Summer squash, Cucurbita pepo, is a popular vegetable crop in Mississippi, where it is harvested at a tender, immature stage and valued as a source of vitamins A and C as well as potassium and iron. Small farms in the state typically sell summer squash directly to consumers through local farmers’ markets, which means disease outbreaks can affect growers’ livelihoods in a direct and immediate way. The crop belongs to the gourd family Cucurbitaceae, is native to the Americas, and there is evidence of its domestication in the lower Mississippi River region. The field observation that set the study in motion occurred in July 2025, during a field trial in which seeds of ‘Black Beauty’, a widely grown commercial summer squash cultivar, had been purchased from a local store in Natchez, Mississippi, and direct seeded in May 2025 at the Alcorn Experiment Station, located at 31 degrees 52 minutes 23.412 seconds north and 91 degrees 7 minutes 46.739 seconds west.

Of the five plants in the research plot, only one developed disease, corresponding to a disease incidence of 20 percent. The affected fruit displayed mushy, water-soaked spots on its exterior, with visible oozing of tissue, while the interior revealed wet, rotten, slimy brown tissue characteristic of bacterial soft rot. These symptoms prompted the team to isolate and identify the causal organism, with the goals of isolating and identifying the soft rot bacterium from symptomatic squash fruit, confirming its pathogenicity, and re-isolating and identifying it to fulfill Koch’s postulates.

To recover the bacterium, the researchers excised small sections of internal fruit tissue measuring five by five millimeters from the margin of symptomatic areas. The tissue was macerated for two minutes in a microcentrifuge tube containing 100 microliters of sterile one-times phosphate-buffered saline, and the resulting bacterial suspension was streaked onto nutrient agar medium. Plates were incubated at 25 degrees Celsius in the dark for 24 hours, after which a single colony was restreaked on the same medium to obtain a pure culture. The strain recovered through this process was designated 25ASUB12, and its colonies showed a consistent morphology when viewed from both the top and bottom of the agar plate one day after streaking from the initial single colony.

Molecular characterization began with the extraction of genomic DNA from a two-day-old bacterial culture grown on nutrient agar, using a commercial fungal and bacterial miniprep kit and approximately 50 milligrams of harvested colonies scooped from the plate with a sterile plastic loop. The team used the primers 27F and 1492R to amplify the 16S ribosomal RNA gene, a standard genetic marker for bacterial identification, in a GeneAmp PCR System 9700 thermal cycler. The polymerase chain reaction product was visualized by gel electrophoresis, purified with a commercial cycle-pure kit, and sent for sequencing to an external service. The resulting sequence was then analyzed with the National Center for Biotechnology Information’s Basic Local Alignment Search Tool against both the core nucleotide collection and the dedicated 16S ribosomal RNA database.

Against the core nucleotide database, the strain’s 16S rRNA sequence showed greater than 99 percent nucleotide identity to more than thirty strains of Pectobacterium brasiliense. However, alignment against the 16S ribosomal RNA database revealed equally high similarity to Pectobacterium aroidearum strain SCRI 109 and Pectobacterium brasiliense strain 212, leaving the species-level identity ambiguous. To resolve this ambiguity completely at the species level, the researchers turned to whole genome sequencing, which was carried out and reported separately. That analysis confirmed strain 25ASUB12 as Pectobacterium brasiliense, supported by an average nucleotide identity of 96.27 percent with the Pectobacterium brasiliense type strain 1692. The 16S rRNA gene sequence of the strain was deposited in GenBank under accession number PX884501, and the whole genome sequence under accession JBUQDA010000001.1.

With the identity established, the team set out to demonstrate that the isolated bacterium could actually cause the disease observed in the field. The pathogenicity assay was conducted on summer squash fruits of the same ‘Black Beauty’ cultivar. The fruits were surface disinfected by spraying with 70 percent ethanol and wiping with sterile paper towels. An inoculum was prepared from a two-day-old nutrient agar culture and adjusted to an optical density of 0.2 at 600 nanometers, corresponding to approximately one times ten to the eighth colony-forming units per milliliter. A 500-microliter volume of this bacterial suspension was injected into each of three test fruits using a sterile syringe and needle, while two control fruits received the same volume of sterile phosphate-buffered saline. The inoculated and control fruits were then placed in a tray lined with sterile paper towels moistened with sterile water and covered loosely with a lid, where they were kept in the dark at 25 degrees Celsius for four days.

At the end of this period, the bacterium-treated fruits exhibited symptoms resembling those seen on the original field specimen, while the buffer-inoculated controls remained healthy. This contrast between treated and control fruits provided direct experimental evidence that strain 25ASUB12 is capable of causing soft rot in summer squash, rather than merely being associated with diseased tissue. To complete Koch’s postulates, the researchers re-isolated the bacterium from the symptomatic assay fruits using the same procedure applied to the original sample, and referred to these recovered organisms as daughter strains in contrast to the original mother strain. Molecular identification of the daughter strains was again performed using the 16S rRNA gene, and sequencing produced six distinct daughter strain sequences, deposited in GenBank under accession numbers PX884502 through PX884507.

The percentage nucleotide identity between the mother strain and the daughter strains ranged from 99.86 to 99.93 percent. The authors note that the one to two base pair differences observed among these 16S rRNA sequences could stem from intragenomic heterogeneity, meaning polymorphisms among the multiple 16S rRNA gene copies within a single genome, or from sequencing artifacts, polymerase errors, or natural microevolution. The daughter strains also matched the colony morphology of the mother strain, closing the loop between the field isolate, the experimental infection, and the recovered organism. Together, these results satisfy the classical requirements for attributing the disease to the isolated bacterium in this host.

The significance of the report lies in what it adds to the known distribution of this pathogen in the United States. Pectobacterium brasiliense is a highly virulent, pectinolytic bacterium, meaning it produces enzymes that degrade pectin and break down plant tissue into the soft, slimy rot characteristic of these infections. It was first reported causing blackleg of potato in Brazil, and within the United States it has previously been documented as a pathogen of kale, mizuna, pak choi, potato, and avocado. This brief report adds summer squash to the diversity of crops affected by Pectobacterium brasiliense in the country. The authors emphasize that, to the best of their knowledge, this is the first documented case of the bacterium causing soft rot of summer squash in Mississippi, and that further investigation into the prevalence of this soft rot disease in the region, along with the development of disease management practices, will be warranted.

Subject of Research: First report of the soft rot bacterium Pectobacterium brasiliense on summer squash in Mississippi, USA

Article Title: First report of Pectobacterium brasiliense associated with soft rot of summer squash in Mississippi, USA

Article References: Brooks, L., Bista, P., Clark, E., Mrema, F., & Bhatta, B. P. (2026). First report of Pectobacterium brasiliense associated with soft rot of summer squash in Mississippi, USA. Discover Plants, 3(1), Article 498. https://doi.org/10.1007/s44372-026-00948-8

Image Credits: AI Generated

DOI: 10.1007/s44372-026-00948-8

Keywords: Pectobacterium brasiliense, summer squash, soft rot, plant pathology, bacterial disease, Koch's postulates, whole genome sequencing, 16S rRNA, Mississippi, Cucurbita pepo, pathogenicity assay, emerging plant pathogen