Identification of Non-Pseudomonas Blotch Pathogens Using High Throughput Isolation

View the project final report

Commodities

• Miscellaneous: mushrooms

Practices

• Pest Management: biological control

Bacterial blotch refers to a group of diseases characterized by the formation of spots and discoloration on the mushroom cap. Blotch diseases are common threats in mushroom production and were once thought to have been caused by a few Pseudomonas species. Several non-Pseudomonads, including Mycetocola spp., Burkholderia cepacia, and Ewingella americana, have been identified as causal agents of bacterial blotch and found in different combinations with other species on the surface of mushroom caps. The complete diversity of pathogens, both Pseudomonas and non-Pseudomonas, causing blotch on white button mushroom caps is still unknown. One barrier to discovering the complete diversity of blotch diseases is the labor-intensive nature of pathogen isolation and identification. Using traditional low-throughput schemes, bacteria are isolated on non- or semi-selective media followed by multiple rounds of purification and storage of individual isolates prior to intensive pathogenicity testing. This results in high demand for supplies, hands-on effort, time, and space to conduct the study.

In this study, a high-throughput bacterial isolation system and traditional isolation scheme will be used to recover pathogenic bacteria from symptomatic mushrooms. The Prospector System ™ (manufactured by General Automation Laboratory Technologies (GALT)) will be used for high-throughput isolation, cultivation, and screening of bacteria from symptomatic mushrooms in Pennsylvania. High-throughput isolation and standard isolation, followed by pathogenicity tests, will be used to build a collection of blotch pathogens. 16S rDNA sequence analysis will determine the preliminary identification of the isolates, additional methods will be used to confirm the 16S rDNA results.

Aim 1: Determine whether non-Pseudomonas species are responsible for causing bacterial blotch in mushroom production in Pennsylvania using two distinct approaches. The first approach will use a high-throughput system to exhaustively isolate non-fluorescent organisms from a limited number of symptomatic mushrooms. The second more traditional approach, will be labor-intensive and isolate a few individual organisms from many different symptomatic mushrooms. Non-fluorescent isolates will be tested for pathogenicity using Koch’s Postulates. 

Aim 2: Determine the identity of the non-Pseudomonas blotch pathogens from aim 1. 16S rDNA sequence analysis will provide preliminary identification of the pathogenic organisms isolated in aim 1. If appropriate, other identification techniques such as MLSA or phenotypic tests will be used to confirm the identity of organisms.