Phytopathology®

Xanthomonas wilt (XW) of banana caused by Xanthomonas vasicola pv. musacearum (Xvm) does not spread to all plants physically interconnected through the rhizome when one or a few are diseased. However, the factors behind this incomplete systemic spread of Xvm are not fully known yet could inform XW management. This study explored the effect of Xvm inoculum amounts, number and size of suckers, sucker positioning on mother plant corms and other mother plant corm attributes on sucker colonization. A shorter (p <0.05) incubation period (17.9 vs 21.1 days) and higher (p<.001) cumulative number of symptomatic leaves (5.2 vs 1.6 leaves) was observed when all (high inoculum) compared to two leaves (low inoculum) were inoculated. Xvm was recovered in corms at 29 days post inoculation (dpi) in both treatments with no differences (p >0.05) in proportions of corms with Xvm between the treatments. However, Xvm was recovered earlier and at a higher frequency in suckers when all leaves were inoculated. Lower Xvm recoveries occurred in the lower corm sections to which most suckers were attached relative to the middle and upper corm sections. Xvm incidence in corms increased with the number of attached maiden suckers, and the dpi while it declined with increasing mother plant and corm height. Thus, Xvm spread within mats is influenced by the amount of inoculum and the physiological stage (e.g., height) of the plant and attached suckers. The position of suckers, predominantly at the bottom of corms also protects them from infection. Measures that reduce Xvm inoculum build-up in mats are thus crucial for minimizing within mat XW spread.

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.

The continuous changes in the lineage proportions of populations in the clonal plant pathogen Phytophthora infestans on potato and tomato crops have been perplexing to researchers and disease managers. Sudden outbreaks of newly emergent genotypes are often associated with these rapid composition changes. Modelling can predict the persistence and displacement of pathogen genotypes with differential fitness among hosts. Building upon previous models, we combined analytical and simulation methods to model the outcome of interactions between competing lineages on multiple hosts. Model inputs include pathogenesis parameters, and the outputs are fitness and lineage proportions within each host. Analytical solutions yielding complete displacement, partial coexistence-displacement, and complete coexistence were described. In a retrospective study, the lesion growth rate and sporulation density of P. infestans lineages on potato and tomato from pathogenicity trials were used as inputs. Output lineage frequencies were compared with historical epidemiological situations to check model accuracy. The results showed that pathogenesis traits measured from empirical trials could simulate lineage constituents on potato and tomato, and estimate genotypic fitness with reasonable accuracy. The model also showed promise in predicting ongoing lineage displacements in the subsequent year or few years, even when the displaced lineage was still highly prevalent during the time of isolation. However, large uncertainties remain at temporal-spatial scales owing to complex meta-population dynamics in some regions and adaptation to local environmental factors. This simulation model provides a new tool for forecasting pathogen compositions, and can be used to identify potentially problematic genotypes based on pathogen life-history traits.

Management of widespread plant pathogens is challenging as climatic differences among crop growing regions may alter key aspects of pathogen spread and disease severity. Xylella fastidiosa is a xylem-limited bacterial pathogen that is transmitted by xylem sap feeding insects. Geographic distribution of X. fastidiosa is limited by winter climate and vines infected with X. fastidiosa can recover from infection when held at cold temperatures. California has a long history of research on Pierces disease, and significant geographic and climatic diversity among grape-growing regions. This background in combination with experimental disease studies under controlled temperature conditions can inform risk assessment for X. fastidiosa spread and epidemic severity across different regions and under changing climate conditions. Californias grape growing regions have considerable differences in summer and winter climate. In northern and coastal regions, summers are mild and winters cool, conditions favoring winter recovery of infected vines. In contrast, in inland and southern areas summers are hot and winters mild, reducing likelihood of winter recovery. Here, winter recovery of three table grape cultivars (Flame, Scarlet Royal, and Thompson seedless) and three wine grape cultivars (Sauvignon Blanc, Cabernet Sauvignon, and Zinfandel) were evaluated under temperature conditions representative of the San Joaquin Valley, an area with hot summers and mild winters that has been severely impacted by Pierces disease, and contains a large portion of California grape production. Mechanically inoculated vines were held in the greenhouse under one of three warming treatments to represent different seasonal inoculation dates prior to being moved into a cold chamber. Winter recovery under all treatments was generally limited, but with some cultivar variation. Given hot summer temperatures of many grape-growing regions worldwide, as well as increasing global temperatures overall, winter recovery of grapevines should not be considered a key factor affecting X. fastidiosa spread and epidemic severity in the majority of cases.

In South Asia, bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) impose major constraints on eggplant, tomato, and pepper production. To improve the efficacy of bacterial wilt management, the goals of this study were to (1) conduct a survey of RSSC pathogens in Bangladesh and Nepal, (2) characterize the genetic diversity of these isolates, and (3) screen 37 tomato, eggplant, and pepper accessions for resistance to six representative isolates from South Asia. We isolated 99 isolates from Bangladesh and 20 isolates from Nepal and determined that all are phylotype I isolates of the Ralstonia pseudosolanacearum species. We sequenced and assembled draft genomes for 25 isolates. Phylogenomic analyses suggest that there is a wide diversity of endemic phylotype I isolates in South Asia, and possible introductions of two clonal phylotype I lineages into Bangladesh and Nepal. We contextualize our newly described isolates based on prior reports of RSSC diversity in South Asia and global reports of RSSC pathogens on eggplant and pepper. Greenhouse trials revealed multiple tomato, eggplant, and pepper accessions that exhibit promising levels of resistance to six phylotype I isolates from South Asia.

Canada thistle (Cirsium arvense (L.) Scop., CT) is one of the worst weeds threatening temperate regions of the world. A host-specific rust fungus, Puccinia punctiformis (F. Strauss) Rohl., is known to cause systemic disease of CT, ultimately killing individuals and reducing stand densities. In 2013, it was demonstrated that fall inoculation of rosettes with coarsely ground leaves bearing P. punctiformis telia can successfully initiate epiphytotics. In the same year, a cooperative project between the Colorado Department of Agriculture and United States Department of Agriculture was initiated, in which CT patches across the state of Colorado (USA) were inoculated and tracked over subsequent years for changes in stem density. Here, we report our findings from 8 years (2014-2021) of monitoring effort. At most sites (N = 87), CT stem densities declined, from a mean ({+/-} SE) of 87.9 ({+/-} 6.5) stems to 44.7 ({+/-} 4.2). These declines however were spatially-autocorrelated, and likely attributable to local growing conditions, as mean annual daily maximum temperature and standard deviation of elevation, as well as climatic conditions around the times of both treatment and monitoring, were found to be important predictors of CT decline. Further, we observed that the amount of inoculum deployed, timing since last release, and method in which it was spread locally at a site were also associated with the magnitude of CT stem decline. These results are indicative of the value of P. punctiformis as a CT biological control agent. The name Cirsium arvense dieback (CADB) is proposed herein to describe the agriculturally important decline in CT stem densities attributable to this previously un-named systemic disease.

Tar spot, a disease caused by the ascomycete fungal pathogen Phyllachora maydis, is considered one of the most significant yield-limiting diseases of maize (Zea mays L.) within the United States. P. maydis may also be found in association with other fungi, forming a disease complex with characteristic fish eye lesions. Understanding how P. maydis colonizes maize leaf cells is essential for developing effective disease control strategies. Here, we used histological approaches to elucidate how P. maydis infects and multiplies within susceptible maize leaves. We collected tar spot-infected maize leaf samples from four different fields in northern Indiana at three different time points during the growing season. Samples were chemically fixed and paraffin-embedded for high-resolution light and scanning electron microscopy. We observed a consistent pattern of disease progression in independent leaf samples collected across different geographical regions. Each stromata contained a central pycnidium that produced asexual spores. Perithecia with sexual spores developed in the stomatal chambers adjacent to the pycnidia, and a cap of spores formed over the stromata. P. maydis reproductive structures formed around but not within the vasculature. In our samples containing fish eye lesions, P. maydis is associated with two additional fungi, one of which is likely a member of the Paraphaeospheria genus; the other is an unknown fungi. Our data provide fundamental insights into how this pathogen colonizes and spreads within maize leaves. This knowledge can inform new approaches to managing tar spot, which could help mitigate the significant economic losses caused by this disease.

Downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is the most economically important disease of spinach. In the past 30 years, 14 new races and 13 strains with novel virulence have been identified. However, the mechanism(s) driving the rapid evolution of virulence remains unknown. To understand reproductive strategies potentially driving the emergence of new races in P. effusa, 67 composite isolates (a collection of symptomatic leaves from a single cultivar grown in a defined area) of P. effusa obtained from 13 states between 2010 and 2018 were used to analyze the population genetic diversity hierarchically. Genotypes at 33 SNP loci of 719 lesions from these 67 isolates were determined by targeted sequencing. Diversity was then evaluated among individual lesions within the composite isolates, between isolates, host cultivars, geographic locations, and years of isolates collected. A total of 380 genotypes were identified from 719 individual lesions. Of the 380 genotypes, 350 (92%) were unique while the most common genotype was identified in 110 lesions of 16 isolates collected from 13 cultivars from CA and AZ in 2016. Variation within composite isolates ranged from none (a single genotype among lesions from a composite isolate) to 38 unique genotypes recovered from 39 lesions of a composite isolate. An index of association analysis suggested asexual (clonal) and sexual reproduction play important roles in population structure. Based on discriminant analysis of principal components, four distinct subpopulations were identified. Host cultivar, origin, and time of collection had an effect on population differentiation, and genotypes specific to a certain location or collection period were identified. Some subpopulations were unique to certain areas, and were only detected after 2014-2016. The co-existence of sexual and asexual reproduction strategies may partially explain the rapid emergence and spread of new races and novel strains of P. effusa.

Phytophthora are eukaryotic microbes that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via sexual reproduction. These resting spores can survive in the soil for several years in the absence of a host plant, thus providing a long-term inoculum for disease. The ability to quantitatively evaluate oospore viability is an important part of many phytopathology studies. Here, we tested six fluorescent viability dyes for their ability to differentially stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate (FDA), 5-carboxyfluorescein diacetate (CFDA), propidium iodide, and TOTO-3 iodide. Each dye was first tested individually with untreated or heat-treated oospores as proxies for viable and non-viable oospores, respectively. SYTO9, FUN-1, CFDA and propidium iodide stained untreated and heat-treated oospores indiscriminately. In contrast, FDA (a green-fluorescent viable cell stain) and TOTO-3 (a red-fluorescent non-viable cell stain) differentially stained untreated or heat-treated oospores with no cross-fluorescence. We then tested the efficacy of dual viability staining and in conjunction with a pipeline for automated image analysis. To validate the method, untreated and heat-treated oospores were mixed at specific ratios, dual-stained, and analyzed using the pipeline. Linear regression of the resulting data showed a clear correlation between the expected and measured oospore ratios (dy/dx=0.95, R2=0.88). Overall, the combination of dual-fluorescence staining and automated image analysis provides a high-throughput method for quantitatively assessing oospore viability and therefore can facilitate further studies on this key part of the Phytophthora disease cycle.

Needle pathogens cause the discoloration, death, or premature abscission of conifer foliage, which reduce growth and vigor, and repeated defoliation may eventually result in tree mortality. Since 2016, forest managers in the southeast United States have reported an increasing scale, frequency, and severity of needle disease outbreaks on the regions principal timber species, loblolly pine (Pinus taeda L.). These recent outbreaks are raising concern throughout the region, as needle diseases are not traditionally considered a threat to P. taeda. Lecanosticta acicola (Thum.) Syd., the native causal agent of brown-spot needle blight, has been recovered from some outbreaks, however, the full array of fungi associated with symptoms has not been explored. In this research, P. taeda foliage was collected from affected stands throughout the region and analyzed to identify fungi associated with needle disease symptoms. We employed both targeted molecular diagnostics, to confirm the presence or absence of L. acicola, and DNA metabarcoding, to characterize the foliar mycobiome and screen for other potential pathogens. Lecanosticta acicola was detected among symptomatic needles from multiple states, particularly in western portions of the P. taeda range but rarely from stands in eastern states. Fungal ITS1 metabarcoding revealed multiple pathogens in symptomatic needles and identified associations between known pathogens fungi and differing symptoms. Additionally, the fungal community composition of needles varied with patterns of symptom presentation. This study is the first regionwide assessment of fungi associated with recent large-scale needle disease outbreaks on P. taeda and identifies multiple pathogens that warrant study in greater detail.

Field Pennycress (Thlaspi arvense) is gaining attention in the US Midwest as a potential oilseed cover crop for their corn-soybean systems. Field research with breeding trials have shown that pennycress is susceptible to major fungal diseases affecting Brassica crops. In this study, we identified two pathogens: Alternaria japonica and Sclerotinia sclerotiorum which cause Alternaria black spot and Sclerotinia white mold diseases, respectively. Both fungi infect pennycress leaves and pods, with S. sclerotiorum also able to infect stems of the two pennycress accessions tested. We found accession 2032 to be more susceptible than MN106. Traditional visual methods to estimate disease severity could not capture the differential progression of Alternaria black spot in the two pennycress accessions. To address this, we developed a cost-effective DNA-based qPCR-based assay that can detect differential growth of both A. japonica and S. sclerotiorum on leaves and pods of two pennycress accessions. This method also provided more precise quantification of A. japonica and S. sclerotiorum at early infection stages than was possible visually. This assay could be helpful in evaluating various pennycress cultivars and other crops affected by these pathogens.

O_LICurrent legislation enforces the implementation of intensive surveillance programs for quarantine plant pathogens. After an outbreak, surveys are implemented to delimit the geographic extent of the pathogen and execute disease control. The feasibility of control programs is highly dependent on budget availability, thus it is necessary to target and optimize surveillance strategies. C_LIO_LIA sequential adaptive delimiting survey involving a three-phase and a two-phase design with increasing spatial resolution was developed and implemented for the Xylella fastidiosa outbreak in Alicante, Spain. Inspection and sampling intensities were optimized using simulation-based methods and results were validated using Bayesian spatial models. C_LIO_LIThis strategy made it possible to sequence inspection and sampling considering different spatial resolutions, and to adapt the inspection and sampling intensity according to the information obtained in the previous, coarser, spatial resolution. C_LIO_LIThe proposed strategy was able to delimit efficiently the extent of Xf improving efficiency of the current in terms of survey efforts. From a methodological perspective, our approach provides new insights of alternative delimiting designs and new reference sampling intensity values. C_LI

Heterobasidion annosum sensu lato comprises some of the most devastating conifer pathogens conifers. Exploring virocontrol as a potential strategy to mitigate economic losses caused by these fungi holds promise for the future. In this study, we conducted a comprehensive screening for viruses in a 98 H. annosum s.l. specimens from different regions of Czechia aiming to identify viruses inducing hypovirulence. Initial examination for dsRNA presence was followed by RNA-Seq analyses using pooled RNA libraries constructed from H. annosum and Heterobasidion parviporum, with diverse bioinformatic pipelines employed for virus discovery. Our study uncovered 25 distinct ssRNA viruses, including two ourmia-like viruses, one mitovirus, one fusarivirus, one tobamo-like virus, one cogu-like virus, one bisegmented narna-like virus and one segment of another narna-like virus, and 17 ambi-like viruses, for which hairpin and hammerhead ribozymes were detected. Coinfections of up to 10 viruses were observed in six Heterobasidion isolates, while another six harbored a single virus. 73% of the isolates analyzed by RNA-Seq were virus-free. These findings show that the virome of Heterobasidion populations in Czechia is highly diverse and differs from that in the boreal region. We further investigated the host effects of certain identified viruses through comparisons of the mycelial growth rate and proteomic analyses and found that certain tested viruses caused growth reductions of up to 22% and significant alterations in the host proteome profile. Their intraspecific transmission rates ranged from 0% to 33%. Further studies are needed to fully understand the biocontrol potential of these viruses in planta. ImportanceO_LIFirst report of a fusarivirus, a tobamo- and a cogu-like virus in Heterobasidion C_LIO_LICertain viruses caused mycelial growth reduction in H. annosum host strains C_LIO_LIViral infections lead to proteome changes in Heterobasidion C_LI

Verticillium wilt, Verticillium stem striping, and Verticillium stripe, are common disease names that all denote infection caused by Verticillium longisporum, on canola, or other Brassica crops. In this study, a quantitative PCR (qPCR) assay and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-{micro} L reaction or 1,400 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg.

In 2010, a Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) incursion into New Zealand kiwifruit orchards devastated susceptible Actinidia chinensis cultivars. In contrast, many Actinidia species maintained in germplasm collections were resistant to Psa3 and showed limited symptoms. Recent genome biosurveillance revealed the emergence of widespread leaf spot symptoms in Psa3-resistant Actinidia germplasm. Surprisingly, few Psa3 isolates were recovered from symptomatic tissues, despite the frequent isolation of phenotypical Pseudomonas isolates on selective agar. Despite the poor recovery of Psa3 isolates, Psa3 was found in all symptomatic leaf tissue through qPCR and metabarcoding analysis. Metabarcoding revealed stark differences in bacterial community composition from samples taken from lesion-carrying or lesion-free material from the same leaf. Host genotype also appeared to influence community composition but to a lesser extent. Whole genome sequencing of diverse Pseudomonas spp. isolates revealed that many belonged to the P. syringae species complex. Curiously, the kiwifruit-associated P. syringae pv. actinidifoliorum (Pfm), was never recovered, nor were any other phylogroup 1 pathovars. Pathogenicity and competitive assays revealed that while individually diverse Pseudomonas isolates were not more pathogenic than Pfm or Psa3 on resistant Actinidia hosts, they could interact with Psa to sometimes improve their growth, suggesting that these isolates may form pathogenic consortia in these disease-associated phyllosphere communities on typically Psa3-resistant hosts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/635981v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e48d68org.highwire.dtl.DTLVardef@1ffa8d7org.highwire.dtl.DTLVardef@1a5c47corg.highwire.dtl.DTLVardef@6bd39_HPS_FORMAT_FIGEXP M_FIG C_FIG

The wheat curl mite (WCM)-transmissible wheat streak disease complex is the most serious disease of wheat in the U.S. Great Plains. In the current study, we determined the genetic variability in WCM and mite-transmitted viruses in Colorado and identified sources of resistance in Colorado wheat germplasm to wheat streak disease complex. We identified two distinct genotypes of WCM, Type 1 and Type 2 based on the ribosomal ITS1 region. Both genotypes were found to co-exist throughout the wheat producing regions of Colorado. Analysis of the whole genome and partial coat protein sequences revealed rich diversity of wheat streak mosaic virus (WSMV) and High Plains wheat mosaic virus (HPWMoV) isolates collected from Colorado, whereas triticum mosaic virus (TriMV) showed low sequence variability. Analysis of WSMV isolates revealed two novel isolates and one that was 100% similar to a new variant of WSMV from Kansas. Interestingly, between 2-4 genotypes of all 8 RNA segments of HPWMoV were identified, which suggests new variants of emaraviruses and co-occurrence of multiple strains within host populations. Several novel viruses including mycoviruses were identified for the first time in Colorado. We found variation in WSMV resistance among wheat varieties; however a variety that harbored dual resistance to mite and WSMV had lower virus titer compared to varieties that contained single resistance gene. This suggests that pyramiding genes will ensure improved and durable resistance. Future research may be aimed at elucidating the dynamics, diversity, and distribution of the new WSMV and HPWMoV isolates and their responses to wheat genotypes.

Beyond the significant impact of Cassava witches broom disease (CWBD), caused by the fungus Rhizoctonia (syn. Ceratobasidium) theobromae on cassava cultivation in French Guiana and Brazil, this disease also poses a potential threat to cacao trees in the region, since the fungus is responsible for Vascular Streak Dieback (VSD) of cacao in South East Asia. Cross-pathogenicity trials were conducted in several cassava fields in French Guiana by planting young cacao plants adjacent to diseased cassava plants. Vascular necrosis was observed in some cacao plants, and the presence of R. theobromae in the cacao tissues was confirmed through PCR diagnostics using primers specific to the fungus. Sequence analysis indicated 100% similarity between samples from both hosts and 97.53 to 99.74% identity with R. theobromae isolates previously reported from cassava in the Americas and Southeast Asia. Additionally, symptomatic cacao in a mixed cacao-cassava farm yielded R. theobromae-positive PCR results, suggesting a natural infection. Ongoing work includes artificial inoculations and controlled cross-pathogenicity trials under screenhouse conditions to attempt reproduction of the symptoms. While current data do not yet establish definitive causality, the findings indicate potential host jump and warrant rapid communication to researchers, policy makers, and farmers to safeguard cacao production and Theobroma biodiversity in the Amazon region.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola, is one of the most devastating diseases of grapevine worldwide. Primary inoculum (i.e. oospores) play a decisive role in downy mildew epidemics, but we still know very little about its abundance in vineyard soil. This study presents a novel molecular method for quantifying P. viticola oospore concentration in vineyard soil using digital droplet PCR (ddPCR). The development of this method enabled characterization of both the abundance and spatial distribution of oospores in a vineyard at the onset of the growing season. Following a regular grid, a total of 198 soil samples (0-15cm horizon) were collected in March 2022 in grapevine rows in a 0.22 ha vineyard planted with cv. Merlot and conducted according to French organic viticulture specifications. Additional samples were collected from the same field within five nested sampling plots with three distance levels, including samples collected in the inter-rows. Using ddPCR, we found P. viticola DNA in all soil samples except one, and we estimated that oospore concentration ranged from 0 to 1858 oospores per gram of soil (303 {+/-} 308 on average). The distribution of oospores at field scale was not random but characterized by 15 m-diameter patches of concentrically increasing oospore concentration. Oospores accumulated 5 times more below the vine stocks than in the inter-row. Using a leaf disc bioassay, we found that soil infectious potential significantly increased with oospore concentration assessed by ddPCR. However, the low coefficient of determination of the relationship indicated that DNA-based oospore quantification lacked clear epidemiological significance. Both ddPCR and bioassay methods are valuable tools that could be used to assess reservoirs of P. viticola primary inoculum across different agroclimatic contexts, thereby bringing greater genericity. Further methodological improvement will also help refine the accuracy of DNA-based assessment of primary inoculum reservoir and improve our understanding of the relationship between primary inoculum reservoir and epidemic dynamics. Ultimately, these data will be essential for improving epidemic risk models and evaluating new preventive disease management strategies targeting the primary inoculum. ImportanceGrapevine downy mildew caused by the oomycete Plasmopara viticola, affects leaves and bunches, and leads to important economic losses for viticulturists. Recently, evidences have accumulated that soilborne primary inoculum (i.e. oospores in the soil) importantly contributes to disease progress. The significance of our work is in presenting a direct and sensitive method for assessing soil oospore concentration, as well as quantitative and spatially-explicit data on downy mildew primary inoculum. This opens the way to new research, the evaluation of new disease control strategies based on primary inoculum management and the improvement of epidemic risk models, which will potentially contribute to lower fungicide use in viticulture in fine.

Most Ralstonia solanacearum species complex strains cause bacterial wilts in tropical or subtropical zones, but the group known as Race 3 biovar 2 (R3bv2) is cool virulent and causes potato brown rot at lower temperatures. R3bv2 has invaded potato-growing regions around the world but is not established in the United States. Phylogenetically, R3bv2 corresponds to a subset of the R. solanacearum phylotype IIB clade, but little is known about the distribution of the cool virulence phenotype within phylotype IIB. Therefore, genomes of 76 potentially cool virulent phylotype IIB strains and 30 public genomes were phylogenetically analyzed. A single clonal lineage within the sequevar 1 subclade of phylotype IIB that originated in South America has caused nearly all brown rot outbreaks worldwide. To correlate genotypes with relevant phenotypes, we quantified virulence of ten Ralstonia strains on tomato and potato at both 22{degrees}C and 28{degrees}C. Cool virulence on tomato did not predict cool virulence on potato. We found that cool virulence is a quantitative trait. Strains in the sequevar 1 pandemic clonal lineage caused the most disease, while other R3bv2 strains were only moderately cool virulent. However, some non-R3bv2 strains were highly cool virulent and aggressively colonized potato tubers. Thus, cool virulence is not consistently correlated with strains historically classified as R3bv2 group. To aid detection of sequevar 1 strains, this group was genomically delimited in the LINbase web server and a sequevar 1 diagnostic primer pair was developed and validated. We discuss implications of these results for the R3bv2 definition.

Cucurbit yellow vine disease (CYVD), caused by the bacterium Serratia ureilytica, is a phloem-associated disease of cucurbits. This study characterized the spatial and temporal distribution of S. ureilytica in Cucurbita pepo cultivar Delicata plants under greenhouse conditions using a GFP-tagged isolate (P01). Seedlings were sampled weekly for four weeks. Transverse sections from the stem, petiole, leaf, shoot apex, and root were imaged by laser scanning confocal and fluorescent dissecting microscopy. In parallel, bacterial abundance in each plant tissue was assessed by quantifying colony-forming units (CFU) via droplet plating over a 4-week time course. Across plant tissues and time points, S. ureilytica fluorescent signal was primarily concentrated in the inner and outer periphery of the bicollateral vascular bundles, with higher magnification images revealing mainly symplastic localization within phloem-associated cells. Consistent with the imaging results, bacterial quantification data showed a high abundance of CFUs in the main stem across weeks, with an irregular pattern of presence in the distal tissues at later time points. These results suggest that S. ureilytica is predominantly localized within phloem-associated cells and spreads both acropetally and basipetally during infection.