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

Abstract：【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.