Progress report for GNE24-321
Project Information
Cucurbit yellow vine disease (CYVD) affects most commercially available cucurbits. Disease symptoms include yellowing, leaf scorching, and stem discoloration. CYVD is caused by the bacterium Serratia marcescens and is vectored by the squash bug (Anasa tristis), a common pest of cucurbits. Squash bugs acquire S. marcescens by feeding on the vascular tissue of infected plants, and the bacterium can persist in the insect's hemocoel through molting and overwintering events. Our project aims to determine the distribution, overwintering, and recent CYVD entry through genetic diversity studies of S. marcescens isolates collected in New York and across the Northeast. Furthermore, we seek to investigate the percentage of squash bugs that carry S. marcescens in a field at different time points, starting with the overwintering adults; we will collect squash bugs from different farms and test them for the presence of S. marcescens using molecular techniques. Lastly, we seek to determine identify the host range of S. marcescens isolates collected from cucurbits to determine the risk of infection with the presence of these strains in the state. Overall, this research will provide information needed to develop an integrated pest management program for this emergent disease.
- Collect Serratia marcescens isolates from NY and the different Northeastern states for whole genome sequencing to understand pathogen overwintering and geographic distribution
Hypothesis
There will be low genetic diversity between S. marcescens isolates collected from different states in the Northeast due to pathogen overwintering and not introduction of new isolates.
- Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S marcescens and how it changes over time
Hypothesis:
There will be a percentage of squash bugs that carry S. marcescens overwinter and this percentage will increase over the growing season as newly hatched nymphs feed on infected plants.
- Host range study of S. marcescens isolates into other crops of economic importance to understand the risk of disease spread into other specialty crops in the Northeast
Hypothesis:
Isolates of S. marcescens coming from cucurbit can infect other crops of economic importance in New York.
The purpose of this project is to assess the need for sustainable management techniques for cucurbit yellow vine disease (CYVD) caused by Serratia marcescens and transmitted by the squash bug Anasa tristis. Cucurbits are grown across the state of New York, with more than 12,000 acres on 1170 farms in cultivation annually (USDA-NASS 2022). The main cucurbit crops grown are squash (including zucchini) (1109 farms, 5,582 acres), pumpkins (1170 farms, 4,577 acres), cucumbers and pickles (869 farms and 1917 acres), cantaloupe, muskmelon, and honeydew (407 farms and 289 acres), and watermelon (362 farms and 265 acres). Cucurbits are grown on large and small operations, and CYVD can be found on both. Symptoms of this disease include leaf yellowing, stunting, decaying, discoloration of the stem, and scorching of margins; losses due to this disease are up to 100% (Zhang et al. 2003). CYVD is an emerging threat in New York and was only recently reported in the state (Rodriguez-Herrera et al. 2023). However, CYVD has been present in surrounding states, including Massachusetts and Connecticut, for a long time (Boucher 2005; Wick et al. 2001). Therefore, the disease could be common in New York but might be confused with other diseases like bacterial wilt caused by Erwinia tracheiphila since symptoms are similar, which has been the case in other states (Boucher 2005). Therefore, learning about pathogen distribution and diversity is critical. I have contacted pathologists and diagnosticians in four states (NJ, CT, PA, and MA), and they will provide squash bugs and diseased cucurbits for this study. Clonality of the population will indicate that the same isolate is causing disease across the Northeast (and perhaps across the country) and is surviving on a farm from one season to the next. On the other hand, if S. marcescens isolates are genetically diverse, this indicates that new isolates have been introduced to the region. Based on available literature, there are no reports of seed transmission of the bacterium.
Squash bugs transmit the bacterium, causing CYVD when it feeds on cucurbit plants. Adults of squash bugs overwinter and start feeding and laying eggs on cucurbit plants in the spring and early summer (Bonjour and Fargo 1989). Currently, there is no information on the percentage of adult squash bugs that may be carrying S. marcescens after overwintering in northern climates. Additionally, while there are no reports of A. tristis eggs harboring S. marcescens, nymphs can be infected if they feed on an infected plant. Determining the percentage of early-season adults infected with S. marcescens, as well as the percentage of nymphs and adults over the course of the season, will elucidate not only the risk of infected overwintering insects but also the rate of spread of the pathogen during an outbreak. This information is critical to develop a sustainable disease management strategy. Current control strategies rely on insecticides for squash bug control.
Serratia marcescens is a common environmental bacterium. Some strains can cause human and insect diseases, while others are present in the environment, and some are known to be rice endophytes (Tavares-Carreon et al. 2023). There are several reports of S. marcescens causing disease in crops other than cucurbits including corn, bell pepper carrots, onions, tobacco, and alfalfa (Bruton, Fletcher, et al. 1998; Bruton, Pair, et al. 1998; Gillis, Rodríguez, and Santana 2014; Luo et al. 2006; Ovcharenko et al. 2010; Wang et al. 2015). There is no evidence in the literature that one strain of S. marcescens coming from a single host can infect others, except one doctoral thesis from 2006 suggesting that some environmental strains can cause disease in other crops like carrots, onions, and tobacco (Luo et al. 2006). This proposal seeks to better understand S. marcescens host range by testing isolates from cucurbits on other hosts as well as testing isolates from the environment, insect, or other plant hosts to see if they can infect cucurbits. This will help us measure the risk of disease outbreaks in crops of economic importance in New York and the Northeast.
Research
Objective One: Collect Serratia marcescens isolates from NY and the different Northeastern states for whole genome sequencing to understand pathogen overwintering and geographic distribution
Collection of isolates: The stems of symptomatic plants will be collected. Each stem will be cut into small pieces (2 to 3 mm), surface sterilized with 70% ethanol for 60 s and 10% bleach for 60 s and rinsed with sterile water. The tissue will be macerated in sterile water, and the resultant suspension will be streaked on King’s B (KB) medium (King et al. 1954). Plates will be incubated at 28°C for 24 h. When development of white, round bacterial colonies that are smooth and creamy in appearance, single colonies will be transferred to new KB plates and incubated for 24 h. Storage will be done by growing a single colony in KB liquid and grown overnight. 1000 ul of bacteria will be put in 250 ul of 16% glycerol and stored at -80.
Bacterial DNA will be extracted using the Wizard Genomic DNA Purification Kit Protocol (Promega, Madison, WI). PCR will be carried out using YV1 and YV4 primers specific to the 16S rDNA region of S. marcescens (Zhang et al. 2005). DNA concentrations will be determined with a Nanodrop One at 600 nm (Thermo Fisher, Waltham, MA).
Whole-genome sequencing: DNA libraries will be prepared by the Institute of Biotechnology at Cornell University from bacterial gDNA using Illumina Nextera DNA prep kits with six PCR cycles. Illumina NovaSeq 2x150-bp sequencing will be conducted on this library to generate raw reads. Genome sequencing raw reads will be downloaded to BioHPC servers at Cornell University, and md5 hashtags will be checked for integrity. raw reads will be assembled and annotated for computation of average nucleotide identity (ANI) and phylogenic analysis to resolve species identity.
Phylogenetic analysis: The bacterial genome Genbank format will be generated using the Prokka v1.14.5 annotation tool. Other published Serratia marcescens Genbank accessions will be downloaded and compared with the sequenced isolates. Single copy orthology gene and single nucleotide polymorphism (SNP) analyses will be conducted to infer the evolution and epidemiology of S. marcescens isolates.
Objective two: Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S. marcescens and how it changes over time
Squash bug samples will be collected at the farm that will be visited over the summer and samples will include eggs, nymphs, and adults. squash bugs will be placed in plastic bags and will be taken to the lab to store them at -80 °C. To test the percentage of squash bugs carrying CYVD pathogen, DNA will be extracted from the insects using a commercial kit (Qiagen, Hilden, Germany). After DNA extraction, A PCR will be carried out using CYVD pathogen specific primers (YV1-YV4) (Zhang et al. 2005), and the presence or absence of S. marcescens will be determined after running amplification products on an agarose gel. We expect to optimize a protocol to determine the presence of bacteria in the insect.
Objective three: Host range study of S. marcescens isolates into other crops of economic importance to understand the risk of disease spread into other specialty crops in the Northeast
Bacterial strains and growth conditions: Bacterial strains that will be used include several strains of S. marcescens, a nonpathogenic control, Escherichia coli DH5a. Bacteria will be stored at 80ºC in KB broth containing 50% glycerol. For the experiment, S. marcescens strains will be streaked in KB media and incubated overnight at 28ºC. E. coli strain will be grown in LB and incubated overnight at 37ºC
Plants. Plant species were selected based on a literature review indicating that S. marcescens could infect these species. Tobacco (Nicotiana tabacum), carrot (Daucus carota), bell pepper (Capsicum annuum), onion (Allium cepa), hemp (Cannabis sativa), eggplant (Solanum melongena), potato (Solanum tuberosum) and squash (Cucurbita maxima) will be used for this experiment; plants will be grown under greenhouse conditions. All seeds will be started in 72-cell plastic trays and then transplanted to 5-inch pots. Greenhouse conditions will be set at 16h light and 8hr dark (23-35°C). squash and pepper will be inoculated two weeks after planted. For tobacco and hemp, plants will be inoculated four weeks later. Carrots will be inoculated at six weeks. Onion, eggplants, and potatoes will be purchased in the store for fruit inoculation.
Bacterial inoculation: Overnight-grown bacterial plates will be used for inoculation. For squash, hemp, and pepper, a mounting tip will be used to grab a single colony and insert it into the junction between the cotyledon and stem up to five times. Each bacterial strain (22211, 22212, 22213, RO1, and PO1) will be inoculated into 40 plants from each species. The test will be performed three times. Four weeks after inoculation in the greenhouse, the stems will be sliced horizontally and examined visually for phloem necrosis. For each of the 10 fruits, onion, carrot, bell pepper, and eggplant will be used. The overnight bacteria isolate grown in KB and adjusted 1.2 OD will be used. Fruit will be punctured, and 10 ul of the bacterial suspension will be applied. Fruit will be placed in a petri dish lined with moistened paper towels and incubated at 28°C for symptom development. Symptoms will be observed one and two days after inoculation.
Bacterial DNA will be extracted using the Wizard Genomic DNA Purification Kit Protocol (Promega, Madison, WI). PCR will be carried out using YV1 and YV4 primers specific to the 16S rDNA region of S. marcescens and 79F/R primers specific for S. marcescens causing CYVD (Zhang et al. 2005).
Objective One: Collect Serratia marcescens isolates from NY and the different Northeastern states for whole genome sequencing to understand pathogen overwintering and geographic distribution
Progress to date: Isolates were collected from the research plots at Cornell AgriTech Research Station at the end of the field season 2024. Extension educators and partners were also contacted to look out for CYVD-like symptoms. Cucurbit crowns (3 to 5 inches from the root to the stem) were requested. To date, six plant samples were sent from two different farms in Orange County. I have also scouted fields in Erie County in Western NY and have received samples from colleagues in Suffolk County on Long Island. One of the samples from Orange County was positive for CYVD. These samples are being processed for Illumina sequencing at the beginning of Spring 2025.
Objective two: Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S. marcescens and how it changes over time
Progress to date: Squash bugs were collected during the field season in 2024. A number of 30 squash bugs, 30 squash bug nymphs, and 30 cucumber beetles (a common pest that may also transmit S. marcescens) were collected over 8 weeks. These insects will be tested for the presence of S. marcescens in 2025.
Objective three: Host range study of S. marcescens isolates into other crops of economic importance to understand the risk of disease spread into other specialty crops in the Northeast
Progress to date: Since there has not been an inoculation method that could yield uniform diseased plants, we are currently testing inoculation methods.
Education & Outreach Activities and Participation Summary
Participation Summary:
I plan to share the results of this project with farmers, gardeners, industry leaders, and academic peers throughout the time of this project and after it. Collaborators and extensions will be key to accomplishing this goal. Having Steve Reiners as a collaborator is key for maintaining relationships with growers and extensionists. I have already engaged in meetings to inform about Cucurbit Yellow Vine Disease in New York and plan to expand that knowledge at field days, vegetable meetings, and professional conferences. I will also use the huge impact of social media to share my results. I plan on doing YouTube videos that will talk about our research and findings.
As a Latina, I understand what it is to be at a disadvantage when it comes to finding opportunities, whether for higher education or funding my project. Therefore, as a native Spanish speaker, I intend to use this skill to translate all the information from English to Spanish to reach a broader and underseen audience. I have already done this, as I created videos about common problems in cucurbits last year along with Chris Smart and Steve Reiners. The videos were done in English, which I translated and included captions in Spanish, and they have more than 1,800 views on YouTube. I will keep working with the Hispanic communities to make all the outcomes from this project available to them as they are a working force in the US, and having access to this information is a right that we can give to them. Specifically, I will create at least three YouTube videos talking about 1. Overwintering of Squash Bug 2. Management of CYVD 3. Prevalence of CYVD in NY. These videos will be made in English and Spanish. A factsheet will be made to talk about the CYVD disease cycle and management in both English and Spanish.
Specific Outreach
Empire State Producers’ Expo (January 2025 or January 2026): Here I will reach an audience of farmers across the state and northeast region.
Winter Vegetable Meeting in Western NY (February 2025 or 2026): I plan on attending this meeting and talking about current and future projects organized by Cornell Cooperative Extension
American Phytopathological Society (APS) Meeting (Summer 2025): I will share my work whether through a talk or poster to reach an audience of plant pathologists and people from the industry
Cornell Cooperative Extension Factsheet and Newsletter: I will write a factsheet that describes CYVD diagnosis and management practices and will be translated into Spanish.
Video submission on the Cornell SIPS YouTube Channel (18k subscribers): I will record videos about CYVD and cucurbits, which will also have Spanish captions.
Peer review journal article: I will write a manuscript article based on the outcomes of this project that will be submitted to a Journal like Plant Disease or Plant Health Progress
Annual and Final Reports: I will submit annual reports and a final report to Northeast SARE
Project Outcomes
We expect that this project will bring awareness about a yield-limiting disease like cucurbit yellow vine disease. We hope to bring new management tools that will help growers avoid disease in their fields. Our goal is to write a fact sheet that we can share with the farmers and extension educators before the summer starts so we can bring awareness about the disease and make an impact by providing research-based solutions. We also hope to give talks to farmers throughout the year.
At the end of the project, we hope we can learn the primary survival method used by the pathogen that causes CYVD, as well as the % of squash bugs that carry the CYVD pathogen in an infected field. Finally, we hope to learn the host range of the causal agent of CYVD.
During this period of the grant, I have been able to contact research educators and faculty at different universities to ask for samples of cucurbit yellow vine disease (CYVD). This will increase people's awareness about this disease and has molded my communication skills. This experience also creates awareness of the importance of sustainable agriculture by consciously using the farmer's resources destined for research and yielding results that will positively impact their lives and the economy.
We expect that during this grant we can learn more about CYVD and have the opportunity to build relationships with growers. We hope to use this knowledge to develop management techniques for growers.