First Report of Root Rot on Strawberry Caused by Binucleate Rhizoctonia AG-K in Spain

Spain is the second largest strawberry (Fragaria × ananassa Duch.) producing country in Europe. In January 2018 in a strawberry fruit production field in Lepe, Huelva (southwestern Spain), strawberry cultivar Rociera FNM plants showed varying degrees of poor growth, stunting, black necrosis in basal petioles, brown to dark brown discoloration in roots, few new roots emerging from the crown, and brown necrotic areas of crown tissue. These plants came from a nursery in Ávila Province (Spain) with a high clay content soil and transplanted nearly a month later than usual. Two foci and six plants per focus were surveyed. Petiole, crown, and root pieces of symptomatic plants were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed in sterile distilled water, and air dried in a laminar flow cabinet. Disinfested pieces were transferred to Petri dishes containing potato dextrose agar (PDA) and incubated for 7 days at 25°C. A total of two Rhizoctonia-like colonies were observed. These isolates were purified by cutting hyphal tips from margins. Colonies of the isolates developed dark brown mycelia on PDA at 25°C. Microscopic examination showed morphological characters coinciding with Rhizoctonia genus (Sneh et al. 1991). Nuclear staining with acridine orange solution (20 μl/ml) prepared in acetic acid buffer (pH 7.2) for 15 min (Seema et al. 2012) showed two nuclei per cell in the hyphae of the two isolates. Hyphal diameter was less than 5 μm. No sclerotia were formed after 20 days on PDA medium at 25°C. All these morphological characteristics of the isolates resembled those of binucleate Rhizoctonia (BNR). Species identity was confirmed for one isolate by sequencing the intergenic spacer (ITS) region of ribosomal DNA with the primers ITS1 and ITS4 (White et al. 1990). Identities of the resulting sequences were confirmed by BLAST analysis. The sequences were 99% identical to several Ceratobasidium sp. AG-K isolates (e.g., MF070698) and were deposited as MH666074 at GenBank. Hyphal anastomosis tests were conducted by paring isolates with seven known tester isolates of BNR AG on 2% water agar. Anastomosis was observed with AG-K tester isolate. The isolates were identified as BNR AG-K. A bioassay to confirm the pathogenicity of the isolate was developed with runner strawberry plants Rociera FNM and Rábida FNM (five plants per cultivar). Plants were inoculated by placing PDA colonized plugs of 1-cm diameter at the crown base. Five noninoculated plants were used as negative controls per cultivar. Plants were grown in 200-ml pots with peat in a growth chamber at 25/22°C (day/night) with a 12-h photoperiod. All inoculated plants showed the same symptoms described above and had lower dry weight than control plants. The fungus was reisolated and confirmed to be Rhizoctonia sp. The BNR AG-K has been associated with root rot on strawberry in Israel and Australia (Fang et al. 2013). To our knowledge, this is the first report of BNR AG-K causing root rot of strawberry in Spain. Root rot disease caused by BNR can increase in cumulative years of strawberry monoculture. Therefore, growers should consider it in their management strategies.