Seed infection rate, but not pathogen titer, positively correlates with disease index of Cephalosporium stripe in winter wheat

Authors: SHENG, HONGYAN - Washington State University; Esvelt Klos, Kathy; MURRAY, TIMOTHY - Washington State University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2022
Publication Date: 3/26/2023
Citation: Sheng, H., Esvelt Klos, K.L., Murray, T.D. 2023. Seed infection rate, but not pathogen titer, positively correlates with disease index of Cephalosporium stripe in winter wheat. Phytopathology. 113(3):436-447. https://doi.org/10.1094/PHYTO-06-22-0211-R.
DOI: https://doi.org/10.1094/PHYTO-06-22-0211-R

Interpretive Summary: Wheat infected with the Cephalosporium gramineum fungus can produce seed colonized by the fungus. This seed may be a source of disease when planted. The purpose of this study was to improve upon existing assays for detection of Cephalosporium gramineum within wheat seed for both accuracy and suitablity to a high-throughput laboratory environment. A PCR-based detection method was modified for single-seed assays. Three winter wheat cultivars were selected for assay validation to represent a range of disease reactions. To validate the assay in relation to disease intensity in the field, field-grown seed was collected over 4 years from replicated plots after visual scoring of disease intensity. Assay performance was compared against the traditional culture-based method wherein seed samples are placed on a semi-selective fungal growth media and incubated to allow time for fungi to grow and emerge from infected seed. Diagnosis of Cephalosporium gramineum was made after visual examination of spores. The improved assay was more accurate and more sensitive than the traditional culture-based method; and was more suited to high-throughput testing that the previous PCR-based assay.

Technical Abstract: Cephalosporium gramineum survives primarily in colonized plant residue but is also transmitted by seed at low frequency. Factors influencing seed transmission are not well understood, in part due to the difficulty of accurately assessing the seed infection rate. The purpose of this study was to improve the existing PCR-based seed detection assay for accuracy and adaptation to high-throughput assessment, to validate it in relation to disease intensity in the field, and examine the effect of host-resistance on seed infection rate. Field-grown seeds of three winter wheat cultivars were collected over 4 years from replicated plots with known disease intensity. Culture-based determination of percent infected seed was done by isolation of C. gramineum on a semi-selective medium from 2016 seeds per seed lot. DNA of 384 seeds from the same seed lot was extracted individually and the PCR-based assay for detecting C. gramineum was performed on each seed. C. gramineum was isolated from 0.12% of the seed on average (range 0 to 0.74%), whereas the PCR-based assay averaged 3.7% of the seed (range 1.3 to 7.6%). The PCR products from 25 positive seeds had 99 to 100% sequence similarity to the ITS sequence of C. gramineum in the GenBank. The high-throughput single-seed PCR-based assay was more accurate and more sensitive than the culture-based method. Quantitative real-time PCR (qPCR) on DNA of 751 seeds that tested positive for C. gramineum was used to quantify the pathogen load. Positive seed contained 0.017 to 77.1 pg/seed with a mean of 3.0 pg of C. gramineum DNA. Disease intensity of affected plots was positively correlated to % detection by PCR (R2 = 0.3471, p = 0.0157). This high-throughput single seed detection method, along with qPCR should facilitate studies to improve our understanding of seed transmission of C. gramineum in winter wheat.