Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China

Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jzzhang@zju.edu.cn †E-mail:jzzhang@zju.edu.cn jzzhang@zju.edu.cn Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Strawberry anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry. This study identifies 31 isolates of Colletotrichum spp. which cause strawberry anthracnose in Zhejiang Province and Shanghai City, China. Eleven isolates were identified as C. acutatum , 10 as C. gloeosporioides and 10 as C. fragariae based on morphological characteristics, phylogenetic and sequence analyses. Species-specific polymerase chain reaction (PCR) and enzyme digestion further confirmed the identification of the Colletotrichum spp., demonstrating that these three species are currently the causal agents of strawberry anthracnose in the studied regions. Based on analysis of rDNA internal transcribed spacers (ITS) sequences, sequences of all C. acutatum were identical, and little genetic variability was observed between C. fragariae and C. gloeosporioides . However, the conservative nature of the Mvn I specific site from isolates of C. gloeosporioides was confirmed, and this site could be used to differentiate C. gloeosporioides from C. fragariae . Fragaria × ananassa Taxonomic identification Colletotrichum acutatum C. gloeosporioides C. fragariae Introduction 1 Colletotrichum species cause economically significant diseases including wilts, rots and anthracnose in production areas of strawberry ( Fragaria × ananassa Duch.) worldwide (Buddie et al., 1999 1999 ; Debode et al., 2009 2009 ). Three species have been reported as causal agents of strawberry anthracnose: C. acutatum Simmonds (teleomorph Glomerella acutata J.C. Guerber & J.C. Correll), C. gloeosporioides (Penz.) Penz. & Sacc. in Penz. [teleomorph Glomerella cingulata (Stoneman) Spauld. & Schrenk], and C. fragariae Brooks (Gunnell and Gubler, 1992 1992 ; Denoyes-Rothan et al., 2003 2003 ). C. gloeosporioides and C. acutatum are distributed worldwide on a number of hosts, while C. fragariae has a very narrow host range (MacKenzie et al., 2008 2008 ). The disease causes up to 80% of plant deaths in nurseries and over 50% of yield losses in strawberry production fields (Sreenivasaprasad and Talhinhas, 2005 2005 ). Likewise, in the Zhejiang region, nearly 50% of seedling deaths in nurseries and over 40% of yield losses in strawberry production fields are caused by this disease according to our investigations. Strawberry is cultivated extensively in some districts of China and severe levels of strawberry anthracnose have been prevalent for many years. Shao ( 1992 1992 ) first observed strawberry anthracnose disease in Shanghai on cv. Lego introduced from Japan and the pathogen was identified as C. acutatum (Dai et al., 2006 2006 ). Subsequently, Ye et al. ( 1997 1997 ) reported that the pathogens responsible for strawberry anthracnose in Shanghai belonged to C. fragariae and C. gloeosporioides , based on pathogen morphological characteristics. However, based on a combination of morphology and rDNA internal transcribed spacers (ITS) sequence analysis, C. acutatum and C. gloeosporioides were identified as the causal agents (Ren et al., 2008 2008 ). Zhejiang Province, adjacent to Shanghai, is an important strawberry-producing area with about 6 000 ha under cultivation. Although the disease occurs every year, isolation and molecular identification to clarify which Colletotrichum spp. are responsible for anthracnose disease in the area have not been performed. The main purpose of this study was to establish which species are currently causing anthracnose disease in strawberry in Zhejiang Province and to compare them with isolates from strawberry plants from Shanghai. Materials and methods 1 Fungal isolates 2 Colletotrichum spp. were isolated from diseased strawberry plants (fruit, vegetative parts, and root crowns) from different cultivars and sites from the summer of 2006 to the spring of 2007. Infected plant organs were surface-sterilized [0.5% (w/v) sodium hypochlorite] for 2–5 min before they were placed onto potato dextrose agar (PDA) containing streptomycin. Isolates selected were transferred to PDA, and single conidial isolates were prepared from each isolate. Cultures were grown on PDA at 25 °C. Plugs (5 mm) were cut from the margin of fresh colonies and stored in 20% (w/v) glycerol in a freezer at −70 °C. Morphological examination 2 Isolates representative of different sites were selected for morphological species identification. These isolates were cultured on PDA in darkness at 25 °C for 5 d. Mycelial discs (5 mm) were taken from the growing edge of colonies and transferred onto plates of strawberry leaf agar (SLA) (Gunnell and Gubler, 1992 1992 ). The SLA plates were placed in an incubator under continuous fluorescent light at 25 °C. Conidia were harvested after 14 d, and setae after 22–30 d, from acervuli produced on the leaf pieces. Conidia were mounted in water, and examined for size and shape, using a Zeiss Axiophot 2 microscope with an Axiocam CCD camera and AxioVision digital imaging software (AxioVision Software Release 3.1, version 3-2002; Carl Zeiss Vision Imaging Systems). Colony color was determined after 7 d on PDA at 25 °C under conditions of alternating cycles of 12 h light and 12 h darkness. DNA extraction 2 Fungal isolates were cultured in darkness at 25 °C for 5 d. Mycelial disks were excised from the margins of colonies and inoculated into 100 ml of liquid growth media potato dextrose broth (PDB) in 250 ml flasks, shaken at 200 r/min at 25 °C for 5 d. Mycelia were harvested by filtration, freeze-dried, ground to a fine powder in liquid nitrogen, and then stored at −70 °C. About 50 mg of mycelial powder was removed into a sterile 1.5-ml tube, rehydrated in 600 μl of 2× cetyltrimethylammonium bromide (CTAB) buffer [100 mmol/L Tris (pH 8.0), 1.4 mol/L NaCl, 30 mmol/L ethylenediaminetetraacetic acid (EDTA), 2% (w/v) hexadecyltrimethylammonium bromide], and incubated in a water bath at 65 °C for 30–60 min. Following a phenol/chloroform extraction, the genomic DNA was precipitated by isopropanol in the presence of sodium acetate. Genomic DNA was visualized in 1% (w/v) agarose gels after ethidium bromide staining. DNA concentration was determined by ultraviolet (UV) spectroscopy. Amplification of rDNA-ITS regions 2 The ITS regions of rDNA were amplified using the primers ITS6 (5′-GAAGGTGAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) for direct sequencing (Cooke and Duncan, 1997 1997 ). Polymerase chain reaction (PCR) amplifications were performed in a total volume of 50 μl containing 40 mmol/L of Tris-HCl (pH 8.4), 100 mmol/L of KCl, 3 mmol/L of MgCl 2 , 200 μmol/L of each dNTP, 1 μmol/L of each primer, and 0.5 U of Taq (Sangon Co., Shanghai, China). PCR amplifications were carried out using a PTC-150 MiniCycler (MJ Research Corp., America). Following an initial denaturation at 95 °C for 4 min, the DNA templates were amplified for 35 cycles. Each cycle consisted of a denaturation step at 95 °C for 1 min, an annealing step at 52 °C for 1 min, and an extension step at 72 °C for 1 min. A final extension step (72 °C for 10 min) was included at the end. Aliquots of 4 μl of the amplification products were electrophoresed on 1% (w/v) agarose gels, and the PCR products were subsequently stained with ethidium bromide. Successful PCR amplification products resulted in a single DNA band (corresponding to ~560 bp). The PCR products were purified using a Biolight PCR Purification Kit (Shanghai Biolight Technology Co. Ltd., China) according to the manufacturer’s instructions. Sequencing and analysis of rDNA-ITS regions 2 The purified PCR products were submitted to the Hangzhou Genomics Institute for sequencing in both directions. Sequence files were assembled and edited, and consensus sequences were constructed using DNAMAN 4.0 (Lynnon bioSoft). The ITS sequences were submitted to the GenBank database. Sequences were deposited in GenBank (accession numbers given in Table 1 1 ) and compared against those sequences already found in the databases using the BLAST search. The ITS sequences for reference isolates were downloaded from the GenBank database (Table 1 1 ), along with that of Neurospora crassa (M13906) as an outgroup. Sequences were aligned using CLUSTAL X (Thompson et al., 1997 1997 ). Phylogenetic analyses were performed using test version 4.0b10 (PPC) of PAUP (Swofford, 2002 2002 ). Phylogenetic trees were inferred from the ITS sequence data set using parsimony analysis and all characters were weighted equally and gaps were treated as missing data. Clade stability was assessed using a bootstrap analysis with 1 000 replicates, each with 10 replicates of random stepwise addition of taxa. Cultivar/Organ from which fungus isolated Isolate/strain Species Host plant Geographical origin GenBank accession No. Reference 6 Isolates used WY01 C. gloeosporioides Toyonoka/leaf * * Jiande City, Zhejiang Province FJ608625 In this study WY02 C. gloeosporioides Toyonoka/petiole Jiande City, Zhejiang Province FJ608626 In this study WY03 C. gloeosporioides Toyonoka/leaf Jiande City, Zhejiang Province FJ608627 In this study WY04 C. gloeosporioides Toyonoka/leaf Jiande City, Zhejiang Province FJ608628 In this study WY05 C. gloeosporioides Toyonoka/leaf Zhaotun, Shanghai City FJ608629 In this study WY06 C. gloeosporioides Toyonoka/crown Zhaotun, Shanghai City FJ608630 In this study WY07 C. gloeosporioides Hongjia/leaf Hangzhou City, Zhejiang Province FJ608631 In this study WY08 C. gloeosporioides Toyonoka/leaf Hangzhou City, Zhejiang Province FJ608632 In this study WY09 C. gloeosporioides Toyonoka/crown Jiande City, Zhejiang Province FJ608633 In this study WY10 C. gloeosporioides Toyonoka/petiole Hangzhou City, Zhejiang Province FJ608634 In this study WY11 C. fragariae Toyonoka/crown Jiande City, Zhejiang Province FJ608635 In this study WY12 C. fragariae Toyonoka/leaf Jiande City, Zhejiang Province FJ608636 In this study WY13 C. fragariae Toyonoka/crown Zhaotun, Shanghai City FJ608637 In this study WY14 C. fragariae Toyonoka/leaf Zhaotun, Shanghai City FJ608638 In this study WY15 C. fragariae Toyonoka/crown Zhaotun, Shanghai City FJ608639 In this study WY16 C. fragariae Tochiotome/crown Hangzhou City, Zhejiang Province FJ608640 In this study WY17 C. fragariae Toyonoka/leaf Hangzhou City, Zhejiang Province FJ608641 In this study WY18 C. fragariae Akihime/petiole Hangzhou City, Zhejiang Province FJ608642 In this study WY19 C. fragariae Toyonoka/leaf Hangzhou City, Zhejiang Province FJ608643 In this study WY20 C. fragariae Toyonoka/crown Jiande City, Zhejiang Province FJ608644 In this study WY21 C. acutatum Toyonoka/leaf Zhaotun, Shanghai City FJ608645 In this study WY22 C. acutatum Toyonoka/fruit Zhaotun, Shanghai City FJ608646 In this study WY23 C. acutatum Toyonoka/leaf Zhaotun, Shanghai City FJ608647 In this study WY24 C. acutatum Toyonoka/fruit Zhaotun, Shanghai City FJ608648 In this study WY25 C. acutatum Toyonoka/fruit Jiande City, Zhejiang Province FJ608649 In this study WY26 C. acutatum Toyonoka/leaf Jiande City, Zhejiang Province FJ608650 In this study WY27 C. acutatum Toyonoka/leaf Jiande City, Zhejiang Province FJ608651 In this study WY28 C. acutatum Toyonoka/fruit Jiande City, Zhejiang Province FJ608652 In this study WY29 C. acutatum Toyonoka/leaf Jiande City, Zhejiang Province FJ608653 In this study WY30 C. acutatum Toyonoka/leaf Jiande City, Zhejiang Province FJ608654 In this study WY31 C. acutatum Toyonoka/petiole Jiande City, Zhejiang Province FJ608655 In this study 6 Reference isolates IMI 345047 C. fragariae Strawberry USA AJ536223 Martinez-Culebras et al., 2003 2003 IMI 348167 C. fragariae Strawberry USA AJ536222 Martinez-Culebras et al., 2003 2003 CG 231 C. gloeosporioides Strawberry USA AF272780 Freeman et al., 2001 2001 IMI 324983 Glomerella cingulata Strawberry USA AJ536226 Martinez-Culebras et al., 2003 2003 IMI 347765 G. tucumanensis Saccharum sp. Bangladesh AJ536231 Martinez-Culebras et al., 2003 2003 IMI 345051 G. cingulata Strawberry Canada AJ536224 Martinez-Culebras et al., 2003 2003 IMI 345027 G. acutata Strawberry France AJ536199 Martinez-Culebras et al., 2003 2003 IMI 348160 G. acutata Strawberry USA AJ536200 Martinez-Culebras et al., 2003 2003 IMI 360086 G. acutata Strawberry Japan AJ536205 Martinez-Culebras et al., 2003 2003 IMI 345575 G. acutata Strawberry New Zealand AJ536215 Martinez-Culebras et al., 2003 2003 MAFF 511343 G. graminicola unknown Japan AB057436 Moriwaki et al., 2002 2002 CBS 151.28 G. lindemuthiana Phaseolus vulgaris unknown AJ301946 Nirenberg et al., 2002 2002 BBA 70709 C. trifolii Trifolium sp. USA AJ301941 Nirenberg et al., 2002 2002 UQ 343 C. destructivum Unknown Australia AF451908 Unpublished STE-U 5304 C. capsici Arachis hypogaea Tanzania AY376526 Lubbe et al., 2004 2004 STE-U 5301 C. coccodes Lycopersicon esculentum Zimbabwe AY376528 Lubbe et al., 2004 2004 STE-U 5299 C. dematium Peperomia sp. Unknown AY376531 Lubbe et al., 2004 2004 BBA 70535 C. fuscum Digitalis lanata unknown AJ301938 Nirenberg et al., 2002 2002 STE-U 5296 C. orbiculare Xanthium spinosum Argentina AY376541 Lubbe et al., 2004 2004 STE-U 5294 G. truncata Arachis hypogaea Gambia AY376543 Lubbe et al., 2004 2004 PCR with C. acutatum -specific primers 2 PCR primers for detection of the pathogen included the ITS4 primer (5′-TCCTCCGCTTATTGATATGC-3′) coupled with the specific primer for C. acutatum ( Ca Int2) (5′-GGGGAAGCCTCTCGCGG-3′) (Sreenivasaprasad et al., 1996 1996 ; White et al., 1990 1990 ). The genomic DNA of Trichoderma virens was used as a negative control. C. acutatum -specific PCR reactions were performed in a total volume of 50 μl, containing 50 ng of genomic DNA, 50 mmol/L KCl, 10 mmol/L Tris-HCl, 100 μmol/L of each dNTP, 1.5 mmol/L MgCl 2 , 1 U of Taq DNA polymerase (TaKaRa Corp., Japan), and 0.5 μmol/L of each primer. The reaction mixtures were incubated in a PTC-150 MiniCycler (MJ RESEARCH Corp., America) for 35 cycles consisting of 1 min at 95 °C, 30 s at 54 °C, and 1 min at 72 °C. Amplification products were separated in 1% (w/v) agarose gels, stained with the nucleic acid stain GoldView™ (Shanghai SBC Genetch Co. Ltd., China), and viewed under UV light for detection of amplification products. PCR reaction and DNA digestion 2 The 5.8S-ITS regions of representative isolates of C. gloeosporioides and C. fragariae , identified by morphological characteristics, were amplified by PCR using universal primers ITS1 (5′-GCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (Martinez-Culebras et al., 2000 2000 ). The genomic DNA of Trichoderma virens was used as a negative control. PCR reactions were performed in a total volume of 50 μl, containing 150–200 ng of DNA, 50 mmol/L KCl, 10 mmol/L Tris-HCl, 200 μmol/L of each dNTP, 1 μmol/L of each primer, 1.5 mmol/L MgCl 2 , and 2.5 U of Taq DNA polymerase (Sangon Co., Shanghai, China). The reaction mixtures were incubated in a PTC-150 MiniCycler (MJ RESEARCH Corp., America). Following an initial denaturation at 95 °C for 4 min, the DNA templates were amplified for 35 cycles. Each cycle consisted of a denaturation step at 95 °C for 1 min, an annealing step at 56 °C for 1 min, and an extension step at 72 °C for 1 min. A final extension step (72 °C for 10 min) was included at the end. Amplification products were separated on 1% (w/v) agarose gels in Tris-acetate-EDTA (TAE) buffer. To specifically identify strawberry isolates of C. gloeosporioides , PCR products from the ITS1/ITS4 primer pair were digested with the restriction enzyme Mvn I (supplied under the name Bsh1236I by Fermentas, MBI, Republic of Lithuania) at 37 °C for 16 h (Martinez-Culebras et al., 2003 2003 ). PCR products for species-specific identification and restriction fragments from the PCR fragment primed with ITS1 and ITS4 were separated on 1% and 3% (w/v) agarose gels, respectively, with 0.5× Tris-borate-EDTA (TBE) buffer. After electrophoresis, gels were stained with ethidium bromide (0.5 μg/ml), and the DNA bands were visualized with UV light. DNA sizes were estimated by comparison with a DNA length standard (100–3 000 bp molecular marker, Bio Basic Inc., Canada), as described by Martinez-Culebras et al. ( 2003 2003 ). Results 1 Morphological comparisons 2 Isolates of Colletotrichum spp. obtained from fruit, vegetative parts, and root crowns from different cultivars showing typical anthracnose symptoms, were selected for morphological identification. Based on taxonomic criteria of morphological characteristics (colony characters, conidial and setal morphology and production of a teleomorph) (Gunnell and Gubler, 1992 1992 ), 10 C. gloeosporioides , 10 C. fragariae and 11 C. acutatum isolates were recognized among 31 isolates (Table 1 1 ). On SLA, most of the conidia of C. fragariae isolates were narrowly obovate, straight or occasionally slightly curved, [(13.0–) 14.5–16.5 (–19.0)] μm×[(3.5–) 4.2–4.8 (–5.5)] μm (mean=16.0 μm×4.5 μm) (Fig. 1a 1a ). Conidia of C. acutatum isolates were elliptic to fusiform, [(11.0–) 11.2–15.5 (–16.0)] μm×[(3.8–) 4.1–4.7 (–5.0)] μm (mean=13.5 μm×4.4 μm) (Fig. 1b 1b ). The conidia of C. gloeosporioides isolates were oblong with obtuse ends, straight (Fig. 1c 1c ), and were shorter and broader than those of C. fragariae and C . acutatum , [(11.8–) 12.5–14.8 (–16)] μm×[(4.6–) 5.0–6.0 (–6.8)] μm (mean=13.5 μm×5.5 μm). The shapes of the conidia of C. fragariae , C. gloeosporioides and C . acutatum agreed with previous descriptions (Gunnell and Gubler, 1992 1992 ), but the average conidial size of each of the three species was slightly different from the range described by Gunnell and Gubler ( 1992 1992 ), namely 18 μm×4 μm, 15.5 μm×3.7 μm, and 15 μm×4.3 μm for C. fragariae , C. acutatum , and C. gloeosporioides , respectively. Mature setae of C. fragariae were dark brown, uniform in width except for the apical cells which functioned as phialides, and produced conidia in individual isolates (Figs. 1e and 1f 1e and 1f ). Setae of C. gloeosporioides were dark brown, gradually tapered along their entire length to their apices, straight, produced singly, and did not produce conidia (Fig. 1g 1g ). Setae of C. acutatum were brown to dark brown, tapered, generally aseptate (Fig. 1h 1h ), did not produce conidia, and were shorter than those of C. fragariae and C. gloeosporioides . The isolates of C. gloeosporioides readily produced perithecia containing asci and ascospores (Fig. 1d 1d ), but none of the isolates of C. fragariae or C. acutatum produced perithecia. On PDA, the colony color of C. fragariae isolates ranged from beige to dark grey. Colonies of C. gloeosporioides isolates showed a dense, white mycelial growth turning to a dark olive-grey color. The colony color of C. acutatum isolates was white for 4–5 d but later became grey-brown. Sequence analysis of the ribosomal DNA spacer sequence 2 Sequence alignment showed that among 11 isolates (WY21–WY31), all sequences were identical to that of a strawberry isolate of C. acutatum (IMI 360086) from Japan. Among the other isolates (WY01–WY20), all sequences were very similar. Reference sequences were selected cautiously from GenBank, with only those from voucher specimens being selected. The final ITS/5.8S dataset included 52 sequences with 530 characters after alignment. Phylogenetic analysis showed that the 31 isolates in this study were clustered into two groups, group I and group II (Fig. 2 2 ). In group I, 11 isolates, identified as C. acutatum based on morphological characteristics, were clustered together with 4 reference isolates of C. acutatum (teleomorph G. acutata ) from strawberry. The isolate IMI 345575 (New Zealand) formed a single subgroup and did not group with any others. While 11 isolates produced a clade with the reference sequence of C. acutatum IMI 360086 from Japan, they were clustered with the isolates IMI 348160 (USA) and IMI 345027 (France) to form another subgroup with 93% bootstrap support, as described in Table 1 1 of Sreenivasaprasad and Talhinhas ( 2005 2005 ). In group II, 20 isolates (WY01–WY20), classified as C. fragariae and C. gloeosporioides based on morphology, were in a poorly resolved group that included the reference isolates of C. fragariae and C. gloeosporioides (teleomorph G. cingulata ) from strawberry (Fig. 2 2 ). Data were analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Bootstrap values ≥50% are shown above or below branches. The numbers near each branch represent percentages out of 100 bootstrap replications However, by analyzing 5.8S-ITS sequences, an Mvn I restriction site was found to be present within the ITS1 region in only 10 isolates (WY01–WY10) and no Mvn I restriction sites were found in the other isolates. Previous studies have shown an Mvn I restriction site present within the ITS1 region of the 5.8S-ITS sequences of only C. gloeosporioides isolates but not in other isolates of Colletotrichum spp. from strawberry plants (Martinez-Culebras et al., 2000 2000 ; 2003 2003 ). So in group II, 10 isolates (WY01–WY10) were related to C. gloeosporioides and the other isolates were attributed to C. fragariae , consistent with morphological identification (Table 1 1 ). The ITS1-5.8S-ITS2 rDNA sequences of the 10 C. gloeosporioid es isolates fell into two groups: the first group comprised sequences from eight isolates (WY01, WY02, WY03, WY04, WY07, WY09, WY10 and WY06) and the second group comprised sequences from the remaining two (WY05 and WY08). Sequences within each group were identical but the two groups differed by three base pairs. Sequences of the C. fragariae isolates fell into three groups comprising four (WY18, WY20, WY12 and WY13), three (WY14, WY15 and WY16), and three (WY19, WY17 and WY11) isolates, respectively. Again, sequences within each group were identical but the three groups differed by three base pairs. Analysis of the rDNA ITS sequences revealed that the Mvn I restriction site in C. gloeosporioides isolates was conservative and a useful tool for distinguishing C. gloeosporioides from C. fragariae from strawberry plants. It also showed the genetic diversity of population structures in C. gloeosporioides and C. fragariae . Based on 5.8S-ITS sequences and phylogenetic analysis, the 31 isolates could be classified clearly into three species. Eleven isolates (WY21–WY31) were identified as C. acutatum (all identical), 10 (WY01–WY10) as C. gloeosporioides (8 clustering in a group that is 3-bp different from the remaining 2) and 10 (WY11–WY20) as C. fragariae (4 clustering in a group that is 3-bp different from the remaining 6 that are in two groups of 3), consistent with the results of morphological identification (Table 1 1 ). Based on host origin, 7 isolates of C. acutatum , 8 of C. gloeosporioides and 7 of C. fragariae were isolated from the Zhejiang area, and 4, 2 and 3, respectively, from the Shanghai region. This confirms that the species of Colletotrichum causing anthracnose disease of strawberry in Zhejiang and Shanghai are the same. Species-specific PCR and enzyme digestion 2 Representative isolates, WY01, WY06 and WY08 for C. gloeosporioides , WY12, WY19 and WY14 for C. fragariae , and WY27, WY29 and WY31 for C. acutatum , were selected for species-specific PCR and enzyme digestion. The nine representative isolates were detected by using the C. acutatum -specific primer Ca Int2 in conjunction with the ITS4 primer. About 490-bp fragments were amplified from the genomic DNA of the C. acutatum isolates but no amplification products were produced from the isolates representative of C. gloeosporioides or C. fragariae , along with the negative control ( Trichoderma virens ) (Fig. 3 3 ). This species-specific PCR result further confirmed the species identity of C. acutatum , consistent with phylogenetic and morphological identification. Lanes 1–3 correspond to representative C. acutatum isolates; Lanes 4–6 are the representative C. gloeosporioides isolates; Lanes 7–9 correspond to representative C. fragariae isolates; Lane 10 is a negative control ( Trichoderma virens genomic DNA); Lane M corresponds to the 100–3 000 bp molecular weight marker (Bio Basic Inc., Canada) The isolates representative of C. fragariae and C. gloeosporioides were analyzed using the endonuclease Mvn I. The restriction endonuclease Mvn I cut all C. gloeosporoides PCR products amplified by PCR using the universal primers ITS1 and ITS4 to produce two fragments of about 480 and 100 bp. The species-specific restriction pattern could be obtained from the C. gloeosporioides isolates but this endonuclease did not cut any of the C. fragariae isolates (Fig. 4 4 ). This result confirms the established method for species identification using Mvn I digestion data (Martinez-Culebras et al., 2000 2000 ; 2003 2003 ). Lanes 1–3 correspond to representative C. fragariae isolates; Lanes 4–6 correspond to representative C. gloeosporioides isolates; Lane 7 is a positive control ( Trichoderma virens genomic DNA); Lane M corresponds to the 100–3 000 bp molecular weight marker (Bio Basic Inc., Canada) Discussion 1 Most strawberry cultivars grown in Zhejiang Province and Shanghai City were introduced from Japan and the cv. Toyonoka is presently the principal cultivar. Almost all of the isolates of Colletotrichum spp. responsible for the anthracnose disease originated from this cultivar. In this study, we identified the Colletotrichum spp. causing strawberry anthracnose, and aimed to determine whether the same or different species occur in strawberry nurseries and production fields in Zhejiang and Shanghai. Traditionally, three species from strawberry were considered to be differentiated by morphological characters (Gunnell and Gubler, 1992 1992 ). However, morphological criteria can be unreliable and non-predictive, because of highly variable morphological characteristics (Sreenivasaprasad et al., 1996 1996 ; Sutton, 1992 1992 ; Johnston and Jones, 1997 1997 ). So Ye et al. ( 1997 1997 ) possibly failed to distinguish C. gloeosporioides from C. fragariae from strawberry plants from Shanghai. Molecular genetic studies have provided useful data for clarifying the systematics of the genus Colletotrichum (Martinez-Culebras et al., 2003 2003 ). However, our studies showed that the divergence of 5.8S-ITS region sequences among isolates of C. fragariae and C. gloeosporioides was too low to separate them confidently as different species, confirming the observations of Sherriff et al. ( 1995 1995 ), Sreenivasaprasad et al. ( 1996 1996 ) and Martinez-Culebras et al. ( 2003 2003 ). Similarly, Ren et al. ( 2008 2008 ) found that the ITS sequences of 17 isolates from strawberry were 99% identical, but they all were mistaken for C. gloeosporioides . Although the similarity between the nucleotide sequences of C. gloeosporioides and C. fragariae prevented the development of specific primers for these species based on the 5.8S-ITS region, Martinez-Culebras et al. ( 2000 2000 ) found that there was an Mvn I specific site among ITS1 region of isolates of only C. gloeosporioides and not C. fragariae . This trait was confirmed subsequently by different original sequence data and could be used for differentiating C. gloeosporioides from C. fragariae (Martinez-Culebras et al., 2003 2003 ). Martinez-Culebras et al. ( 2003 2003 ) pointed out that this conclusion still needed to be confirmed by studying a higher number of isolates of C. fragariae and C. gloeosporioides from strawberry and also C. gloeosporioides from different hosts (Martinez-Culebras et al., 2003 2003 ). Our results provide new evidence that the Mvn I site within the ITS1 region from isolates of C. gloeosporioides from strawberry is conservative. In C. acutatum , phylogenetic analysis based on 5.8S-ITS region sequences indicated that different original isolates of pathogens of strawberry form a polyphyletic taxon, consistent with other observations (Martinez-Culebras et al., 2003 2003 ; Freeman et al., 2000 2000 ; 2001 2001 ). The sequences of our 11 isolates of C. acutatum from Zhejiang and Shanghai, however, were identical to that of a strawberry isolate of C. acutatum (IMI 360086) from Japan. Although currently there are over 600 rDNA-ITS sequences from C. acutatum isolates available in public databases, a BLAST search showed that our sequences were identical to those of AL-9 (GenBank accession No. AB219025), AL-5 (GenBank accession No. AB219024) and IMI 360086 found only on strawberry in Japan. This suggests that this species has been introduced into Zhejiang and Shanghai from Japan through infected propagation material and has rapidly become established locally. However, analysis of sequences showed that there were two sequence types in C. gloeosporioides and three in C. fragariae . These may reflect the genetic diversity of population structures within them. Within C. gloeosporioid es, two sequence types were distributed in Zhejiang and Shanghai but based on the frequency of isolation, and the sequence types of the isolates WY01, WY02, WY03, WY05, WY06, WY07, WY09 and WY10 might be the predominant forms. Within C. fragariae , there were three sequence types of which two were distributed in Zhejiang and Shanghai and one, from isolates WY19, WY17 and WY11, was found only in Zhejiang (Table 1 1 ). Based on isolation data (Table 1 1 ), C. acutatum can be isolated from fruits, leaves and petioles, C. gloeosporioides from leaves, petioles and root crowns, and C. fragariae from root crowns, leaves and petioles. Differences in pathogenicity among them, however, still need to be evaluated by pathogenicity tests. Although pathogenicity tests had been performed and typical symptoms could be seen, control transplant seedlings also could produce similar symptoms. Apparently, Colletotrichum spp. may be present in the form of latent infections in strawberry. The presence of latent infections of Colletotrichum spp. in strawberry was detected by a freeze treatment method and Colletotrichum spp. could be found on up to 50% of leaves and 10% of petioles in transplant seedlings (Mertely and Legard, 2004 2004 ). For this reason, currently we still have not obtained reliable results from pathogenicity tests. However, this may be resolved in future studies by using pathogen-free strawberry plants obtained by propagating plantlets from meristem culture. Based on morphological characters, phylogenetic analysis, species-specific PCR and enzyme digestion, three species, C. acutatum , C. gloeosporioides and C. fragariae , are considered the cause of anthracnose in strawberry-growing areas of Zhejiang and Shanghai. Although molecular studies are helpful for resolving relationships within the genus Colletotrichum , studies to date can be regarded as only preliminary (Photita et al., 2005 2005 ). Many questions remain regarding the interspecies relationships within the genus, such as between C. gloeosporioides and C. fragariae . 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First report on species of strawberry anthracnose pathogen in Shanghai suburb and resistance of some strawberry cultivars to anthracnose 10.1631/jzus.B0900174 Project supported by the National Natural Science Foundation of China (No. 30571208) and the Key Scientific and Technological Project of Hangzhou City (No. 200432239), China 2009-06-22 2009-12-15 2009-11-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 11