Progress report for GNE21-267
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.
Cultivated edible mushrooms have been on the rise since the late 1800s (Royse et al., 2017). Agaricus bisporus, or the button mushroom, is one of the most common edible mushrooms produced and consumed in the US (Robinson et al., 2018). Between 2019-2020, Agaricus crop production valued at an estimated $1.15 billion US dollars. In the US, Pennsylvania is responsible for leading the country in button mushroom production (USDA, 2020). Bacterial blotch is a complex of diseases threatening the mushroom industry, resulting in discoloration and deformation of the mushroom cap (Tolaas, 1915). Blotch diseases can lead to severe economic loss by reducing the marketability of the crop by lowering the expected shelf life after harvesting, reducing the standard of the product, and reducing crop yield (Navarro et al., 2018). Diverse species of Pseudomonas have been identified as being the causal agent of blotch diseases in mushroom production (Martins et al., 2020). In 2016, the mushroom industry identified bacterial blotch as one of the most important diseases of mushrooms during a Penn State University Industry Strategic Planning meeting. Management of blotch caused by Pseudomonas became one of the primary goals of a subsequent USDA OREI grant.
Bacterial blotch results in the formation of brown spots and sunken lesions on the surface of mushroom caps (Munsch & Alatossava, 2002). As part of the OREI project described above, Martins and Bull identified 10 distinct species of Pseudomonas that cause blotch in Pennsylvania (Martins & Bull, 2019). Similar results were found in Europe (Taparia et al., 2020), all shown to vary in symptoms and disease progression. P. agarici can cause drippy gills or yellow blotch on the mushroom cap (Young, 1970), while P. costantinii and P. tolaasii have been shown to cause brown blotching on the mushroom cap, and P. gingeri have been shown to cause discoloration on the mushroom cap (Osdaghi et al., 2019). These findings are just a couple of examples that highlight the great level of diversity within Pseudomonads that cause bacterial blotch. In recent collaboration between Hamidizade and our team, novel non-Pseudomonads pathogens from Iran were identified but currently no similar study has been conducted in the US (Hamidizade et al., 2020). The spectrum of blotch pathogens causing this disease in US mushroom growing regions is still something that needs to be explored. Understanding which blotch pathogens are responsible for causing this disease will help in targeting the spectrum of pathogens responsible in future management practices.
Diseased mushrooms will be collected from three commercial mushroom houses in Pennsylvania - Marlboro Mushrooms, Phillips Mushrooms, and an additional mushroom to be identified by the American Mushroom Institute (AMI; see letters of support). Two mushrooms per facility will be collected for deep sampling using the Prospector System ™ (approach A). In addition, one hundred mushrooms will be collected for traditional isolation (approach B) from each facility. Each mushroom cap will be stored in a sterile specimen cup in a cooler and transported from facilities in Chester or Burks County to the Penn State University Park campus for processing.
Approach A – High-throughput deep sampling
I have employed the unique features of the Prospector System ™, a high-throughput cultivation platform, to isolate non-fluorescent organisms present in the symptomatic tissue. The Prospector System ™ allows for the isolation of up to a thousand colonies from each sample with minimal manual labor. The mushroom lesions were cut, surface sterilized, macerated in buffer, and passed through a filter to remove lingering mushroom tissues from the sample. The sample was then inserted into the Prospector System ™ array where individual cells will grow in isolation. Pseudomonas blotch pathogens fluoresce in King’s Medium B (KMB) while no other bacteria are fluorescent in this medium (King et al., 1954). The Prospector System™ will be programmed to select non-fluorescent isolates and transfer them onto a 96 well plate for long-term storage and subsequent pathogenicity testing. No additional labor was required after the initial sample preparation in order to obtain large numbers of purified cultures of non-fluorescent isolates from symptomatic mushrooms. Preliminary data indicated an average of 27 non-fluorescent isolates were recovered from each sample using this approach (Herschlag et al., unpublished results). It is anticipated that a library of 150 isolates will be generated from these mushrooms.
The Prospector System ™ is designed to enhance the process of studying microbial organisms by scaling up the number of isolates retrieved within a given sample compared to traditional approaches. This technology has never been used to study the diversity of blotch causing pathogens, symptomatic mushrooms were used to troubleshoot the system’s ability to select fluorescent and non-fluorescent organisms. Seven mushrooms were collected August 2021 from three different mushroom facilities in CA – Royal Oaks Mushroom Farm, South Valley Mushroom Farm, and Morgan Hill Mushroom Farm. From this sampling a total of 870 isolates were retrieved, 684 selected to be fluorescent and 186 selected to be non-fluorescent organisms as shown in table 1.
Approach B – Traditional sampling
An additional 100 mushrooms from each of the three organic mushroom facilities will be sampled. The lesions from the diseased mushrooms will be removed, surface sterilized, macerated in buffer, and the bacterial sample will be streaked for single colonies on KBM with cycloheximide to screen for non-fluorescent colonies. Pure cultures will be generated by multiple rounds of isolation, starting with single colonies. Each isolate will be stored at -80°C in 50% nutrient broth 50% glycerol stocks. This approach is labor-intensive and would require inoculating at least 300 agar plates for processing the samples from a single facility. Likewise, to test for pathogenicity, isolates must be grown on individual petri dishes for one or more rounds to prepare inoculum.
48 isolates were collected from the five mushrooms collected from Marlboro Mushrooms. From this sample, 40 fluorescent and 8 non-fluorescent isolates were identified. Phillips Mushrooms and an additional mushroom farm identified by AMI will be used to collect future samples.
To test bacterial isolates for pathogenicity, 10 µl of bacterial suspensions will be spotted onto surface-sterilized mushroom caps where the surface has been removed with a sterile knife. Mushrooms will be incubated in a moisture chamber for a 72-hour period with observations recorded every 24 hrs. A disease scale will be developed by documenting symptoms caused by known blotch pathogens from a current strain collection and be used to rank the level of disease severity by blotch pathogens. Pathogenicity will be confirmed via Koch’s Postulates. Pathogenic isolates will be identified in aim 2.
One potential pitfall would be to not find non-Pseudomonas pathogens from these facilities, if that is the case mushrooms will be sampled from additional facilities.
16S rDNA sequence analysis will be used for the preliminary identification of the isolates. The 16S rDNA for each non-fluorescent mushroom pathogen will be amplified using universal primers for 16S rDNA (Weisburg et al., 1991). Amplicons will be Sanger sequenced at the Penn State Genomics Core facility. 16S sequences will be compared and matched to sequences in the Ribosomal Database Project (Cole et al., 2014). This will identify the pathogens to the genus and provide a hypothesis for species.
Once the organisms are identified by 16S sequence analysis, the taxonomic literature for that group will be explored to identify the phenotypic and sequence-based assays for distinguishing organisms at the species level. This approach was used by Hamidizade et al. (2020) in collaboration with our team to identify non-fluorescent blotch pathogens from Iran.
Education & Outreach Activities and Participation Summary
The mushroom industry is currently not using the Prospector System ™. The first presentations will present the potential to use this approach for their industry. Research findings will be presented to mushroom growers and other industry professionals as they become available. Oral presentations were presented at the 62nd & 63rd annual Penn State Mushroom Short Course meeting September 2021 and 2022. The Mushroom Short Course is an annual event designed to connect mushroom growers to current research addressing issues threatening mushroom production. An oral presentation was delivered in September 2021 to the International Society for Mushroom Science (ISMS) which is an industry-run conference designed to connect growers on an international level (Richardson et al., 2021). An article summarizing the findings will be presented to the American Mushroom Institute (AMI) through their magazine, Mushroom News, which reports issues ranging from management strategies, production approaches, and updates in changes to government regulations to growers across the country. Published articles summarizing the findings will be shared in media using the Penn State Mushroom Research Extension news board provides growers access to crucial information on topics relating to the mushroom industry. Lastly, the completed work was presented during the Plant Health 2022 annual meeting to members in the American Phytopathological Society.
In addition to extension directed at the industry and scientists, I will present to students from historically underrepresented backgrounds. Through a long-standing collaboration with Lincoln University, the oldest degree-granting HBCU in the country and located in the same counties as the mushroom farms, I will present my thesis research and co-present a professional development activity to microbiology students. I will present research plans and findings to the annual Society Advancing Chicanos/Hispanics & Native Americans in Science (SACNAS) national conference. Students will learn about exciting research and careers in the mushroom industry.
The results from the sampling conducted in CA indicate the Prospector System ™ has the potential to be a useful tool for studying the diversity of blotch causing pathogens by selecting a large set of isolates compared to traditional approaches and enhancing the diversity blotch pathogens captured within a given sample.
814 of the isolates processed using the Prospector System ™ were submitted to be Sanger sequenced at the Penn State Genomic Core facility and are currently undergoing sequence analysis. In addition to the identification of blotch pathogens using high-throughput isolation techniques, I along with a Penn State undergraduate Millennium Science Scholar have worked on assessing the host range of known blotch pathogens of Agaricus bisporus on a variety of exotic mushrooms. We evaluated the virulence of 11 species of Pseudomonas blotch pathogens previously demonstrated to cause bacterial blotch disease on white button mushrooms grown in Pennsylvania by the Bull lab. The known blotch pathogens were inoculated onto four healthy varieties of exotic mushrooms, Lentunula edodes (shiitake), Pleurotus ostrearus (oyster), Pleurotus eryngii (king oyster), and Grifola frondose (maitake), all of which were collected from a commercial mushroom farm in Pennsylvania. Disease severity was assessed daily over three days using a visual scale and the pathogen population levels were estimated using a serial dilution assay performed on rifampicin amended media. Our results showed that disease severity and pathogen population levels for oyster and king oyster mushrooms increased more by 1 million fold above the level of inoculation compared to maitake and shiitake mushrooms. Shiitake and maitake mushrooms disease severity and population levels either remained the same or were less than the level of inoculation, potentially indicating differences in the susceptibility of mushroom variety. We are currently investigating whether these exotic mushrooms (shiitake and maitake) contain any inhibitory compounds to blotch pathogens.
Additional processing of the isolates using the Prospector System ™ needs to be conducted to determine the most effective way to use this technology for studying the diversity of blotch pathogens. Our results from the exotic mushroom experiment suggest there could be some level of pathogen inhibition or difference in susceptibility being observed with shiitake and maitake mushrooms.