeosporioides was epitypified by Cannon et al. (2008) it has been possible to compare strains of Colletotrichum with that of the epitype, using multigene analysis (Cannon et al. 2012; Damm et al. 2012; Weir et al. 2012; Doyle et al. 2013; Gunjan et al. 2013; Hyde et al. 2014). Several Colletotrichum species have been reported as causing disease of V. vinifera including C. acutatum (Shiraishi et al. 2007; Whitelaw-Weckert et al. 2007; Greer et al. 2011), C. aenigma (this paper), C. crassipes (Speg.) Arx (Hyde et al. 2009b), C. fructicola (Peng et al. 2012), C. hebeiense (this paper), C. vitis (Saccardo 1913) and C. viniferum (Peng et al. 2012). Some of these records were published before epitypification of C. gloeosporioides; therefore these species must be treated with caution unless they are confirmed by molecular data.
Thirty-four Colletotrichum strains were isolated from the diseased grape plants. Two strains of the new species C. hebeiense were isolated from stem lesions and from grape fruits. Five strains of C. aenigma and 27 strains of C. viniferum were isolated from anthracnose lesions on grape fruits. None of these species grew at 5 ° C and 40 ° C. In most previous studies the causal agents of grape ripe rot disease were reported to be C. acutatum and C. gloeosporioides (Whitelaw-Weckert et al. 2007; Suzaki 2011; Steel et al. 2011), but in this study these two species were not recorded. This paper provides the first record of C. aenigma as a causal agent of ripe rot disease of grapes and indicates the wide host range of this species. C. aenigma had previously been isolated from lesions of avocado fruit in Israel and Pyrus pyrifolia (ICMP 18686) in Japan (Weir et al. 2012). C. gloeosporioides was previously listed as infecting many fruits, especially in the tropics (Paull et al. 1977; Freeman et al. 1998; AfanadorKafuri et al. 2003; Sangeetha and Rawal 2008). However it has been demonstrated that this species rarely caused anthracnose of tropical fruits and is not a common pathogen in the tropics (Prihastuti et al. 2009; Phoulivong et al. 2010; Rojas et al. 2010; Udayanga et al. 2013). Multigene phylogenetic analyses showed that 27 strains belonged to C. viniferum, but with various genotypes. Peng et al. (2012) introduced this as a single species, while our study suggests that C. viniferum has a phylogenetic divergence. Further studies on C. viniferum are needed to clarify this. Silva et al. (2012) emphasized the use of ‘Powerful genes’ in identifying the Colletotrichum species. There is however, no consensus among mycologists as to which gene markers should be used to define and delimit a species within the species complex (Doyle et al. 2013). In this study, the virulence of different isolates towards the Red Globe cultivar, were significantly different between species and interspecies. Among the three species recorded in the study, C. viniferum is the most virulence species with virulence ranging from high to low, while C. hebeiense and C. aenigma showed mild virulence. The results of this study are important as they show that C. acutatum and C. gloeosporioides were not the causal agents of grape rip rot disease in China, as has been previously understood. In China three species are responsible for grape rip rot disease. C. viniferum contains the most virulent strains and appears to be responsible for most grape ripe rot disease in China. C. viniferum has a certain phylogenetic divergence and is most likely a species complex; further work is required with more discerning genes. C. aenigma was rarely found to cause grape ripe rot disease, while C. hebeiense is a new species, and only caused disease in Hebei and Shandong provinces. Both species cause disease but the disease affect does not appear to be significant.
Fungal Diversity Acknowledgments The study is funded by CARS-30. There are many people to thank for their assistance with this work. The authors would like to thank Miss. Qin Tian for the help given to perform the pathogenicity tests and Miss. K. W. T. Chethana for the help given in phylogenetic analysis. Authors would like to thank the grape cultivators who provided the diseased fruits. K.D. Hyde thanks the National Research Council of Thailand, Colletotrichum grant number 54201020003 and The Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. This work was also supported by a grant from the National Plan of Science and Technology, King Abdulaziz City of Science and Technology, Riyadh, Saudi Arabia (10-Bio-965-02). M.M.R.S. Jayawardena would like to thank H. A. Ariyawansa, D. Udayanga for the support given.
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