Print version ISSN 1982-5676
Trop. plant pathol. vol.37 no.3 Brasília May/June 2012
RESEARCH ARTICLE ARTIGO
New species and notes of Colletotrichum on daylilies (Hemerocallis spp.)
Youlian YangI,II; Zuoyi LiuI; Lei CaiIII; Kevin D. HydeIV
IGuizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou 550006, P. R. China
IIDepartment of Life Science, Liupanshui Normal University, Shuicheng, Guizhou 553006, P. R. China
IIIState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No.10, North 4th Ring Road West, Beijing 100190, P. R. China
IVInstitute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand; Visiting Professor, Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
Nine Colletotrichum strains were isolated from diseased and dead stalks of Hemerocallis species (daylilies) from Guizhou, Guangxi, and Liaoning provinces in China. Morphological characteristics and multilocus phylogenetic analysis of ACT, CHS I, GPDH, ITS, and TUB 2 indicate that these strains represent four taxa. Colletotrichum hemerocallidis is a new species that is described, illustrated, and compared with similar species. Colletotrichum gloeosporioides, C. liriopes, and C. spaethianum are also recorded on Hemerocallis species.
Key words: anthracnose, multilocus phylogeny, systematics.
Hemerocallis species (including H. fulva (L.) Linn., H. citrina Baroni, and other species or cultivars) are economically important as food plants in China, Japan, Korea, Thailand, and Vietnam, being known as "yellow flower vegetables" or "golden needles" in China (Zhou et al., 1994; Staples & Kristiansen, 1999; Zhang & Chen, 2008). Species are also cultivated and bred worldwide for their showy flowers and ability to adapt to a wide range of soils and climates (Munson, 1989; Tomkins et al., 2010), and are used as Traditional Chinese Medicine (Zhu et al., 2008; Ma et al., 2010).
Hemerocallis production has often been limited by anthracnose disease (Jiang et al., 1993). Disease outbreaks can be severe with 100% of some ornamental Hemerocallis species being infected (Jiang et al., 1993). Six Colletotrichum species have previously been reported as causal agent of anthracnose of Hemerocallis including C. dematium (Pers.) Grove on Hemerocallis sp. in the United States (Farr & Rossman, 2011), C. gloeosporioides (Penz.) Penz. & Sacc. on H. citrina in China (Gu et al., 2007), C. liliacearum Ferraris on H. fulva var. kwanso Regel in China (Jiang et al., 1993; Farr & Rossman, 2011), C. lilii Plakidas ex Boerema & Hamers on Hemerocallis sp. in the United States (Farr & Rossman, 2011), C. spaethianum (Allesch.) Damm, P.F. Cannon, & Crous, on Hemerocallis sp. in New Zealand, and Colletotrichum sp. (CBS 125338) on H. fulva in Canada (Damm et al., 2009). There is, however, little knowledge concerning the Colletotrichum species associated with Hemerocallis in China. The objective of this paper was to characterize Colletotrichum species associated with these plants in China based on morphology and multilocus DNA sequence data.
MATERIALS AND METHODS
Isolation of Colletotrichum
Dead leaves and stalks of Hemerocallis citrina, H. fulva, and H. fulva var. kwanso with anthracnose lesions were collected in Guizhou, Guangxi, and Liaoning provinces in China from 2008 to 2011 (Table 1). Single-spore isolates were obtained using the procedure described by Choi et al. (1999) and Chomnunti et al. (2011). Pure cultures were stored at 4ºC on PDA slants. Isolates are deposited in Guizhou Academy of Agricultural Sciences, China, and the China General Microbiological Culture Collection Center (CGMCC).
Morphological and cultural characterization
Starter cultures were prepared by growing each isolate on PDA at 25ºC in darkness for five days. Five replicate cultures of each isolate were prepared by aseptically cutting disks from the actively growing edge of the starter culture using a sterile cork borer. Each plug was placed onto PDA plates (90 mm × 15 mm) and grown in alternating light and dark at 25ºC (Sutton, 1980). To induce sporulation, plugs of actively growing mycelium were placed on to the surface of synthetic nutrient-poor agar medium (SNA: 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 20 g agar, 1 L tap water) with autoclaved filter paper and double-autoclaved stems of Sium suave Walt. [(Apiaceae; comp. method proposed by Damm et al. (2009), using stems of Anthriscus sylvestris (L.) Hoffm., Apiaceae)] and incubated in the same conditions. Colony diameter was measured at day seven. After 7-10 days, the size and shape of 50 conidia harvested from the cultures were measured, and their mean and standard deviations (SD) were calculated. The colour of the conidial masses and zonation were recorded at day seven (Than et al., 2008). Mycelial appressoria were produced and measured using a slide culture technique (Sutton, 1980). Conidial appressoria were also induced by placing conidia in two drops of distilled water (about 1 × 1012-14conidia/mL) on a microscope slide, then placing the slide inside a Petri dish containing cotton moistened with distilled sterile water, and incubated at 25ºC in darkness. After incubation for 24 hours, conidial appressoria formed by germ tubes were characterized.
DNA extraction and sequencing
DNA was extracted from the isolates grown on PDA at 25ºC for 8-10 days using a modified protocol of Chen et al. (2007). The partial sequence of the actin (ACT), beta-tubulin (TUB2), chitin synthase 1 (CHS I), glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, and 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS) were amplified and sequenced using the primer pairs ACT-512F/ACT-783R (Carbone & Kohn 1999), T1/ Bt-2b (O' Donnell & Cigelnik 1997; Glass & Donaldson 1995), CHS-79F/CHS -354R (Carbone & Kohn 1999), GDF1/GDR1 (Guerber et al., 2003), and ITS-1/ITS-4 (White et al., 1990), respectively. The PCR amplifications were performed in a 25 µL mixture containing 9.5 µL ddH2O, 12.5 µL 2×PCR Master Mix (TIANGEN Co. China), 1 µL of DNA template, 1 µL of each primer (10 µM). The reactions were performed with a thermal cycler (MyclerTM, Bio-Rad, Hercules, CA, USA) using the thermal program described by Yang et al. (2009). PCR products were sequenced using the above-mentioned PCR primers and ABI BigDye v3.1 terminator sequencing chemistry according to the manufacturer's instructions of a BigDye® Terminator v3.1Cycle sequencing kit (Applied Biosystems, CA, USA) in an Applied Biosystems 3730xl DNA Analyzers at Sinomax Co., China.
Molecular phylogenetic analysis
Phylogenetic analysis was performed using the five gene regions cited above. The accession numbers of sequences generated are listed in Table 1. Multiple sequence alignments were generated using ClustalX 2.0.10 (Larkin et al., 2007) and manually adjusted to give the best fit with BioEdit 7.0.8.
A partition homogeneity test (PHT) was performed with 1000 replicates in PAUP 4.0b10 (Swofford, 2003) to evaluate statistical congruence among the five gene regions and each of the single and combined sequence alignments were analyzed using maximum parsimony (MP) in PAUP* 4.0b10. Ambiguously aligned regions were excluded from all analyses, and gaps were treated as missing data. Trees were inferred using the heuristic search option with tree bisection-reconnection (TBR) branch swapping and 1000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability of the trees resulting from the parsimony analyses were assessed by bootstrap analysis with 1000 replicates. Trees were visualized in Treeview. When analyzing single and combined sequences, some reference sequences were obtained from GenBank (Table 1). Sequences obtained in this study were submitted to GenBank (accession No: ACT, JQ399989- JQ399995; CHS I, JQ399996-JQ400002; ITS, JQ400003- JQ400009; GPDH, JQ400010- JQ400016; TUB 2, JQ400017-JQ400023), the alignment in TreeBASE (http://www.treebase.org/treebase/index.html, ID: 12294), and taxonomic novelties in MycoBank (Crous et al., 2004).
Isolation of Colletotrichum species
Nine isolates of Colletotrichum were obtained from recently dead or infected stalks and leaves of Hemerocallis citrina, H. fulva, and H. fulva var. kwanso in Guiyang, Nanning, and Dandong, China.
The partition homogeneity test (P = 0.01) suggested that the individual gene partitions were not highly incongruent (Farris et al., 1995; Cunningham, 1997), thus the five gene datasets (ACT, CHS I, GPDH, ITS, TUB 2) from the Colletotrichum species plus datasets obtained from GenBank were combined for phylogenetic analysis. The combined datasets comprise 1797 characters after alignment, of which 700 characters are parsimony-informative, 991 constant, and 106 parsimony-uninformative. Parsimony analysis generated eight trees; SH test verified that they were similar, one of which (tree length = 2131 steps, CI = 0.605, RI = 0.88, RC = 0.532, HI = 0.395) is shown in Figure 1. Tree topologies obtained from the individual alignment of five genes and from the combined alignment are similar to each other, with only slight differences in bootstrap values, e. g. Colletotrichum spaethianum and C. lilii were not distinguished in two (ACT and ITS) of five phylogenies.
The phylogram constructed using combined datasets shows that the Chinese Hemerocallis isolates cluster into four distinct clades with high bootstrap support, presumably representing different Colletotrichum species. Sequences of the cultures CDLG2 and CDLG3 cluster with sequences of Colletotrichum spaethianum (CBS 167.49) and C. liriopes (CBS 119444) with 100% bootstrap support, respectively.
Sequences of CDLG1, CDLG4, and Colletotrichum gloeosporioides epitype (CBS 953.57) are nested in a clade with 100% bootstrap support. Sequences of CDLG5, CDLN6, CDLN7, and CBS 125338 form a distinct clade with 100% bootstrap value (Figure 1).
The nine strains isolated from Hemerocallis spp. represent four species based on DNA sequence analysis and morphological characteristics. Three strains represent one new species. The other six isolates represent three known Colletotrichum species which are presented with comments.
Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., Atti Inst. Veneto Sci. lett., ed Arti, Sér. 62: 670 (1884) Colletotrichum gloeosporioides has been epitypified and can now be identified using sequence data (Cannon et al., 2008; Cai et al., 2009; Hyde et al., 2009; Phoulivong, 2011). In the present study this species was isolated from dead stalks of Hemerocallis citrina and H. fulva. Acervuli are black with pink conidia masses and setae are sparse. Based on morphological identification, Gu et al. (2007) reported that C. gloeosporioides caused severe anthracnose on Hemerocallis citrina leaves and this is confirmed here using morphological and molecular data. Fruit rots (anthracnose) have often been attributed to C. gloeosporioides with identifications based on morphological characteristics, but C. gloeosporioides is not a common pathogen on tropical fruits as shown by a recent study by Phoulivong et al. (2010).
Material examined: China, Guizhou province, Guizhou Academy of Agricultural Sciences, on recently dead flower stalk of Hemerocallis citrina, 10 June 2008, Y. L. Yang (GZAAS 080055, ex-living culture CDLG1); China, Guizhou province, Guiyang Botanical Garden of Medicinal Plants, on recently dead flower stalk of H. fulva, 1 July 2008, Y. L. Yang (GZAAS 080058, ex-living culture CDLG4).
Colletotrichum hemerocallidis Y. L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai, sp. nov.
MycoBank: MB 564162
Etymology: Named after its host, Hemerocallis sp.
Holotype: China, Guizhou Province: Guiyang. On dead stalk of Hemerocallis fulva var. kwanso Regel, 1 July 2008, Y. L. Yang (GZAAS 080059; ex-holotype living culture CDLG5 = CGMCC 3.14971, CBS 130642).
On host, acervuli elliptical to circular, arranged irregularly, subepidermal, disrupting outer epidermal cell wall of host, setae present (Figure 2A). Setae 71.5-130.5 × 7-12 µM, dark brown, opaque, 2- to 4-septated, base inflated, tip acute (Figure 3). Conidiophores hyaline, pale brown at base, cylindrical, 1- to 2-celled, branched, 1219.5(-25.5) × 3-5 µM, mean ± SD = 15.6 ± 3.4 × 4.2 ± 0.5 µM (n = 20) (Figure 3), conidiogenous cells cylindrical to ampulliform, hyaline, 9-19 (-24) × 3.5-5 µM, mean ± SD = 14.1 ± 3.5 × 4.3 ± 0.5 µM (n = 20). Conidia slightly curved, often straight on one side and slightly curved on the other, hyaline, (17.5-) 20.5-27 × 3.5-5 µM, mean ± SD = 23.2 ± 2 × 4.1 ± 0.3 µM (n = 30), base truncate, apex acute (Figure 2C).
In culture: Colonies on PDA, attaining 4.9-6.7 cm, mean ± SD = 6.1 ± 0.5 cm (n = 15) diam. in seven days at 25ºC. Aerial mycelium sparse, white to grey, flat with entire margin, reverse greenish black. Sclerotia present, globose to subglobose, without setae. Conidia not produced. Colonies on SNA, attaining 4.8-6.1 cm, mean ± SD = 5.5 ± 3.7 cm (n = 15) diam. in seven days at 25ºC. Aerial very sparse, grey. Sclerotia absent; Conidia not produced.
On Sium suave stem: acervuli abundant (Figure 2B), setae dark brown to black, opaque, smooth, septation hardly visible, 76.5-152.5 × 5-11.5 µM, tapered from base to apex. Conidiophores pale brown, 1-to 3-septate, branched, 16.540 (-44.5) × 3.5-5 µM, mean ± SD = 28.1 ± 8.3 × 4.3 ± 0.5 µM (n = 20) (Figure 4). Conidiogenous cells pale brown, cylindrical to elongate ampulliform, 7-16.5 (-19) × 3.5-5.5 µM, mean ± SD = 12.3 ± 3.3 × 4.5 ± 0.6 µM (n = 20). Conidia in white to yellowish masses, hyaline, smoothwalled, aseptate, one side straight and the other slightly curved, apex acute or slightly rounded, base truncate, 23-31.5 (-33.5) × 3.5-5.5 µM, mean ± SD = 27.8 ± 2.3 × 4.6 ± 0.4 µM (n = 150) (Figure 2 I). Mycelial appressoria clavate, brown, margin entire, sometimes slightly lobed, 6.5-16 (-18.5) × 5-9 µM, mean ± SD = 11.7 ± 2.9 × 6.5 ± 1.1 µM (n = 50) (Figures 2D, E), usually in loose groups; Conidial appressoria clavate to irregular, brown, margin entire to crenate, sometimes deeply lobed, 6.5-13 × 4-9.5 µM, mean ± SD = 9.4 ± 1.4 × 6.4 ± 1.2 µM (n = 60) (Figures 2F, G, H).
Known hosts and distribution: Hemerocallis fulva, Hemerocallis fulva var. kwanso, Guizhou and Guangxi provinces, China.
Additional specimens examined: China, Guangxi province, Nanning, on leaf spot of Hemerocallis fulva var. kwanso, 19 June 2008, Y. L. Yang (GZAAS 080040, living culture CDLN6); China, Guangxi province, Nanning, on leaf spot of Hemerocallis fulva, 19 June 2008, Y. L. Yang (GZAAS 080041, living culture CDLN7).
Notes: The conidial shape of C. hemerocallidis is similar to that of C. anthrisci Damm, P.F. Cannon & Crous, and C. lineola Corda, while the conidial width and mycelial appressoria of C. hemerocallidis are different from those of C. anthrisci and C. lineola. The conidia of C. hemerocallidis are wider than those of the latter (3.5-5.5 µm vs. 3-4 µm). The mycelial appressoria of C. hemerocallidis are clavate with entire or sometimes slightly lobed margins, while those of C. anthrisci are navicular, bullet-shaped to clavate, and those of C. lineola ellipsoidal to clavate (Damm et al., 2009). In multilocus phylograms, sequence data of C. anthrisci, C. hemerocallidis, and C. lineola indicate positions nested in different clades (Figure 1).
Colletotrichum liriopes Damm, P.F. Cannon & Crous
This taxon was isolated from a dead stalk of Hemerocallis fulva, acervuli are small with short black setae.
Material examined: China, Guizhou Province: Guiyang Botanical Garden of Medicinal Plants. On recently dead flower stalk of Hemerocallis fulva, 1 July 2008, Y. L. Yang (GZAAS 080057, living culture CDLG3).
Note: This taxon was first reported from Liriopes muscari (Decne.) L. H. Bailey in Mexico (Damm et al., 2009). We also collected this species from anthracnose on Eria coronaria (Lindl.) Rchb. F. (Orchidaceae) and a healthy root of Pleione bulbocodioides (Franch.) Rolfe (Orchidaceae) (Yang et al., 2011), so this species is not host-specific.
Colletotrichum spaethianum (Allesch.) Damm, P.F. Cannon & Crous
This species was isolated from anthracnose of Hemerocallis fulva, causing brown spots on leaves and having small acervuli containing black setae.
Material examined: China, Guizhou Province: Guiyang Botanical Garden of Medicinal Plants. On leaf spot of Hemerocallis fulva, 1 July 2008, Y. L. Yang (GZAAS 080056, living culture CDLG2). China, Liaoning Province: on leaf spot of Hemerocallis citrina, 4 July 2011, Y. L. Yang (GZAAS 110007, living culture CDLL1; GZAAS 110008, living culture CDLL2).
Note: Damm et al. (2009) reported Colletotrichum spaethianum from dead stems of Hosta sieboldiana (Lodd.) Engl., leaf spot of Hemerocallis sp., and infected leaf of Lilium sp. Yang et al. (2009) also isolated this species from a leaf spot of Hymenocallis americana (Jacq.) Salisb. This suggests a broad host range for this species.
Six species of Colletotrichum have previously been reported from Hemerocallis species, but with the exception of C. spaethianum (CBS 101631) and Colletotrichum sp. (CBS 125338) which have been sequenced, the identifications were based on morphological characteristics (Table 2). In the context of the present study, the species of Colletotrichum on Hemerocallis spp. in China are accurately identified and data are provided extending our knowledge on the host range and distribution of four species. One new species is proposed. Several studies have shown the importance of using sequence data when identifying Colletotrichum species, because wrong diagnosis may otherwise result (Phoulivong et al., 2010; Damm et al., 2010; Cai et al., 2011; Ko Ko et al., 2011).
Colletotrichum hemerocallidis apparently is saprobic and pathogenic on H. fulva. This suggests that C. hemerocallidis is similar to some other Colletotrichum species (e.g. C. gloeosporioides, C. liriopes, C. spaethianum) in having more than one biological life strategy (Damm et al., 2009; Rojas et al., 2010; Yang et al., 2011; Phoulivong, 2011). As we gain more knowledge on the distribution and host range of Colletotrichum species, it appears that many species may be saprobes, endophytes, or pathogens, having a wide host range and distribution. The new strains of Colletotrichum gloeosporioides obtained during this study were isolated from symptomatic tissues of Hemerocallis thus suggesting these strains are pathogens of this genus.
This project was supported by the National Natural Science Foundation of China (No. 31070025) and Guizhou Science and Technology Department [No. (2010) 4002-1]. The National Research Council of Thailand awarded grant No. 5420102003 to study the genus Colletotrichum. Lei Cai acknowledges grants CAS KSCX2-YW-Z-1026 and NSFC 31110103906. 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).
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Received 16 January 2012
Accepted 25 April 2012
Author for correspondence: Kevin D. Hyde, e-mail: firstname.lastname@example.org
Section Editor: Meike Piepenbring