Agricultural Research Service

Colletotrichum camelliae Massee, a member of Colletotrichum gloeosporioides species complex (CGSC), is an economically important fungal pathogen that causes significant damage to Camellia sinensis (tea) and other Camellia species that include ornamental crops. Although the occurrence of C. camelliae in the United States has been documented, to date most of the reports and all available genome sequences of this pathogen are from strains isolated outside the United States. Here, we report the isolation of four C. camelliae isolates (GCC14, PS22-1, PS23, and PS24) from symptomatic Camellia japonica plants in George County, Mississippi. Whole genome sequencing of all four isolates was performed as paired-end reads of 150 bp using NovaSeq X Plus. Clean reads were de novo assembled, mapped to C. camelliae reference genome sequence, LS-19, and genome-wide variants were analyzed. Sequencing data were submitted to NCBI GenBank BioProject PRJNA1273064, and the following accession numbers were assigned: SRR34108282 (GCC14), SRR33953793 (PS22-1), SRR33955217 (PS23) and SRR33957315 & SRR33956925 (PS24). The genome sequence data reported here for C. camelliae are valuable for developing diagnostic assays for the identification, tracking, and management of this pathogen on Camellia species that include tea and ornamental crops. In addition, comparative genomics, phylogenetics, and evolutionary studies using these and other available genomes can aid in deriving insights on the diversity and pathogenicity of this pathogen, CGSC, and the Colletotrichum genus.

The Thielaviopsis paradoxa species complex comprises fungal pathogens that cause both bud (heart) and trunk rot of palms. Thielaviopsis spp. are soilborne pathogens that infect all plant parts (i.e., roots, stem, leaves, and fruits) of primarily monocot hosts, including palms, banana, sugarcane, and pineapple. However, very little is known about the T. paradoxa species complex in the United States and the palm-Thielaviopsis pathosystem. Our previous analysis showed that the T. paradoxa complex comprises at least two species in Florida, T. ethacetica and a novel, undescribed Thielaviopsis species. To study the interaction, a detached leaflet assay for palms was established to test the pathogenicity of the species in the T. paradoxa complex. Lesion size was measured quantitatively and used to assess pathogenicity of the fungal isolates and host response. Wounding the detached leaflets promoted lesion development, and disease symptoms were similar to whole plants. A comparison of the two Thielaviopsis species showed that all T. ethacetica isolates consistently produced larger lesions and were more aggressive than the undescribed Thielaviopsis species in detached leaflet assay on foxtail palms (Wodyetia bifurcata). Subsequently, T. ethacetica was used to screen 262 individuals from 29 species across 17 genera of palms using the detached leaflet assay. Lesion development in individuals from at least five palm species across four genera was significantly different from the negative control, suggesting that these palm species may be susceptible to T. ethacetica. Additionally, variation in lesion development was also observed at varietal level in coconut palms. This detached leaflet assay would facilitate dissection of the Thielaviopsis-palm interaction and accelerate screening of palm germplasm repositories to identify sources of resistance to Thielaviopsis.

Consumption of poultry products continues to rise in the United States, highlighting the importance of mitigating foodborne pathogens during poultry processing. Developing new methodologies to improve the detection of pathogens within processing plants is essential to protect food safety. Growing demand for a more representative sample and desire to improve bacterial detection at low levels have led to the evaluation of new sample collection tools. Our objective was to evaluate one such tool, the MicroTally® Mitt, a polymer fabric that has shown improved microbial recovery in beef trim and turkey carcasses, by comparing it to the traditional carcass or parts rinse method for detecting total aerobic microorganisms (rapid aerobic count; RAC), Enterobacteriaceae (EB), Campylobacter, and Salmonella before and after antimicrobial interventions. Additionally, the mitt was tested for its utility in collecting a composite sample (multi-carcass mitt). Three commercial broiler processing establishments were visited twice. Whole carcass samples were collected at hot rehang (n=150) and post-chill (n=150). Wing samples were collected before and after antimicrobial application (n=136 and n=147, respectively). A total of 233 whole carcass rinses (WCRs), 233 single-carcass mitts (SC-mitts), and 117 multi-carcass mitts (MC-mitts) were collected. As expected, recovery was greatest at hot rehang for all microbial counts (RAC, EB, Campylobacter, and Salmonella) and lowest post-chill. Deep serotyping of Salmonella culture-positive samples (n=100) from hot rehang showed multiple serovars were detected in 53% (20/38) WCR, 58% (22/38) single-carcass mitt, and 50% (12/24) of multi-carcass mitt samples. For all microbial counts, the single-carcass mitt had comparable bacterial recovery to rinses in hot rehang samples. Low bacterial recovery following antimicrobial interventions prevented further sample type comparisons at other processing stages but showed processing intervention efficacy.

Quantitative PCR (qPCR) is a common, culture-independent method to estimate gene copies at levels as broad as Kingdom and specific as guild, function or species; however, it is dependent upon several factors that affect the amplification efficiency. Amplification efficiency of an individual qPCR reaction is dependent on template type, qPCR product, and assay optimization including primer design. Although qPCR reactions can be optimized, the design of primers to meet guidelines for relatively short amplicon length (75–150 base pairs) is often unfeasible especially when targeting a broad phylogeny of organisms or functional genes. The addition of exogenous non-target plasmid DNA (0.05 ng µL-1) as a PCR additive significantly increases the amplification efficiency for long target amplicons up to 723 bp. The method is applicable for different template types including circular and linearized plasmids (24–30% increase) and cell-free DNA extracts (up to 26%), as well as different thermocyclers and qPCR products. As an exogenous DNA source, the pUC19 plasmid provides flexibility with a reduced chance of non-target amplification due to the relatively simple, short, and known DNA sequence. Overall, this method enhances amplification efficiency, reduces template bias, and lowers qPCR variability with a strong benefit for long amplicon assays.

Understanding the ecology of fungi in Aspergillus section Flavi is necessary to identify factors that contribute to aflatoxin accumulation risk and to improve interventions to reduce contamination. For example, interactions between non‑aflatoxigenic and aflatoxigenic Aspergillus species play a key role in determining the aflatoxin-producing potential of crop-associated fungi. The non-aflatoxigenic species A. tamarii is often associated with agricultural soils and occasionally with crops, prompting the question as to whether A. tamarii and aflatoxigenic A. flavus compete on these crops. In this study, interactions between A. tamarii and A. flavus during co-colonization and sporulation on maize kernels were assessed. In co-culture experiments, mycelial and conidial growth was measured for each species using a custom quantitative PCR assay, and aflatoxin was measured via lateral flow. Both A. tamarii and A. flavus exhibited reduced growth under co-culture, confirming these species compete for shared resources in maize kernels. However, their responses diverged in ways that suggest distinct life history strategies. In competition, there was a significant relationship between mycelial growth and production of conidia for A. tamarii. In contrast, for A. flavus isolates mycelial biomass in kernels and conidia production during co-colonization of maize kernels with A. tamarii were not correlated. This suggests differences in resource allocation between growth and reproduction between the two species. Additionally, isolate identity influenced competitive outcomes and aflatoxin production, underscoring the importance of genotype-level variation in fungal interactions. Understanding these species-specific interactions provides insight into fungal dynamics on crops and may inform strategies for managing aflatoxin contamination events.

Olive pomace, a byproduct of olive oil production, is a rich source of bioactive phenolic compounds with potential health benefits. This study aimed to characterize the chemical composition and evaluate the metabolic effects of a subcritical water extract from California olive pomace (SWE COP) obtained from Arbequina olives. The extract was mainly composed of carbohydrates (72.81%) and contained 66.62 ± 1.22 mg gallic acid equivalents/g of phenolics, with 3,4-DHPEA-EDA, hydroxytyrosol, and verbascoside identified as the predominant compounds. Male C57BL/6N mice were fed a standard diet (SD; n = 7), a high-fat and high-sugar diet (HFSD; n = 7), which was used to induce features of diet-associated metabolic syndrome, or an HFSD supplemented with 3% (w/w) SWE COP (n = 7) for 16 weeks. Supplementation with SWE COP significantly reduced plasma triglycerides and increased HDL cholesterol levels compared with the HFSD group. Moreover, SWE COP improved glucose tolerance, enhanced insulin sensitivity, and reduced mesenteric and epididymal adiposity. Histological analysis showed that SWE COP alleviated hepatic steatosis and lowered the NAFLD activity score. These findings demonstrate that phenolic-rich SWE COP exerts beneficial effects on glucose and lipid metabolism and reduces liver fat accumulation in diet-induced obese mice. Overall, SWE COP represents a promising functional ingredient derived from olive industry byproducts for mitigating metabolic dysfunctions associated with obesity.

Delivery of biomolecules into plant vascular tissues remains a barrier to managing diseases caused by insect vector-borne pathogens and to modifying phenotypes of established perennial crops. Inspired by the vascularized growth of crown galls induced by Agrobacterium tumefaciens, we repurposed the bacterium’s plant growth regulator (PGR) genes to engineer autonomously dividing, transgene-expressing plant cell structures termed symbionts. A plant transformation vector (pSYM) incorporating the IaaM, IaaH, Ipt and gene5 cassette from A. tumefaciens strain C58 together with a gene of interest on the same transfer DNA was delivered to stems of herbaceous and woody dicots using disarmed A. tumefaciens strain EHA105. Symbiont morphology, vascular differentiation, transgene expression, molecular mobility and protein secretion were evaluated using microscopy, fluorescent reporters, dye tracing, RNA silencing assays and mass spectrometry-based proteomics. pSym inoculation reproducibly generated symbionts across diverse host plant species that were vascularly integrated into their host plants and transgene expression ranging from heterogeneous niches to more uniform patterns. Small molecules moved between symbionts and host vascular tissues, whereas larger proteins exhibited more restricted mobility. Post-transcriptional gene silencing signals moved freely throughout the symbiont and slightly into adjacent stem tissue. Under tested field and greenhouse conditions in potato and tomato, respectively, gall or symbiont formation had no negative impacts on plant growth or tuber and fruit yield. In vitro, symbiont cultures abundantly secreted recombinant protein into surrounding media. Together, these results establish symbionts as a modular, plant bioengineering platform capable of producing and potentially delivering biomolecules without modifying the host plant genome, providing a foundation for vascular-targeted therapeutics and phenotype modulation in crops.

Reproductive performance is a key determinant of overall livestock productivity. In both beef and dairy systems, reproductive failure represents a leading cause of cow culling. Reproductive traits are complex in nature and present a low heritability in general. Additionally, the collection of such phenotypes usually relies on indirect measures of fertility, such as conception success. Therefore, further investigation into genetic and non-genetic factors of reproductive traits in cattle is necessary. The hosts' microbiome plays a crucial role in vertebrate biology, including reproduction. We, therefore, hypothesize that microbiome indicators may serve as a biomarker of fertility. This study explored the relationship between vaginal microbiome profiles and pregnancy among three beef cattle genetic groups using field data. Vaginal swabs were collected from 74 cows at Fort Keogh, MT, including 23 Angus, 23 Hereford Line 1, and 28 crossbreds, and DNA was extracted and analyzed via 16S rRNA gene amplification. Significant differences in alpha diversity (p < 0.05) were found among Line 1 cows compared to Angus and crossbreds in many indicators of alpha diversity. Pregnancy status did not influence alpha diversity of samples significantly, but trends toward significance were observed. PERMANOVA analysis indicated that genetic groups and pregnancy status affected microbial composition (p < 0.05), but their interaction was not significant. Each genetic group showed unique compositions of operational taxonomic units (OTUs), with higher proportions of Ureaplasma and Mycoplasma families in Line 1. Additionally, variations in microbial communities were observed between pregnant and non-pregnant cows, with certain uncultured bacteria more prevalent in non-pregnant cows. While field data are useful for such studies and represent a real production system, better-designed experiments are necessary to validate findings and test hypotheses. These results suggest variation in vaginal microbiomes across breeds and pregnancy status, emphasizing the need for further research to identify factors affecting these changes.