广西杧果炭疽病菌种类的构成与分布

Composition and distribution of Colletitruchm species associated with anthracnose of mango (Mangifera indica L.) in Guangxi

【目的】利用多基因手段结合形态学特征重新确定广西杧果炭疽病菌的分类地位,并统计分析各种类病菌的分布,了解广西杧果炭疽病菌的种类和优势群体,为进一步研究我国杧果炭疽菌及其病害的诊断、防控提供有力依据。【方法】从广西百色市、南宁市、钦州市等杧果产地采集杧果不同部位的炭疽病害样本,采用组织分离法进行分离,对所得分离物进行致病性测定,证明为杧果炭疽病菌。对病菌的ITS和ACT、GPDH、TUB2、CAL多基因位点进行扩增和测序,各基因序列按ITS、GPDH、ACT、TUB2、CAL的顺序相连接形成复合序列,采用MEGA 6.06软件以邻接法(neighbor-joining,NJ)构建系统发育树,结合病菌的形态特征鉴定其分类地位,并统计各种类病菌的总分离率及其在不同地区、植株不同部位的分离率。【结果】获得50株杧果炭疽病菌菌株,分别属于3个种:Colletotrichum asianum、C.fructicola、C.siamense,其中36株为C.asianum、12株为C.fructicola、2株为C.siamense。C.asianum、C.fructicola、C.siamense的总分离率分别为72%、24%、4%;C.asianum在所有检测地区、植株部位均能分离到,且分离率均最高,超过57.1%;C.fructicola能从所有检测地区、枝条以外的植株部位分离到,其中花梗上的分离率最高,为42.9%;C.siamense仅从百色市的果实上分离到,分离率为13.3%。【结论】广西杧果炭疽病菌主要有C.asianum、C.fructicola和C.siamense 3个种,均属于C.gloeosporioides复合种,其中C.asianum为优势种,C.fructicola是我国杧果炭疽病菌的新记录种;病菌的种类与地理来源、侵染部位无明显相关性。

【Objective】Mango（Mangifera indica L.） is an economically important fruit crop in China and other tropical/subtropical regions in the world. Mango is affected by a number of diseases during its growth and development as well as postharvest storage and marketing. Anthracnose, caused by Colletotrichum spp., is the most serious disease effecting mangoes. The disease can significantly lower mango productivity and quality, which can lead to large economic losses in the mango production chain in severe cases. In China, the anthracnose of mango was previously reported to be caused by Colletotrichum gloeosporioides Sensu Lato and C. acutatum Sensu Lato. However, based on recent multigene phylogenetic analyses, both C. gloeosporioides Sensu Lato and C. acutatum Sensu Lato have been treated as species complex. There are few studies on the morphological and molecular characterization of distinct Colletotrichum species associated with mango in the species complex. This study aimed to investigate the populations of the Colletitruchm species associated with mango anthracnose in Guangxi based onfive gene sequences and morphological characteristics, and to determine the predominant Colletotrichum species responsible for mango anthracnose. The results of this study are expected to provide a better understanding on the etiology of the disease and scientific basis for its management.【Methods】Mango leaves,fruits, twigs and pedicels showing anthracnose symptoms were sampled from the major mango-producing areas in Baise, Nanning and Qinzhou cities, Guangxi, China. Colletitruchm cultures were isolated using the routine tissue isolation method and their pathogenicities were tested on detached mango leaves（wounded and unwounded） using mycelial plugs as inocula. Partial r DNA-ITS（ITS） region and glyceraldehyde-3-phosphate dehydrogenase（GPDH）, actin（ACT）, β-tubulin（TUB2）, and calmodulin（CAL） genes were amplified by polymerase chain reaction（PCR）. A concatenated Neighbor-Joining phylogenetic tree was constructed using MEGA 6.06. The collected Colletotrichum strains were incubated on PDA plates at 25 ℃under continuous fluorescent light irradiation and their cultural characteristics were recorded. Conidia and appressoria produced by the strains were morphologically characterized under a microscope. The whole isolation rate（IR）, IR at different sampling sites and in different plant tissues, were calculated for each Colletotrichum spp. isolated to determine the predominant Colletotrichum species causing anthracnose on the mango.【Results】A total of 50 Colletitruchm strains were obtained from various diseased tissues of mango in Guangxi. In the concatenated neighbor-joining phylogenetic tree constructed, there were thirty-six,twelve and two Colletotrichum strains forming a clade with C. asianum, C. fructicola and C. siamense, respectively. The colonies of C. asianum developed on PDA were white to pale grey with dense cottony aerial mycelia and regular margin, growing at a speed of 7.9-12.7（avg. 10.4） mm · d-1. The reverse colonies were pale yellowish. The conidia were straight, unicellular, cylindrical or elongated-obovoid, sometimes slightly constricted near the center, and measured（12.3-18.3） μm×（5.6-8.5） μm（avg. 15.9 μm×6.8 μm） in size, with ends obtuse（sometimes tapering towards the basal end） and oil globules. Mycelial appressoria were grayish black to dark chocolate-brown, clavate or subcircular, with an entire（occasionally irregular）margin and a few broad lobes, and measuring（7.8-11.9） μm×（6.5-9.7） μm（avg. 8.1 μm×6.9 μm） in size.Conidial appressoria were grayish black, clavate or subcircular, with the margin entirely or irregularly lobed, and measuring（5.6-13.5） μm×（4.7-8.3） μm（avg. 9.9 μm×6.8 μm） in size. The colonies of C. fructicola were grown on a PDA at a speed of 7.7-14.2（avg. 11.3） mm · d-1, with a regular margin, and white to green black or grayish, dense, cottony aerial mycelia. Saffron conidial masses developed on the colonies.The reverse side of a colony was grey-yellow to dark green. The conidia were straight, unicellular, cylindrical or elongated-obovoid with both ends rounded with oil globules, and measuring（12.1-19.8） μm ×（4.6-8.5） μm（avg. 15.6 μm × 6.7 μm） in size. Mycelial appressoria were brown to dark brown, clavate with an entire or undulate margin, and measuring（7.5-17.4） μm×（4.9-10.4） μm（avg. 12.7 μm×7.3 μm）.Conidial appressoria were grayish black, ovate or subcircular with entire margin, and measuring（6.7-10.4） μm×（5.5-8.4） μm（avg. 8.4 μm×6.3 μm） in size. The colonies of C. siamense were white to grey on PDA and grown at a speed of 8.9-14.4（avg. 12.4） mm · d-1, with dense, cottony aerial mycelia and regular margins. The reverse side of a colony was pale yellow, and subsequently turned greenish black with age.The conidia were straight, unicellular, cylindrical to elongated-obovoid, obtuse at the ends, with oil globules, and measuring（11.3-19.4） μm×（3.9-7.1） μm（avg. 14.8 μm×5.5 μm） in size. Mycelial appressoria were brown, clabate, occasionally lobed, with an entire margin, and measuring（6.5-14.3） μm×（5.7-9.7）μm（avg. 11 μm × 7.4 μm） in size. The conidial appressoria were grayish black, ovate to clavate, rarely deep lobes, with an entire margin, and measuring（8-14.8） μm ×（5.5-9.9） μm（avg. 10.5 μm×7.2 μm） in size. The whole isolation rates（IR） of C. asianum, C. fructicola and C. siamense were 72%, 24% and 4%,respectively. C. asianum was the most predominant species at all sampling sites and in all tissues evaluat-ed, with an IR above 57.1%. C. fructicola was isolated from all tissues except twigs at all sampling sites,with the highest IR（42.9%） in pedicel. C. siamense was isolated only from mango fruits sampled in Baise city with an IR of 13.3%.【Conclusion】Based on the phylogenetic analyses and morphological characteristics of the 50 strains investigated, three Colletotrichum spp.（C. asianum, C. fructicola, and C. siamense）are the main pathogens causing anthracnose on mango in Guangxi, China, and all of them are members of the C. gloeosporioides species complex. This is the first report of C. fructicola as the causal agent of anthracnose on mango in China. C. asianum is the most widely distributed Colletotrichum species in the mango-producing areas of Guangxi. No obvious correlation exists between the distribution of Colletotrichum species and sampling sites or parts of the mango plants investigated.