RESUMO
Common bean (Phaseolus vulgaris L.), is a grain legume widely cultivated worldwide for its edible dry seeds and pods. In February 2021, root rot symptoms were observed in two common bean (cv. Azufrado Higuera) fields located in Ahome (25º96´19¨N, 109º33´42¨W) and Guasave (25º71´85¨N, 108º78´50¨W) municipalities in Sinaloa, Mexico. Diseased plants showed reduced growth, dark brown canker at the base of the stem, root rot, as well as the absence of secondary roots. The disease incidence was estimated up to 35%. For fungal isolation, symptomatic roots were surface sterilized with 1% sodium hypochlorite for 2 min, rinsed with sterilized distilled water two times, and blotted dry on sterile filter paper. Small fragments of diseased roots were placed on PDA medium and incubated at 25°C in darkness for 3 days. Rhizoctonia-like colonies were consistently obtained and 10 isolates were purified by the hyphal-tip method. Colonies on PDA were white initially and then turned brown after 15 days of incubation. The septate hyphae were 3.9 to 6.3 µm in width and branched at right angles with a septum near the point of branching. Microscopic examination by Safranine-O staining showed two nuclei per cell. The morphological features of the isolates resembled those of Ceratobasidium (Sneh et al. 1991). The two Ceratobasidium isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF395 and FAVF396). For molecular identification, genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region and partial fragments of the second largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with the primer pairs ITS5/ITS4 (White et al. 1990) and RBP2-980F/RPB2-7cR (Liu et al. 1999), respectively. The ITS sequences (accession nos. ON630914 and ON630915) showed 99.66 and 99.01% identity with Ceratobasidium sp. (AG-A) from the USA (OM045887) and Ceratobasidium sp. (AG-G) from China (HM623627), respectively. Whereas, the rpb2 sequences (OM258171 and OM258172) showed 94.10 and 95.74% identity with Ceratobasidium sp. (AG-A) from Serbia (MT1267888) and Ceratobasidium sp. (AG-G) from Japan (DQ301701), respectively. A phylogenetic tree based on Maximum Likelihood and including combined ITS and rpb2 sequences data for Ceratobasidium spp. was generated. The phylogenetic tree grouped the isolates FAVF395 and FAVF396 within the Ceratobasidium sp. AG-A and AG-G clades, respectively. Pathogenicity tests for each isolate were performed by inoculating 10 healthy common bean seedlings (15-day-old) grown in pots. A total of 50 ml of a mycelial suspension adjusted to a concentration of 1 × 105 mycelial fragments/ml were directly placed on the stem base of each plant. Five uninoculated common bean seedlings were used as control. All plants were kept in a greenhouse for 15 days at temperatures ranging from 22 to 32°C. Root rot and stem canker symptoms appeared on inoculated seedlings after 10 days, whereas control plants remained symptomless. Fungi were reisolated from the infected roots and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. Ceratobasidium sp. has been previously reported as the causal agent of root rot of watermelon in Sonora, Mexico (Meza-Moller et al. 2014; Farr and Rossman 2022). To our knowledge, this is the first report of Ceratobasidium sp. causing root rot of common bean in Mexico. Further monitoring should be performed to quantify yield impacts and develop effective management strategies for this disease.
RESUMO
Guar (Cyamopsis tetragonoloba), is an annual legume belonging to the Fabaceae family and it is grown mainly for industrial purposes and also as an ingredient for animal feed. In September 2021, anthracnose symptoms were observed on guar fields distributed in Guasave, Sinaloa, Mexico. Disease incidence was estimated up to 15%. Diseased plants exhibited symptoms on leaves and pods. On leaves, lesions were irregular, necrotic, and often surrounded by a dark brown halo. On pods, necrotic and sunken lesions were developed. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under the accession number FAVF642. Colony on PDA was flat with an entire margin, dense, initially grayish white, then became dark gray with black microsclerotia and setae. Conidia (n= 50) were curved, hyaline, aseptate, with granular content, and measuring 20.4 to 25.8 × 2.8 to 3.9 µm. Setae were dark brown, straight, and septate. Morphological features matched those of Colletotrichum truncatum (Damm et al. 2009). For morphological identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR (Weir et al. 2012), and sequenced. The resulting sequences were deposited in GenBank under the accession nos. OM616022 (ITS), OM630461 (ACT), and OM630462 (GAPDH). BLASTn searches in GenBank showed 100%, 99.49%, and 99.15% identity to MT583079 (ITS), MG198003 (ACT), and MG703491 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on maximum Likelihood method and including published ITS, ACT, and GAPDH sequence data for Colletotrichum truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate FAVF642 was placed in the same clade of C. truncatum. Pathogenicity of the isolate FAVF642 was verified on 10 guar seedlings (15-day-old) by spraying a conidial suspension (1 × 106 spores/mL) onto leaves until runoff. Five plants noninoculated served as controls. All plants were kept in a moist chamber for 2 days, and subsequently transferred to a shade house where the temperature ranged from 20 to 30°C. The experiment was conducted twice with similar results. All inoculated leaves developed irregular and necrotic lesions 8 days after inoculation, whereas no symptoms were observed on the control leaves. The fungus was consistently re-isolated from the diseased leaves, fulfilling Koch´s postulates. Colletotrichum truncatum has been previously reported to cause guar anthracnose in India (Farr and Rossman 2022). To our knowledge, this is the first report of C. truncatum causing guar anthracnose in Mexico. This disease is an emerging problem in guar fields in Sinaloa, therefore further studies are required to understand its occurrence and impact in Mexico.
