First Report of Ilyonectria robusta Associated with Black Foot Disease of Grapevine in the United States

Abstract

In October 2017, a vineyard in southwest Idaho reported that up to 80% of self-rooted Cabernet sauvignon plants were dead or displaying poor growth and reduced vigor. Although this was initially attributed to extreme conditions from the previous winter, inspection of the roots of 10 affected plants showed signs of black foot disease. Symptoms included reduced root biomass, and black discoloration with sunken and necrotic root lesions. To isolate fungi associated with the necrotic root tissue, symptomatic pieces of root approximately 3 mm in length were surface sterilized with sodium hypochlorite solution (2%) and rinsed twice in sterile water prior to plating onto potato dextrose agar (PDA) amended with penicillin G (0.6%) and streptomycin sulfate (2%). Fungal colonies of interest were identified after 5 days and transferred to fresh PDA plates. After 2 weeks of growth on PDA at 20°C, colonies resembling Ilyonectria were frequently isolated. These colonies were light brown in color, had a cotton-like texture with abundant conidia, and were consistent with previous descriptions (Cabral et al. 2012; dos Santos et al. 2014; Martinez-Diz et al. 2018). Species identity was determined through sequencing part of the histone 3 (H3) gene. DNA was extracted using a Wizard DNA purification kit for food (Promega) in conjunction with a Kingfisher ML (ThermoFisher), and primers CYLH3F and CYLH3R were used as described previously (Crous et al. 2004). Polymerase chain reaction (PCR) products were purified using a QIAquick PCR clean up kit (Qiagen) and sent to Eurofins Genomics (Louisville, KY) for sequencing. The resulting sequence had 100% identity with another Ilyonectria robusta sequence in GenBank (JF735518). Therefore, from both sequence and morphological analysis, the isolate was identified as I. robusta. To confirm pathogenicity, cuttings of Cabernet sauvignon approximately 30 cm high were planted in individual pots containing approximately 1 kg of premium potting soil (Scotts, Marysville, OH) and grown for 3 months in the glasshouse at 25°C. Once sufficient root mass had developed, the roots were lightly wounded using a scalpel with four consistent strokes below the soil line in a systematic manner. The soil was then inoculated with 14 ml of a 10⁹ conidial suspension taken from 7-day-old plates of isolate G45. Control plants were inoculated with sterile water. Each treatment was replicated 10 times. After 24 days, the roots were inspected for disease. Black foot symptoms were present on all plants inoculated with isolate G45 with a mean root area affected of 59%. In the plants inoculated with sterile water, four had completely healthy roots, and light root browning was visible in six plants (mean root area affected was 4%). Plants inoculated with isolate G45 demonstrated significantly (t test, P < 0.001) more root discoloration than control plants. I. robusta was consistently reisolated from the affected tissue of inoculated roots, thereby confirming Koch’s postulates. To our knowledge, this is the first report of I. robusta on Vitis vinifera in the United States. I. robusta has previously been found associated with black foot disease in Portugal (Cabral et al. 2012), Brazil (dos Santos et al. 2014), Spain (Martinez-Diz et al. 2018), and British Columbia (Urbez-Torres et al. 2014). I. robusta is part of a species complex of Cylindrocarpon-like asexual morphs causing black foot disease, which causes economic losses to the grapevine industry worldwide. Growers in the United States should consider its presence when planting grapevines.