First report of AG-3 causing black scurf on cv. Superchola in Cotopaxi, Ecuador.

Rhizoctonia solani is a globally significant pathogen causing black scurf and stem canker in potatoes, resulting in considerable yield and quality losses. Among the 14 anastomosis groups (AGs), AG3 is primarily linked to potato diseases (Tsror, 2010). In November 2022, symptoms of black scurf were observed on Solanum tuberosum cv. Superchola tubers in Lasso-Cotopaxi, a major potato-growing region in the Andes of Ecuador (0°41'24.8''S, 78°41'27.7''W). The tubers exhibited dark brown, irregularly shaped sclerotia firmly attached to their surfaces. Harvested tubers showed 15% of disease incidence and exhibiting lesions covering from 10% - 50% of the potato surface, harvested from an area 20000 m2 plot (Fig S1). To isolate the causal agent, 10 symptomatic tubers were collected, surface disinfected (2.5% sodium hypochlorite, followed by 70% ethanol), and thoroughly rinsed with sterile distilled water. Tissue samples (~0.5 cm²) were excised from the margins of healthy and diseased areas, samples were plated on Potato Dextrose Agar (PDA) with gentamycin (160 mg/L). After incubation at 25°C in darkness, isolates were purified using the hyphal-tip method. Three isolates displayed brown colonies with sclerotia, consistent with R. solani. Microscopic examination revealed right-angle hyphal branching, septation near the branch points, and slight base constriction (Fig S1). For molecular identification, DNA was extracted from the three isolates using the Qiagen PowerSoil Kit. The ITS region was amplified with primers ITS5 and ITS4 (White et al. 1990), and the resulting fragments (716 bp) were sequenced and showed 100% identity to each other. The Consensus ITS sequence was deposited under GenBank accession PP532868. Similarly, the rpb2 gene (675 bp; GenBank accession PQ632810) was amplified with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999) and consensus sequence showed 99.7% identity to R. solani GenBank accession PP665463.1. Sequence alignment was constructed using ClustalW with the MEGA 11.0 software package (Tamura et al. 2021). Subsequently, phylogenetic analysis was performed using Bayesian inference using the BEAST version 1.8.4 program (Drummond and Rambaut 2007). The phylogenetic analysis of the sequence revealed that the isolate clustered in the same clade with Rhizoctonia solani AG-3, confirming its identity (Fig S2). Koch's postulates were fulfilled using five replicates of S. tuberosum cv. Superchola mini tubers. Symptomless tubers were sprouted at 18°C for 10 days and planted in pots with sterilized substrate (50% peat moss, 50% perlite). Each pot was inoculated with 20 mg of R. solani colonized rice, while controls received sterile rice (Lopez-Corrales et al. 2023). Plants were maintained under controlled greenhouse conditions for 25 days (15°C/27°C, 70% RH). Inoculated plants exhibited small stem and tuber lesions, while controls remained asymptomatic. The pathogen was successfully reisolated from symptomatic plants and confirmed as R. solani AG-3. To our knowledge, this is the first report of Rhizoctonia solani AG-3 affecting potatoes in the country. Although not a major potato disease in Ecuador, it could become a significant soil pathogen, especially in areas with limited crop rotation and high soil-borne infection risk.