Rhizoctonia solani on potato in New Zealand: pathogen characterisation and identification of double-stranded RNA viruses that may affect their virulence

Abstract

Rhizoctonia solani is an economically important pathogen of potato in New Zealand, causing significant yield and quality reductions in potato crops. Rhizoctonia disease management relies heavily upon fungicide applications, which can lead to increased production costs and potential environmental damage. Identification and utilisation of novel biocontrol agents will provide “environmentally friendly” disease management strategies. Mycoviruses or viral double stranded RNA (dsRNA) elements have been associated with hypovirulence in a number of plant pathogenic fungi including R. solani. This study determined the anastomosis group (AG) composition, genetic variation and virulence of isolates of the pathogen from potato in New Zealand. In addition, dsRNA elements in R. solani isolates of different virulence were characterised. Isolates (129) of R. solani were collected from black scurf on potato tubers of different cultivars from various potato growing localities. Molecular characterisation of the internal transcribed spacer (ITS) regions showed that isolates belonged to AG-3PT (110), AG-2-1 (18) and AG-5 (1). AG-3PT was widely distributed throughout New Zealand, whereas AG-2-1 and AG-5 were confined to distinct locations. Sequence heterogeneity was identified in the ITS regions of 100 isolates belonging to AG-3PT or AG-2-1. Variations in the sequence and length of the intergenic spacer 1 (IGS1) region were also observed for selected AG-3PT and AG-2-1 isolates. Phylogenetic studies found all AG-2-1 isolates belonged to AG-2Nt, a subset of AG-2-1 previously associated with non-potato solanaceous crops overseas. Comparative pathogenicity assessments, in glasshouse and shadehouse experiments, showed that AG-2Nt isolates were consistently more aggressive than AG-3PT isolates. AG-2Nt isolates caused delayed emergence, severe stolon infection, formation of aerial tubers, yield losses, and severe tuber malformation, but little black scurf. Isolates of AG-3PT differed in aggressiveness, causing black scurf on progeny tubers but variable effects on stems and stolons and did not cause tuber malformations. Forty-three isolates of AG-2Nt and AG-3PT differing in virulence were assessed for content of dsRNA elements. All of the isolates contained dsRNA elements, but no association between the presence of dsRNA and virulence was identified. The number of dsRNA elements within individual isolates varied between one and 13, with approximate sizes ranging from 0.23 to 18 kb. Considerable variation in dsRNA banding patterns was found between isolates, but a large dsRNA element (ranging from 11.5 to 18 kb) was detected in all isolates tested. Total dsRNA was randomly amplified from six different R. solani AG-3PT isolates belonging to four different ITS types. Sequence analysis identified a nearly complete sequence for two dsRNA elements in one isolate, of size 2797 nt and 14,694 nt. The 2797 nt element contained a single open reading frame (ORF), putatively encoding a polypeptide of 826 amino acids (aa), including conserved motifs for Mitovirus RNA-dependent RNA polymerase (RdRp). Sequence comparison indicated that this could be a new species within the genus Mitovirus, and was designated Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 14,694 nt element contained a large ORF, putatively encoding a polypeptide (4893 aa) containing conserved domains of viral methyl transferase, helicase, and RdRp. Sequential arrangement of these putative domains was most closely related to those in polyprotein from previously reported members of the genus Endornavirus.This element was designated Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial sequence analyses also indicated the presence of an unknown virus related to Betafexiviridae, a second Mitovirus different from RMV-1-RS002 and a Partitivirus in this isolate. Identical copies of RMV-1-RS002 and RsEV-RS002 were detected in two isolates of the pathogen. In addition, an endornavirus was also identified in three of the R. solani isolates studied. This is the first study on AG composition, genetic variability, virulence and dsRNA composition of R. solani isolates associated with black scurf of potato in New Zealand. This study shows for the first time that large dsRNA elements in R. solani are members of the genus Endornavirus. This is also the first report of occurrence of at least five different mycoviruses in a single isolate of R. solani. These findings will provide the basis for further understanding of mycoviruses and their influence on Rhizoctonia hosts, which will help the development of novel biocontrol strategies for management of Rhizoctonia diseases in potato and other economic crops that are being infected by this fungus.