Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

Abstract

A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology, ecology and epidemiology of an obligate biotroph

“…Definitive aerobiological studies have been limited by the relatively low density of ascospores in vineyard air, and the comparatively high threshold of detection of most mechanical samplers (Gadoury and Pearson, 1990a). However, the recent development of polymerase chain reaction (PCR)‐based detection methods (Falacy et al ., 2007) may allow more precise quantification of airborne ascospore dose at inoculum levels that typify the natural vineyard environment.…”

Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees

“…To overcome these difficulties, we can utilize the findings from several studies that identified specific pathogens or multiple species by using DNA-based high-throughput techniques to establish risk assessment models or to monitor microbial diversity in planta under various environmental conditions; for example, identification and differentiation of the bacterial pathogens of potatoes [9], quantitative assessment of the growth of phytopathogenic fungi on various substrates [10], risk assessment of grapevine powdery mildew [11], diversity pattern of maize leaf epiphytic bacteria in relation to the plants that are genetically resistant to fungal pathogens [12], diagnostic DNA microarray for rapid identification of quarantine bacteria [13], characterization of complex communities of fungi and fungal-like protists [14], proteogenomic analysis of the physiology of phyllosphere bacteria [15], microarray screening of the variability of 16S–23S rRNA internal transcribed spacer region (ITS) in Pseudomonas syringae [16], and analysis of molecular battles between the plant and the pathogenic bacteria in the phyllosphere [17].…”

Development of a grower‐conducted inoculum detection assay for management of grape powdery mildew

“…However, the LAMP assay does not require expensive technology or formal training for DNA extraction and the detection of inoculum (Notomi et al ., ), making it suitable for commercial use with grapevine growers conducting the detection analyses. This study, in conjunction with other studies (Falacy et al ., ; Carisse et al ., , ; Van der Heyden et al ., ), also demonstrates that there may be benefit to managing polycyclic diseases, at least those caused by other Erysiphales, using airborne inoculum detection assays. In both sampling years, the L‐LAMP detection results were not significantly different from that of the qPCR detection results, indicating that the extraction assay was sufficient for detection (Table ).…”