First Report of Colletotrichum chlorophyti Causing Soybean Anthracnose.

Abstract

Anthracnose of soybean [Glycine max (L.) Merr.] is caused by several Colletotrichum spp. (4). Petiole samples were collected from Alabama, Mississippi, and Illinois in 2009. Diseased tissues suspected of being caused by Colletotrichum were cut into 1- to 2-cm lengths, surface-disinfested, and placed on water agar. Pure cultures obtained by picking single spores from sporulating acervuli on tissue or hyphal tips on agar were transferred to acidic potato dextrose agar (APDA) at 24 ±1°C under 12-h cool-white fluorescent lighting. Isolates were grouped by morphological characteristics. One group consisting of six isolates (four from IL and one each from AL and MS) did not morphologically match any reported Colletotrichum spp. causing soybean anthracnose but matched the description of C. chlorophyti S. Chandra & Tandon (1,2). On APDA, colonies were initially pink, turning black after several days with smooth margins and no aerial mycelium. Conidial masses were light salmon in color. Conidia ranged from 15.5 to 21.3 μm long (mean 18.0 μm) × 2.5 to 4.3 wide (mean 3.3 μm) (n = 200). They were curved with tapered ends and a truncated base, aseptate, and hyaline. Chlamydospores were dark brown, clustered or chained together, and 5 to 12 μm wide (n = 30). Setae were straight, dark brown, and septate. Appressoria and perithecia were absent. Soybean plants (cv. Williams 82) at the V2 to V3 stage were atomized with a suspension of fragmented mycelia (40 mg/ml) using one isolate from IL. Plants were kept moist (>90% relative humidity) for 48 h in the dark, then transferred to normal growing conditions. Three days post-inoculation, younger trifoliolate leaf margins and intra- and interveinal lesions were necrotic surrounded by slight chlorosis. Isolations were obtained from symptomatic leaves and confirmed as C. chlorophyti by morphological characteristics. Further comparisons were completed with one isolate (IL1A or BPI 884117) by PCR and BLAST sequencing analyses of the partial ITS rDNA region, actin, β-tubulin, GAPDH, and histone H3 genes (2) (GenBank Accession Nos. JX126475, JX126476, JX126477, JX126478, and JX126479, respectively). The results showed high identity of all the five sequences to two C. chlorophyti isolates, IMI 103806 and CBS 142.79 (Accession Nos. GU227894/GU227895 in ITS = 100%, GU227992/GU227993 in actin = 99%, GU228188/GU228189 in β-tubulin = 99%, GU228286/GU228287 in GAPDH = 99% and 96%, respectively, and GU228090/GU228091 in histone H3 = 99%). Soybean anthracnose, commonly caused by C. truncatum, has curved and truncated conidia that are longer than C. chlorophyti. In addition, the two are distinguished by chlamydospores and lack of appressoria in C. chlorophyti combined with differences in multigene sequence analysis. Isolates of C. chlorophyti were reported to infect Chlorophytum sp. (Liliaceae) in India and Stylosanthes hamate in Australia (3). To our knowledge, there are no previous reports of this species in the United States or of it infecting soybean worldwide (3). This report describes C. chlorophyti as a novel incitant of soybean anthracnose.