Detection and molecular phylogenetic-morphometric characterization of Rhizoctonia tuliparum, causal agent of Grey Bulb Rot of tulips and bulbous iris

Authors: COATS, KATIE - Washington State University; DEBAUW, ANNIE - Washington State University; Lakshman, Dilip; Roberts, Daniel; Ismaiel, Ed - Ed; CHASTAGNER, GARY - Washington State University

Submitted to: The Journal of Fungi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2022
Publication Date: 2/8/2022
Citation: Coats, K., Debauw, A., Lakshman, D.K., Roberts, D.P., Ismaiel, A.A., Chastagner, G. 2022. Detection and molecular phylogenetic-morphometric characterization of Rhizoctonia tuliparum, causal agent of Grey Bulb Rot of tulips and bulbous iris. The Journal of Fungi. 8(2):163. https://doi.org/10.3390/jof8020163.
DOI: https://doi.org/10.3390/jof8020163

Interpretive Summary: Grey Bulb Rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen Rhizoctonia tuliparum and is primarily a problem on field- or landscape-grown tulips and irises, but can infect daffodil, crocus, gladiolus, and lily. A sensitive and accurate method for detection of this pathogen in soil and infected bulbs is needed for management of disease. We designed a unique set of molecular primers targeting R. tuliparum and developed a highly sensitive and specific detection method for its detection based on Polymerase Chain Reaction technology. We also characterized R. tuliparum at the molecular level and described the implications of these findings on disease management. This information will be useful to plant pathologists, agricultural extension agents, and growers.

Technical Abstract: Grey bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen Rhizoctonia tuliparum (Rtul). A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels is needed for management of disease caused by this pathogen. Sclerotia present in infected bulbs as well as overwintering sclerotia in soil and field debris are the primary sources of infection. We designed a unique set of primers targeting the ITS2 region of the Rtul genome and developed a highly sensitive quantitative PCR (qPCR)-based method for Rtul identification where the threshold of detection was around 1 femtogram of Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with sclerotia grown in the lab, and more sensitive with natural-light than natural-dark sclerotia. Also, the detection method was more sensitive when sclerotia were extracted from soil than from bulb tissue. The pPCR method was highly specific as no PCR amplification was detected when genomic DNA from 62 non-Rtul Rhizoctonia isolates from a wide range of anastomosis groups were tested. To understand the evolutionary relationships and genomic diversity of Rtul, we performed phylogenetics of the ITS1-5.8S-ITS2 region and ITS2-molecular morphometric characterization (MMC) of Rtul isolates. The three Rtul isolates whose ITS sequences were available in GenBank formed a distinct phylogenetic clade with Ceratobasidium anceps as the nearest relative. Furthermore, MMC analysis revealed genetic divergence among the three Rtul isolates.