RESUMO
Rhizoctonia solani, is the causal agent of black scurf and stem canker of potatoes (Solanum tuberosum L.) throughout the world. In November 2021, stem canker symptoms were observed in two potato fields located in Ahome, Sinaloa, Mexico. The disease incidence was estimated up to 15%. For fungal isolation, fragments of symptomatic stems were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed with sterilized distilled water, and blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25°C in darkness for 4 days. Rhizoctonia-like colonies were consistently obtained and 12 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially and then turned brown, raised, and with entire or undulate edges. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by staining with 1% safranin O and 3% KOH solution showed multinucleate cells. The morphological features of the isolates resembled those of Rhizoctonia solani (Sneh et al. 1991). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF267, CCLF274, CCLF277, and CCLF279. For molecular identification, genomic DNA from each of the four isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP784258 to OP784261). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the four isolates within the R. solani AG-7 clade with high bootstrap support (100%). For pathogenicity tests, certified pathogen-free potato mini-tuber (cv. Fianna) were placed in a polystyrene pot (1 L) filled with a 5 cm layer of a sterile substrate composed of soil and peat moss (2:1 w/w). One rice grain (20 mg) colonized with each isolate was placed 10 mm above the uppermost sprout tip and covered with the sterile substrate (Inokuti et al. 2019). Control plants were inoculated with sterile rice grains. All pots were transferred to a greenhouse where the temperature ranged from 20 to 32°C. Stem necrosis symptoms were observed on all inoculated plants 25 days after emergence, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected stems and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. The AG-7 has been previously reported to cause potato diseases in South Africa (Truter and Wehner 2004). In Mexico, Carling et al. (1998) reported the presence of an isolate of R. solani AG-7 obtained from a potato tuber-borne sclerotium in Toluca; however, there is no information about the methodology used for the characterization of that isolate. To our knowledge, this is the first confirmed report of R. solani AG-7 causing potato stem canker in Mexico. Our findings improve knowledge about R. solani AGs occurring in potato fields in Mexico. So, further studies should be conducted to investigate the diversity, prevalence, and fungicide sensitivity of AGs distributed in the main potato-producing states in Mexico.
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.
