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 ureilytica and is vectored by the squash bug (Anasa tristis), a common pest of cucurbits. Squash bugs acquire S. ureilytica 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. ureilytica isolates collected in New York and across the Northeast. Furthermore, we seek to investigate the percentage of squash bugs that carry S. ureilytica 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. ureilytica using molecular techniques. Lastly, we seek to determine identify the host range of S. ureilytica 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.
Note: because we now know that the species causing CYVD is S. ureilytica, the name has been changed throughout the proposal for accuracy. This research has shown that isolates that were thought to be S. marcescens are actually S. ureilytica.
- Collect Serratia ureilytica 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. ureilytica isolates collected from different states in the Northeast due to pathogen overwintering and not the introduction of new isolates.
- Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S ureilytica and how it changes over time
Hypothesis:
There will be a percentage of squash bugs that carry S. ureilytica overwinter and this percentage will increase over the growing season as newly hatched nymphs feed on infected plants.
- Host range study of S. ureilytica isolates into other crops of economic importance to understand the risk of disease spread into other specialty crops in the Northeast
We would like to change this objective to: Evaluation of Serratia spp isolates for their potential to cause disease in cucurbits.
Hypothesis:
Isolates of other Serratia spp can survive in cucurbit crops, but will not cause disease
The purpose of this project is to assess the need for sustainable management techniques for cucurbit yellow vine disease (CYVD) caused by Serratia ureilytica 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. ureilytica 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. ureilytica after overwintering in northern climates. Additionally, while there are no reports of A. tristis eggs harboring S. ureilytica, nymphs can be infected if they feed on an infected plant. Determining the percentage of early-season adults infected with S. ureilytica, 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 spp. 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). This proposal seeks to better understand Serratia spp. host range by testing isolates from the environment, insect hosts, or other plant hosts to determine whether they can infect cucurbits. This will help us measure the risk of disease outbreaks 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 were collected. Each stem was 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 was macerated in sterile water, and the resultant suspension was streaked on King’s B (KB) medium (King et al. 1954). Plates were incubated at 28°C for 24 h. When white, round bacterial colonies with a smooth, creamy appearance developed, single colonies were transferred to new KB plates and incubated for 24 h. Storage was performed by growing a single colony in KB liquid 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 were 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. Raw reads from genome sequencing were downloaded to the BioHPC servers at Cornell University, and md5 hashes were checked for integrity. Raw reads were assembled and annotated for the computation of average nucleotide identity (ANI) and phylogenetic analysis to resolve species identity.
Phylogenetic analysis: The bacterial genome in GenBank format was generated using Prokka v1.14.5. Other published Serratia ureilytica Genbank accessions were downloaded and compared with the sequenced isolates. Single-copy orthology gene and single-nucleotide polymorphism (SNP) analyses were conducted to infer the evolution and epidemiology of S. ureilytica isolates.
Objective two: Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S. ureilytica 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 taken to the lab for storage at -80 °C. To test the percentage of squash bugs carrying the 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. ureilytica will be determined by running the amplification products on an agarose gel. We expect to optimize a protocol to detect bacteria in the insect.
Objective three: Evaluation of Serratia spp isolates for their potential to cause disease in cucurbits.
Bacterial strains and growth conditions: The strains used will include several Serratia spp. Bacteria will be stored at 80ºC in KB broth containing 50% glycerol. For the experiment, Serratia spp. strains will be streaked in KB media and incubated overnight at 28ºC.
Plants. 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).
Bacterial inoculation: Overnight-grown bacterial plates will be used for inoculation. Each bacterial strain (n=24) will be inoculated into 4 plants using a syringae at a rate of 1.2 OD, and 300 ul of bacteria will be injected into the stem of two-week-old plants. The test will be performed two times. Four weeks after inoculation in the greenhouse, the stems will be cut horizontally and visually examined for phloem necrosis.
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 ureilytica isolates from NY and the different Northeastern states for whole genome sequencing to understand pathogen overwintering and geographic distribution
Note: because we now know that the species causing CYVD is S. ureilytica, the name has been changed throughout the proposal for accuracy. This research has shown that isolates that were thought to be S. marcescens are actually S. ureilytica.
Whole Genome Sequencing of Serratia ureilytica isolates using Illumina Sequencing
Summary
85 isolates from 13 US states were collected (Fig. 2), collected in different years and hosts, plus three sequences downloaded from NCBI (S07, SER00032, Serratia ureilytica T6) (n=88)
Similarity analysis using average nucleotide identity (ANI)
79 out of 88 isolates used in the analysis fell into the Serratia ureilytica clade (all with white colony morphology), with ANI >95%; isolates 22211, J4_1, R35, and W11 (all with red colony morphology) were classified as Serratia marcescens, and 4CB and 5SB (also white colony morphology) were classified as S. sarumanii.
We performed genotype grouping, assigning isolates with an ANI of 99% or higher to the same group. Using this cutoff, 20 groups were formed (Fig. 1), and 63 of the 88 isolates fell into a single genotype group that included isolates from multiple states (Fig. 3). The rest of the groups had one to three isolates per group.
We know that some isolates from group one are pathogenic to cucurbits from previous studies (i.e., 22212, P01, Z01, 1_1), so we assume all isolates from that group are pathogenic to cucurbits, given their low genetic diversity. However, we do not know if the isolates that fell into other genotypic groups are pathogenic. Our next step would be to phenotype representative isolates from each genotypic group.
The highest genetic diversity was observed in isolates from Illinois, which were divided into seven genotypic groups (Groups 4, 10, 11, 12, 13, 14, and 15).
Note: Group 2 contains S. sarumanii isolates; groups 16 and 17 contain S. marcescens isolates. Group 18 is a rice endophyte S. ureilytica, Group 19 is S. ureilytica from a human sample, and Group 20 is the S. ureilytica type strain
Single Nucleotide Polymorphism (SNP) analysis
The SNPs count for the 88 isolates was 222,467, indicating substantial genetic diversity. A tree was constructed based on the presence or absence of SNPs (Fig. 4), showing that S. ureilytica isolates are distinct from the other Serratia isolates we collected.
We then performed a SNP analysis using only the 63 isolates from genotypic group 1 (Figure 1) and found only 475 SNPs. This supports the theory that isolates in this group have little to no genetic diversity in their sequences, despite being collected across a wide geographic distribution and over a range of years (1999-2024). Furthermore, the isolates do not cluster by the year they were collected. These data point to long-term persistence of S. ureilytica isolates causing CYVD rather than to the evolution of distinct isolates in different geographic regions over time.
Pangenome analysis
Pangenome analysis of the 88 isolates showed a total of 12,233 genes, from which 2,274 were core genes (99-100% isolates), 779 soft core genes (genes present in 95-99% isolates), and 7,102 cloud genes (genes present only in 0-7%)
A core genome tree was built from the data generated from the pangenome analysis. We added metadata on year, state, and host (Fig. 6), and observed the same trend: isolates from genotypic group 1 were placed in a single clade within S. ureilytica. Although they were collected from different years (1999-2024) and different states (N=12), there is no apparent sub-clustering. This lack of structure implies that no distinct lineages are evolving separately in specific locations over time in this dataset. Instead, it points to a highly successful and stable genotype that circulates widely. However, the isolates of the other genotypic groups seem to be more genetically diverse and closer to the human S. ureilytica strains and the Type strain. This could indicate that there is some diversification happening within the genus.
2026_figures_Kensy_SARE_results
Objective two: Collection of squash bugs at different time points during the season to determine the percentage of squash bugs carrying S. ureilytica and how it changes over time
Squash bugs were collected during the 2024 and 2025 field seasons. Each week for eight weeks, 30 adult squash bugs, 30 squash bug nymphs, and 30 cucumber beetles (a common pest that may also transmit S. ureilytica) were collected. These insects will be tested for S. ureilytica in 2026. Our current focus is on identifying a DNA extraction kit that allows reliable detection of S. ureilytica while minimizing inhibition from the insect exoskeleton.
Objective three: Host range study of S. ureilytica isolates into other crops of economic importance to understand the risk of disease spread into other specialty crops in the Northeast
We would like to change this objective to: Evaluation of Serratia spp isolates for their potential to cause disease in cucurbits.
Based on results from our whole-genome sequencing, which revealed no genetic diversity among S. ureilytica isolates causing CYVD but also detected other Serratia species, we will test these isolates for their ability to cause disease in cucurbits. This will allow us to identify genetic factors associated with pathogenic isolates and compare them with those isolates that do not cause disease.
The primary objective of this project was to characterize the genetic diversity of Serratia ureilytica associated with cucurbit yellow vine disease (CYVD) across the United States, and to determine whether genetically distinct lineages are emerging across regions, hosts, or time. Understanding population structure is critical for interpreting disease spread, persistence, and potential risks to cucurbit production systems.
To address this objective, whole genome sequencing was performed on 85 Serratia isolates collected from cucurbit crops across 13 U.S. states over a 25-year period (1999–2024), representing multiple hosts and growing regions. Three additional reference genomes were obtained from NCBI, bringing the total dataset to 88 genomes. Genomic libraries were sequenced using Illumina MiSeq technology, assembled and annotated using standardized bioinformatic pipelines, and analyzed using multiple complementary approaches, including Average Nucleotide Identity (ANI), SNP-based phylogenetics, and pangenome analysis.
The results clearly met the project objective. ANI analysis confirmed that 79 of the 88 isolates belong to the S. ureilytica clade, while a small number were identified as S. marcescens or S. sarumanii. Using a 99% ANI cutoff, isolates were grouped into 20 genotypic groups. Strikingly, 63 isolates (approximately 80% of S. ureilytica samples) clustered into a single dominant genotype that was distributed across 12 states and spanned more than two decades. This genotype includes previously confirmed pathogenic isolates, strongly suggesting that this dominant lineage is responsible for most CYVD cases observed in U.S. cucurbit systems.
SNP analysis further supported this conclusion. While the full dataset exhibited substantial diversity (>222,000 SNPs), analysis of the dominant genotype revealed only 475 SNPs across isolates collected over 25 years, with no clustering by year or location. This indicates remarkable genetic stability and suggests long-term persistence of a highly successful pathogenic genotype rather than repeated local emergence of new strains.
Although this project focused on genomic characterization rather than direct on-farm interventions, the findings have important implications for cooperating farms. The identification of a single, widespread, genetically stable pathogenic genotype suggests that CYVD outbreaks are not driven by rapidly evolving strains, but rather by persistence and movement of the same lineage over time and space. This supports management strategies focused on vector control and sanitation. Additionally, these results provide a strong foundation for ongoing phenotyping work, which will directly inform disease risk assessments and management recommendations for cucurbit growers in the Northeast and beyond (Objective 3).
Overall, this project successfully answered its core research question and established a genomic framework that will support future disease management and epidemiological studies. With the addition of percentage squash bug carrying S. ureilytica (objective two), we will be able to determine whether the vector, in fact, allows this bacterium to survive over time.
Education & outreach activities and participation summary
Participation summary:
I have shared the results of this project with farmers, gardeners, industry leaders, and academic peers throughout the project. Collaborators and extensions have been key to accomplishing this goal. Having Steve Reiners as a collaborator is key to maintaining relationships with growers and extensionists. I have already engaged in meetings to inform about Cucurbit Yellow Vine Disease in New York and have expanded 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 have been 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 recorded in English; I translated them and added Spanish captions, and they have more than 1,800 views on YouTube. I will keep working with Hispanic communities to make all the outcomes from this project available to them, as they are a working force in the US and have a right to access this information. Specifically, I will create at least 3 YouTube videos discussing 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 fact sheet was created to discuss the CYVD disease cycle and management.
Specific Outreach
Consultation: People have sent in samples to be tested for CYVD thorughout the summer season
Tours: We have had 3 groups of people who have come to our field trials and we have shown them our work on CYVD
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): 2025- NY Vegetable Growers Expo February 4, 2025. Cornell AgriTech. 30 minute talk to 80 growers. Contact hours = 40.
American Phytopathological Society (APS) Meeting (Summer 2025): I shared my work on to the whole genome sequencing objective through a poster
Cornell Cooperative Extension Factsheet and Newsletter: I wrote a factsheet that describes CYVD diagnosis and management practices
Video submission to the Cornell SIPS YouTube Channel (18k subscribers): I will record videos about CYVD and cucurbits, with 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
This project has raised awareness of a yield-limiting disease, such as cucurbit yellow vine disease. We hope to introduce new management tools that help growers prevent disease in their fields. We wrote a fact sheet to share with farmers and extension educators to raise awareness about the disease and make an impact by providing research-based solutions. We have been giving talks to farmers and educators
At the end of the project, we hope to learn the primary survival strategy of the CYVD pathogen and the % of squash bugs carrying the CYVD pathogen in an infected field. Finally, we hope to learn Serratia Spp potential to cause disease.
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.