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

Abstract

Strawberry (Fragaria × ananassa Duch.) is an important crop in Spain. In January 2018, ‘Florida Fortuna’ strawberry plants exhibiting poor growth were observed in a fruit production field in Palos de la Frontera, Huelva (southwestern Spain). These plants were from nurseries in Avila Province (Spain) that had soils with a high clay content. Affected plants showed varying degrees of stunting, black necrosis on basal petioles, brown to dark-brown discoloration in roots, and few new roots emerging from the crown. Two foci were found and surveyed (six plants per focus). 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. Five fungal isolates were recovered. Colonies were white to pale brown and had floccose aerial mycelia. Two isolates were purified by cutting hyphal tips from their margins. Microscopic examination showed morphological characters coinciding with Rhizoctonia (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. Mean hyphal diameter was 4.79 µm. No sclerotia were formed after 20 days on PDA at 25°C. The 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 100% identical to those of Ceratobasidium sp. AG-A KC782943 and were deposited as MH666073 in 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-A tester isolate. The isolates were identified as a BNR AG-A. To confirm the pathogenicity of the isolate, five runner strawberry plants ‘Rociera FNM’ and five ‘Rabida FNM’ were inoculated with PDA colonized plugs 1 cm in diameter located at the crown base. Five noninoculated plants were used as negative controls. Plants were grown in 200-ml pots with peat in a growth chamber at 25/22°C (day/night) with a 12-h photoperiod. Symptoms started appearing on inoculated plants 18 days after inoculation. All inoculated plants showed the same symptoms described above. Disease severity was scored as proportion of affected or senescent leaves. Inoculated ‘Rociera FNM’ plants showed 36% more affected leaves than noninoculated ones, while this difference in ‘Rabida FNM’ plants was 29%. Inoculated ‘Rociera FNM’ plants also had significantly lower dry weights (mean value: 3.91 g) than control plants (mean value: 8.02 g). The fungus was reisolated and confirmed to be Rhizoctonia sp. The BNR AG-A is one of the most common groups associated with root rot and a serious threat to strawberry production worldwide (Fang et al. 2013). To our knowledge, this is the first report of BNR AG-A causing root rot of strawberry in Spain. From now, growers will have to consider this pathogen for soil health management and for diseased plant diagnosis.