Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris.

Katie Coats,Annie Debauw,Gary Chastagner,Daniel P Roberts,Adnan Ismaiel,Dilip K Lakshman

doi:10.3390/jof8020163

Abstract

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen, Rhizoctonia tuliparum (Rtul). Sclerotia present in infected bulbs, as well as overwintering sclerotia in soil and field debris, are the primary sources of infection. A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels, is needed for the management of disease caused by this pathogen. 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 using these primers, where the threshold of detection was approximately 1 fg Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with those 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 qPCR 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 these three Rtul isolates.

Highlights

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen Rhizoctonia tuliparum (Rtul) [Whetzel and Arthur; Synonym: Sclerotium tuliparum (Kleb)] [1,2,3,4,5,6]
Tested isolates were from R. solani anastomosis groups (AGs) 2-1, 3, 4, 5, 8, and 9, and Ceratobasidium isolates from AGs A, E, and I, as well as W. circinata and several unidentified Rhizoctonia species
We developed a robust TaqMan® real-time quantitative PCR (qPCR) assay to detect and quantify Rtul DNA based on a unique sequence of the internal transcribed spacer region

Summary

Introduction

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen Rhizoctonia tuliparum (Rtul) [Whetzel and Arthur; Synonym: Sclerotium tuliparum (Kleb)] [1,2,3,4,5,6]. Gray bulb rot is primarily a problem in field or landscape-grown tulips and irises, but the pathogen has been reported in daffodil, crocus, gladiolus, and lily [9]. The fungus forms felty masses between the scales, and infected bulbs rot from the top down. Both the bulbs and roots turn grayish in color and progressively dry and shrivel at the site of infection. Diseased plants tend to occur in patches in field and landscape plantings, but this disease can completely destroy the crop after several rotations if unmanaged

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