Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE: The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue.

Mitigating Emerging and Reemerging Diseases of Fruit and Vegetable Crops in a Changing Climate.

Fruit and vegetable crops are important sources of nutrition and income globally. Producing these high-value crops requires significant investment of often scarce resources, and, therefore, the risks associated with climate change and accompanying disease pressures are especially important. Climate change influences the occurrence and pressure of plant diseases, enabling new pathogens to emerge and old enemies to reemerge. Specific environmental changes attributed to climate change, particularly temperature fluctuations and intense rainfall events, greatly alter fruit and vegetable disease incidence and severity. In turn, fruit and vegetable microbiomes, and subsequently overall plant health, are also affected by climate change. Changing disease pressures cause growers and researchers to reassess disease management and climate change adaptation strategies. Approaches such as climate smart integrated pest management, smart sprayer technology, protected culture cultivation, advanced diagnostics, and new soilborne disease management strategies are providing new tools for specialty crops growers. Researchers and educators need to work closely with growers to establish fruit and vegetable production systems that are resilient and responsive to changing climates. This review explores the effects of climate change on specialty food crops, pathogens, insect vectors, and pathosystems, as well as adaptations needed to ensure optimal plant health and environmental and economic sustainability.

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples.

Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

Survival and dissemination of Escherichia coli O157:H7 on physically and biologically damaged lettuce plants.

The ecology of the vegetable leaf surface is important to the survival of enteric pathogens. Understanding changes in ecological parameters during the preharvest stages of production can lead to development of approaches to minimize the hazard of contamination of fresh fruits and vegetables with foodborne pathogens. In this study, survival levels of Escherichia coli O157 over a 10-day period were compared among traumatically injured, phytopathogen-damaged, and healthy lettuce plants. Leaves from lettuce plants cracked along the central vein, plants infected with Xanthomonas campestris pv. vitians, and healthy plants were inoculated with E. coli O157:H7. The presence of E. coli O157:H7 populations on inoculated leaves and non-inoculated leaves of these same plants was determined for 10 days. The density of E. coli O157:H7 decreased over time on the inoculated leaves regardless of the treatment. The population of E. coli O157:H7 remained higher on traumatically injured leaves than on healthy plants (P < 0.001). E. coli O157:H7 was detected on leaves other than the direct inoculation site of the enteric pathogen in all three treatments groups. Preharvest damage, especially that caused by traumatic injury, impacted the survivability of E. coli O157:H7. Maintaining healthy plants and minimizing physical damage around the time of harvest might improve the safety of fresh produce.

An Immunofluorescence Assay to Detect Urediniospores of Phakopsora pachyrhizi.

An indirect immunofluorescence spore assay (IFSA) was developed to detect urediniospores of Phakopsora pachyrhizi, utilizing rabbit polyclonal antisera produced in response to intact nongerminated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to Phakopsora spp. and did not react with other common soybean pathogens or healthy soybean leaf tissue in enzyme-linked immunosorbent assay (ELISA). SBR1A and SBR2 bound to P. pachyrhizi and P. meibomiae urediniospores were detected with goat anti-rabbit Alexa Fluor 488-tagged antiserum using a Leica DM IRB epifluorescent microscope with an I3 blue filter (excitation 450 to 490 nm, emission 515 nm). The assay was performed on standard glass microscope slides; double-sided tape was superior to a thin coating of petroleum jelly both in retaining spores and in immunofluorescence. The IFSA was used to confirm the identity of P. pachyrhizi urediniospores captured on glass slides from passive air samplers from Georgia, Kentucky, and Ohio during 2006.

Diversity of Ralstonia solanacearum Infecting Eggplant in the Philippines.

ABSTRACT The diversity of Ralstonia solanacearum strains isolated from eggplant (Solanum melongena) grown in five provinces of the Philippine island group of Luzon was assessed using a recently described hierarchical system. All strains keyed to race 1, biovar 3 or 4. Phylotype-specific multiplex polymerase chain reaction (PCR) indicated that, like most other strains of Asian origin, all the strains in our Philippine collection belong to phylotype I. Taxometric and phylogenetic analyses of partial endoglucanase gene sequences of strains from this collection and those previously deposited into GenBank revealed at least four subgroups among the otherwise monophyletic phylotype I strains. Nucleotide polymorphisms within each subgroup were infrequent and, among the subgroups identified in this study, variation was always <1.3%, indicating that the large majority of strains could be assigned to a single sequevar. Genomic DNA fingerprinting using enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed additional fine-scale genetic variation that was consistent with the endogluconase sequence data. Whole-pattern and band-based analyses of the genomic fingerprint data revealed four and eight distinct genotypes, respectively, within our collection. Eggplant from infested fields in different provinces tended to harbor mixed populations of ERIC genotypes, with the predominant genotype varying by location.

Gibberella xylarioides (anamorph: Fusarium xylarioides), a causative agent of coffee wilt disease in Africa, is a previously unrecognized member of the G. fujikuroi species complex.

Tracheomycosis or coffee wilt has emerged as a major disease of robusta coffee in Uganda in the past 10 years. Coffee wilt historically has been associated with Fusarium xylarioides Steyaert (teleomorph Gibberella xylarioides Heim and Sacc.), a species that has been classified as a member of Fusarium section Lateritium. We investigated the molecular phylogenetics of fusarial coffee wilt isolates by generating partial DNA sequences from two protein coding regions, translation elongation factor 1-alpha and beta-tubulin, in 36 isolates previously identified as F. xylarioides and related fusaria from coffee and other woody hosts, as well as from 12 isolates associated with a current coffee wilt outbreak in Uganda. These isolates fell into two morphologically and phylogenetically distinct groups. The first group was found to represent previously unidentified members of the Gibberella fujikuroi species complex (GFC), a clade that replaces the artificial Fusarium section Liseola. This group of isolates fit the original description of F. xylarioides, thus connecting it to the GFC. The second group, which was diverse in its morphology and DNA sequences, comprised four distinct lineages related to Fusarium lateritium. Our finding of unrelated species associated with coffee wilt disease has important implications regarding its epidemiology, etiology and control.