Phylodynamic Insights into Global Emergence and Diversification of the Tomato Pathogen Xanthomonas hortorum pv. gardneri.

The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. Xanthomonas hortorum pv. gardneri is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of X. hortorum pv. gardneri, our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of X. hortorum pv. gardneri strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in X. hortorum pv. gardneri is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of X. hortorum pv. gardneri is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.

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.

Role of Stress-Induced Proteins RpoS and YicC in the Persistence of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plants.

Understanding the functional role of bacterial genes in the persistence of Salmonella in plant organs can facilitate the development of agricultural practices to mitigate food safety risks associated with the consumption of fresh produce contaminated with Salmonella spp. Our study showed that Salmonella enterica subsp. enterica serotype Typhimurium (strain MDD14) persisted less in inoculated tomato plants than other Salmonella Typhimurium strains tested (JSG210, JSG626, JSG634, JSG637, JSG3444, and EV030415; P < 0.01). In-vitro assays performed in limited-nutrient conditions (growth rate, biofilm production, and motility) were inconclusive in explaining the in-planta phenotype observed with MDD14. Whole-genome sequencing combined with non-synonymous single nucleotide variations analysis was performed to identify genomic differences between MDD14 and the other Salmonella Typhimurium strains. The genome of MDD14 contained a truncated version (123 bp N-terminal) of yicC and a mutated version of rpoS (two non-synonymous substitutions, i.e., G66E and R82C), which are two stress-induced proteins involved in iron acquisition, environmental sensing, and cell envelope integrity. The rpoS and yicC genes were deleted in Salmonella Typhimurium JSG210 with the Lambda Red recombining system. Both mutants had limited persistence in tomato plant organs, similar to that of MDD14. In conclusion, we demonstrated that YicC and RpoS are involved in the persistence of Salmonella in tomato plants in greenhouse conditions and, thus, could represent potential targets to mitigate persistence of Salmonella spp. in planta. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytophthora capsici, 100 Years Later: Research Mile Markers from 1922 to 2022.

In 1922, Phytophthora capsici was described by Leon Hatching Leonian as a new pathogen infecting pepper (Capsicum annuum), with disease symptoms of root rot, stem and fruit blight, seed rot, and plant wilting and death. Extensive research has been conducted on P. capsici over the last 100 years. This review succinctly describes the salient mile markers of research on P. capsici with current perspectives on the pathogen's distribution, economic importance, epidemiology, genetics and genomics, fungicide resistance, host susceptibility, pathogenicity mechanisms, and management.

A Quantitative PCR Method to Detect the Tomato Corky Root Rot Pathogens, Pseudopyrenochaeta lycopersici and P. terrestris.

Corky root rot is an important disease in tomato production systems and is caused by Pseudopyrenochaeta terrestris and P. lycopersici (formerly Pyrenochaeta lycopersici Types 1 and 2, respectively). The corky root rot pathogens are slow growing and difficult to isolate and quantify in soil and plant tissue. A multiplex hydrolysis probe-based qPCR assay was designed to allow for simultaneous detection and quantification of P. lycopersici and P. terrestris with a competitive internal control to indicate if qPCR inhibitors are present. Single species and multiplex assays for Pseudopyrenochaeta spp. detected DNA levels above 0.013 pg of DNA per reaction. These highly specific assays had no nontarget amplification of other fungal and oomycete pathogens or rhizosphere-associated fungi of tomatoes that were tested. This assay can be used to quantify Pseudopyrenochaeta populations in roots and soils in tomato production systems to better determine the impacts of disease management strategies on Pseudopyrenochaeta spp. and provides a tool to study the biology of Pseudopyrenochaeta spp.

Discovery of Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Peppers (Capsicum sp.).

Pseudomonas leaf spot (PLS) disease in peppers caused by Pseudomonas syringae pv. syringae (Pss) is an emerging seedborne phytopathogen. Pss infection can severely reduce the marketable yield of peppers in favorable environmental conditions and cause significant economic losses. The intensive use of copper-sulfate and streptomycin-sulfate to control PLS and other bacterial diseases is associated with antimicrobial-resistant Pss strains, making these control methods less effective. So, there is an urgent need to develop novel antimicrobials effective against Pss in peppers. Several studies, including those done in our laboratory, have shown that small molecule (SM) antimicrobials are ideal candidates as they can be effective against multidrug resistant bacteria. Therefore, our study aims to identify novel SM growth inhibitors of Pss, assess their safety, and evaluate their efficacy on Pss-infected pepper seeds and seedlings. Using high-throughput screening, we identified 10 SMs (PC1 to PC10) that inhibited the growth of Pss strains at 200 µM or lower concentrations. These SMs were effective against both copper- and streptomycin-resistant as well as biofilm-embedded Pss. These SMs were effective against other plant pathogens (n = 22) at low concentrations (<200 µM) and had no impact on beneficial phytobacteria (n = 12). Furthermore, these SMs showed better or equivalent antimicrobial activity against Pss in infested pepper seeds and inoculated seedlings compared with copper-sulfate (200 µM) and streptomycin (200 µg/ml). Additionally, none of the SMs were toxic to pepper tissues (seeds, seedlings, or fruits), human Caco-2 cells, and pollinator honeybees at 200 µM. Overall, the SMs identified in this study are promising alternative antimicrobials for managing PLS in pepper production.

Novel Small Molecule Growth Inhibitors of Xanthomonas spp. Causing Bacterial Spot of Tomato.

Bacterial spot (BS) of tomato, caused by Xanthomonas gardneri, X. perforans, X. vesicatoria, and X. euvesicatoria, is difficult to control because of the high prevalence of copper- and streptomycin-resistant strains and the lack of resistance cultivars and effective bactericides. The objective of this study was to identify novel growth inhibitors of BS-causing Xanthomonas (BS-X) species by using small molecules (SM; n = 4,182). Several SMs (X1, X2, X5, X9, X12, and X16) completely inhibited the growth of BS-X isolates (n = 68 X. gardneri, 55 X. perforans, 4 X. vesicatoria, and 32 X. euvesicatoria) at ≥12.5 µM by disrupting Xanthomonas cell integrity through weakening of the cell membrane and formation of pores. These SMs were also effective against biofilm-embedded, copper- and streptomycin-resistant Xanthomonas strains while having minimal impact on other plant pathogenic (n = 20) and beneficial bacteria (n = 12). Furthermore, these SMs displayed equivalent antimicrobial activity against BS-X in seeds and X. gardneri in seedlings compared with conventional control methods (copper sulfate and streptomycin) at similar concentrations while having no detectable toxicity to tomato tissues. SMs X2, X5, and X12 reduced X. gardneri, X. perforans, X. vesicatoria, and X. euvesicatoria populations in artificially infested seeds ≤3.4-log CFU/seed 1 day postinfection (dpi) compared with the infested untreated control (P ≤ 0.05). SMs X1, X2, X5, and X12 reduced disease severity ≤72% and engineered bioluminescent X. gardneri populations ≤3.0-log CFU/plant in infected seedlings at 7 dpi compared with the infected untreated control (P ≤ 0.05). Additional studies are needed to increase the applicability of these SMs for BS management in tomato production.

On-Farm Evaluations of Anaerobic Soil Disinfestation and Grafting for Management of a Widespread Soilborne Disease Complex in Protected Culture Tomato Production.

Tomato production in Ohio protected culture systems is hindered by a soilborne disease complex consisting of corky root rot (Pyrenochaeta lycopersici), black dot root rot (Colletotrichum coccodes), Verticillium wilt (Verticillium dahliae), and root-knot (Meloidogyne hapla and M. incognita). In a survey of 71 high tunnels, C. coccodes was detected in 90% of high tunnels, and P. lycopersici (46%), V. dahliae (48%), and Meloidogyne spp. (45%) were found in nearly half of high tunnels. Anaerobic soil disinfestation (ASD) with wheat bran (20.2 Mg/ha) plus molasses (10.1 Mg/ha) and grafting onto 'Maxifort' or 'Estamino' rootstocks were evaluated in high tunnels on five farms. In post-ASD bioassays of trial soils, root and taproot rot severity were significantly reduced after ASD, and root-knot galling was also reduced by ASD. Soilborne pathogenic fungi were isolated less frequently from bioassay plants grown in ASD-treated soils than control soils. Similar results were observed in tomato plants grown in high tunnels. Root rot was significantly reduced by ASD in nearly all trials. Corky root rot severity was highest in nongrafted plants grown in nontreated soils, and the lowest levels of corky root rot were observed in 'Maxifort'-grafted plants. Black dot root rot severity was higher or equivalent in grafted plants compared with nongrafted plants. Root-knot severity was lower in plants grown in ASD-treated soils in high tunnels compared with plants grown in control soils, but grafting did not significantly decrease root-knot severity. However, soil treatment did not significantly affect yield, and grafting led to inconsistent impacts on yield.

Discovery and Characterization of Low-Molecular Weight Inhibitors of Erwinia tracheiphila.

Plant pathogenic bacteria in the genus Erwinia cause economically important diseases, including bacterial wilt of cucurbits caused by Erwinia tracheiphila. Conventional bactericides are insufficient to control this disease. Using high-throughput screening, 464 small molecules (SMs) with either cidal or static activity at 100 µM against a cucumber strain of E. tracheiphila were identified. Among them, 20 SMs (SM1 to SM20), composed of nine distinct chemical moiety structures, were cidal to multiple E. tracheiphila strains at 100 µM. These lead SMs had low toxicity to human cells and honey bees at 100 µM. No phytotoxicity was observed on melon plants at 100 µM, except when SM12 was either mixed with Silwet L-77 and foliar sprayed or when delivered through the roots. Lead SMs did not inhibit the growth of beneficial Pseudomonas and Enterobacter species but inhibited the growth of Bacillus species. Nineteen SMs were cidal to Xanthomonas cucurbitae and showed >50% growth inhibition against Pseudomonas syringae pv. lachrymans. In addition, 19 SMs were cidal or static against Erwinia amylovora in vitro. Five SMs demonstrated potential to suppress E. tracheiphila when foliar sprayed on melon plants at 2× the minimum bactericidal concentration. Thirteen SMs reduced Et load in melon plants when delivered via roots. Temperature and light did not affect the activity of SMs. In vitro cidal activity was observed after 3 to 10 h of exposure to these five SMs. Here, we report 19 SMs that provide chemical scaffolds for future development of bactericides against plant pathogenic bacterial species.

Anaerobic Soil Disinfestation Reduces Viability of Sclerotinia sclerotiorum and S. minor Sclerotia and Root-Knot Nematodes in Muck Soils.

Experiments were conducted to evaluate potential functional and mechanistic differences in the suppression of Sclerotinia sclerotiorum and S. minor and root-knot nematodes in muck soils by anaerobic soil disinfestation (ASD) using different carbon source amendments. Volatile compounds produced during ASD in muck soil amended with molasses, wheat bran, or mustard greens at 20.2 Mg/ha or a 2% ethanol solution significantly reduced the mycelial growth and number of sclerotia produced by both Sclerotinia spp. compared with the anaerobic control. In amended soils, acetic and butyric acids were detected in concentrations that reduced the viability of sclerotia of both pathogens. Higher concentrations of carbon dioxide were observed in ASD-treated soils, regardless of the amendment, than in the nonamended anaerobic control. Only amendment with wheat bran did not increase the production of methane gas during ASD compared with the controls. Meloidogyne hapla survival was completely suppressed in soils treated with ASD regardless of carbon source. Field trials were conducted in Ohio muck soil to assess survival of sclerotia of both Sclerotinia spp. The viability of sclerotia of both Sclerotinia spp. was significantly reduced in soil subjected to ASD amended with wheat bran (20.2 Mg/ha), molasses (10.1 Mg/ha), or wheat bran (20.2 Mg/ha) plus molasses (10.1 Mg/ha) compared with the controls. A consistent negative correlation between soil reduction and viability of sclerotia of both pathogens was observed. Wheat bran and molasses are both widely available amendments that can be used as ASD carbon sources for the management of soilborne pathogens in muck soils.

Specific Environmental Temperature and Relative Humidity Conditions and Grafting Affect the Persistence and Dissemination of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plant Tissues.

Little is known about the abiotic factors contributing to the preharvest persistence of Salmonella in tomato tissues. Therefore, we investigated the effects of specific environmental conditions and contamination methods on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (JSG626) in tomato plants. When plants were sprayed on the leaves with a JSG626-contaminated solution, JSG626 persistence in the phyllosphere (bacteria located on the surface of the inoculated foliage and stem tissues) was lower at higher temperatures (30°C day/25°C night) than at lower temperatures (20°C day/15°C night). However, wounding cotyledons with contaminated tools improved JSG626 persistence and the internalization rate (2.27%) in planta compared to spray inoculation (0.004%). The systemic dissemination of JSG626 to other tissues increased when contaminated plants were grown under low relative humidity (<40%); however, JSG626 was only detected in the root systems at later sampling times (between 21 and 98 days postinoculation [dpi]). Further, after tomato scions were grafted onto rootstocks using contaminated cutting tools, dissemination of JSG626 was preferentially basipetal and occasionally acropetal in the plants, with higher persistence rates and loads of JSG626 in root systems compared to foliar tissues. JSG626 was detected in the grafting point and root systems up to 242 dpi; however, none of the fruits harvested from contaminated plants between 90 and 137 dpi were positive for JSG626. This study demonstrates that environmental temperature and relative humidity could be good indicators for estimating the persistence of Salmonella enterica in tomato plants. Further, root systems may represent a risk for long-term persistence of Salmonella enterica in tomato plants.IMPORTANCE Tomatoes are one of the most widely produced vegetables around the world; however, fresh tomatoes have been connected to multiple wide-scale salmonellosis outbreaks over the past decades. Salmonella is commonly found in the environment and can persist in hostile conditions for several weeks before being internalized into plant tissues, where it is protected from conventional sanitation methods. In addition to biotic factors (host, inoculum size, and phytobiome), abiotic factors (environmental conditions) may affect the persistence of Salmonella in crop production. This study demonstrates that specific environmental conditions, the inoculation method, and the inoculum density affect the persistence and dissemination of JSG626 in tomato plant tissues. Our findings enhance the understanding of interactions between Salmonella enterica and fresh produce and may lead to the development of novel management practices on farms.

Anaerobic Soil Disinfestation to Manage Soilborne Diseases in Muck Soil Vegetable Production Systems.

Anaerobic soil disinfestation (ASD) was evaluated as a tool for managing the root-knot nematode Meloidogyne hapla in lettuce (Lactuca sativa) and clubroot disease, caused by Plasmodiophora brassicae, in mustard greens (Brassica juncea) produced on Ohio muck soils in Huron and Stark Counties. In two consecutive years of field trials, wheat bran (20.2 Mg ha-1), molasses (10.1 Mg ha-1), and wheat bran (20.2 Mg ha-1) plus molasses (10.1 Mg ha-1) were assessed as ASD carbon sources and compared with nonamended controls. Data were collected from plants grown in the field and from plants grown in field-treated soils in growth chamber-based post-ASD bioassays. Anaerobic conditions developed in ASD-treated soils in both trial years, as indicated by polyvinyl chloride pipes painted with an iron oxide paint. Soil pH did not decrease during ASD at the Huron County site of the mustard greens clubroot trials in either trial year but soil pH decreased significantly during ASD in Stark County soils treated with ASD with either wheat bran or wheat bran plus molasses compared with control soils in both trial years. Impacts of ASD on plant biomass were inconsistent in direct field measurements; however, significantly higher biomasses were observed in lettuce and mustard greens grown in bioassay soils collected from plots treated with ASD with wheat bran-based amendments compared with plants grown in soils from control plots. Based on direct field measurements and bioassays, the use of ASD with any carbon source led to significant reductions in root-knot nematode galling on lettuce compared with controls. Reductions in clubroot severity in mustard greens following ASD were less consistent; however, significant reductions in clubroot severity were observed in the field in one trial year and in both years of bioassays. The results of these studies indicate that ASD is a promising tool for managing soilborne diseases in muck soil vegetable production systems.

Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid.

Rasta is a virus-like disease of unknown etiology affecting tomato (Solanum lycopersicum) plants in Ghana. Symptoms include stunting; epinasty, crumpling, and chlorosis of leaves; and necrosis of leaf veins, petioles, and stems. Leaf samples with rasta symptoms were collected from commercial tomato fields in Ghana in October 2012 and applied to FTA cards, and RNA extracts were prepared. Reverse-transcription polymerase chain reaction (RT-PCR) tests with primers for Columnea latent viroid, which causes rasta-like symptoms in tomato plants in Mali, were negative, whereas tests with degenerate viroid primer pairs were inconclusive. However, tomato seedlings (Early Pak 7) mechanically inoculated with RNA extracts of 10 of 13 samples developed rasta-like symptoms. In RT-PCR tests with RNA from leaves of the 10 symptomatic seedlings and primers for Potato spindle tuber viroid (PSTVd) or Tomato apical stunt viroid (TASVd), the expected size (approximately 360 bp) of DNA fragment was amplified from eight and two seedlings, respectively. Sequence analyses confirmed that these fragments were from PSTVd and TASVd isolates, and revealed a single PSTVd haplotype and two TASVd haplotypes. The PSTVd and TASVd isolates from Ghana had high nucleotide identities (>94%) with isolates from other geographic regions. In a host range study, PSTVd and TASVd isolates from Ghana induced rasta symptoms in the highly susceptible tomato cultivar Early Pak 7 and mild or no symptoms in Glamour, and symptomless infections in a number of other solanaceous species. PSTVd and TASVd isolates were seed associated and possibly seed transmitted.

A Novel Phylogroup of Pseudomonas cichorii Identified Following an Unusual Disease Outbreak on Tomato.

Recently, in Central Florida tomato production fields, tomato foliage and fruit were observed with symptoms similar to bacterial speck. Fluorescent pseudomonads were consistently isolated and the strains were characterized by standard LOPAT tests, pathogenicity tests, and genetic characterization using 16S ribosomal RNA (rRNA) sequences and multilocus sequence analysis (MLSA) of conserved housekeeping genes. LOPAT test results indicated that the strains were likely Pseudomonas cichorii. These strains were pathogenic on tomato and were also pathogenic on lettuce, the host for the type strain of P. cichorii. Likewise, strains of P. cichorii isolated in Florida since the early 1980s from hosts other than tomato, along with the type strain, were also pathogenic on tomato. Genetic characterization using 16S rRNA and MLSA confirmed that the strains were most closely related to P. cichorii but varied significantly from the type strain. The Florida P. cichorii strains formed a separate phylogenetic group along with P. cichorii strains isolated from tomato in Tanzania. These strains were different from the previously described morphotypes and genomovars of P. cichorii. Our results indicate the presence of a genetically distinct group of multihost pathogenic P. cichorii strains that have been present in Florida since at least the early 1980s.

Evaluation of Ralstonia solanacearum Infection Dynamics in Resistant and Susceptible Pepper Lines Using Bioluminescence Imaging.

Bacterial wilt, incited by Ralstonia solanacearum, is a major disease affecting pepper (Capsicum annuum) production worldwide. The most effective management tactic is the deployment of wilt-resistant varieties. However, the lack of a nondestructive method to measure invasiveness and spatio-temporal distribution of R. solanacearum, a vascular pathogen, in planta limits better understanding of pepper resistance and plant-pathogen interactions. We evaluated the resistance of 100 pepper lines using R. solanacearum strain Rs-SY1 (phylotype I, isolated from a sweet pepper in South China). Based on the disease severity index (DSI) values, the elite inbred line BVRC 1 and the small-fruited accessions PI 640435 and PI 640444 were identified as resistant (DSI: 1.2, 1.8, and 1.9 out of 4.0, respectively). In order to evaluate bacterial infection dynamics in planta in real time, we generated seven bioluminescent R. solanacearum strains (BL-Rs1 to BL-Rs7) using vector pXX3 carrying luxCDABE genes, and selected BL-Rs7 for inoculation due to its similarity with parent strain Rs-SY1 in morphology, pathogenicity, and highest light emission in vitro. Luminescence intensity was strongly correlated to bacterial population in planta (R2 = 0.88). The utility of the bioluminescence assay was validated by comparing R. solanacearum infection dynamics in real-time in vivo between resistant line BVRC 1 and susceptible line BVRC 25. The distribution and multiplication of BL-Rs7 strain in resistant line BVRC 1 was conspicuously limited in plants inoculated in either roots or stem compared with susceptible line BVRC 25. These results suggest that pepper line BVRC 1 may resist colonization by interfering with R. solanacearum multiplication in the roots and stem.

Differential Colonization Dynamics of Cucurbit Hosts by Erwinia tracheiphila.

Bacterial wilt is one of the most destructive diseases of cucurbits in the Midwestern and Northeastern United States. Although the disease has been studied since 1900, host colonization dynamics remain unclear. Cucumis- and Cucurbita-derived strains exhibit host preference for the cucurbit genus from which they were isolated. We constructed a bioluminescent strain of Erwinia tracheiphila (TedCu10-BL#9) and colonization of different cucurbit hosts was monitored. At the second-true-leaf stage, Cucumis melo plants were inoculated with TedCu10-BL#9 via wounded leaves, stems, and roots. Daily monitoring of colonization showed bioluminescent bacteria in the inoculated leaf and petiole beginning 1 day postinoculation (DPI). The bacteria spread to roots via the stem by 2 DPI, reached the plant extremities 4 DPI, and the plant wilted 6 DPI. However, Cucurbita plants inoculated with TedCu10-BL#9 did not wilt, even at 35 DPI. Bioluminescent bacteria were detected 6 DPI in the main stem of squash and pumpkin plants, which harbored approximately 10(4) and 10(1) CFU/g, respectively, of TedCu10-BL#9 without symptoms. Although significantly less systemic plant colonization was observed in nonpreferred host Cucurbita plants compared with preferred hosts, the mechanism of tolerance of Cucurbita plants to E. tracheiphila strains from Cucumis remains unknown.

Evaluation of disinfectants to prevent mechanical transmission of viruses and a viroid in greenhouse tomato production.

BACKGROUND: In recent years, a number of serious disease outbreaks caused by viruses and viroids on greenhouse tomatoes in North America have resulted in significant economic losses to growers. The objectives of this study were to evaluate the effectiveness of commercial disinfectants against mechanical transmission of these pathogens, and to select disinfectants with broad spectrum reactivity to control general virus and viroid diseases in greenhouse tomato production. METHODS: A total of 16 disinfectants were evaluated against Pepino mosaic virus (PepMV), Potato spindle tuber viroid (PSTVd), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). The efficacy of each disinfectant to deactivate the pathogen's infectivity was evaluated in replicate experiments from at least three independent experiments. Any infectivity that remained in the treated solutions was assessed through bioassays on susceptible tomato plants through mechanical inoculation using inocula that had been exposed with the individual disinfectant for three short time periods (0-10 sec, 30 sec and 60 sec). A positive infection on the inoculated plant was determined through symptom observation and confirmed with enzyme-linked immunosorbent assay (PepMV, ToMV, and TMV) and real-time reverse transcription-PCR (PSTVd). Experimental data were analyzed using Logistic regression and the Bayesian methodology. RESULTS: Statistical analyses using logistic regression and the Bayesian methodology indicated that two disinfectants (2% Virkon S and 10% Clorox regular bleach) were the most effective to prevent transmission of PepMV, PSTVd, ToMV, and TMV from mechanical inoculation. Lysol all-purpose cleaner (50%) and nonfat dry milk (20%) were also effective against ToMV and TMV, but with only partial effects for PepMV and PSTVd. CONCLUSION: With the broad spectrum efficacy against three common viruses and a viroid, several disinfectants, including 2% Virkon S, 10% Clorox regular bleach and 20% nonfat dry milk, are recommend to greenhouse facilities for consideration to prevent general virus and viroid infection on tomato plants.

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein.

Mild variants of many viruses are able to protect infected plants from subsequent invasion by more severe variants of the same viruses through a process known as cross-protection. In the past, the cross-protective viral variants were commonly derived from mild field isolates that were sometimes genetically heterogeneous, providing variable levels of cross-protection. Here, we report a novel approach to rapidly generate cross-protective variants of the tomato-infecting Pepino mosaic virus (PepMV) independently of the availability of mild field isolates. Our approach sought to attenuate PepMV by mutating less conserved amino acid residues of the abundantly produced capsid protein (CP). These less-conserved amino acid residues were identified through multiple alignments of CPs of six potexviruses including PepMV, and were altered systematically to yield six PepMV mutants. These mutants were subsequently inoculated onto the model plant Nicotiana benthamiana, as well as tomato, to evaluate their accumulation levels, symptom severities, and cross-protection potentials. The mutant KD, in which the threonine (T) and alanine (A) residues at CP positions 66 and 67 were replaced with lysine (K) and aspartic acid (D), respectively, were found to accumulate to low levels in infected plants, cause very mild symptoms, and effectively protect both N. benthamiana and tomato against secondary infections by wild-type PepMV. These data suggest that our approach represents a simple, fast, and reliable way of generating attenuated viral variants capable of cross-protection.

Use of the vital stain FUN-1 indicates viability of Phytophthora capsici propagules and can be used to predict maximum zoospore production.

The fluorescent vital dye FUN®-1 (2-chloro-4-[2,3-dihydro-3-methyl-{benzo-1,3-thiazol-2-yl}-methylidene]-1-phenylquinolinium iodide) was evaluated as a tool to assess Phytophthora capsici sporangia and zoospore metabolic activity and viability. Under aerobic conditions, mycelia, sporangia and zoospores cultured on agar medium and stained with FUN-1 exhibited red fluorescent cylindrical intravacuolar structures (CIVS) that were clearly visible at 100× magnification. Encysted zoospores did not exhibit CIVS after exposure to FUN-1 dye. Over 7 d there was a significant reduction in the percent of sporangia containing CIVS, which corresponded with a significant increase in zoospore formation and release. The decline in the percentage of metabolically active sporangia and increase in the number of zoospores fit both a linear and log regression model. The FUN-1 dye was suitable for distinguishing between live and dead sporangia and effective in monitoring the change in metabolic activity of sporangia over time. It will be useful in determining parameters, including P. capsici culture age, that maximize production of zoospores in vitro.