Population History and Pathways of Spread of the Plant Pathogen Phytophthora plurivora

Human activity has been shown to considerably affect the spread of dangerous pests and pathogens worldwide. Therefore, strict regulations of international trade exist for particularly harmful pathogenic organisms. Phytophthora plurivora, which is not subject to regulations, is a plant pathogen frequently found on a broad range of host species, both in natural and artificial environments. It is supposed to be native to Europe while resident populations are also present in the US. We characterized a hierarchical sample of isolates from Europe and the US and conducted coalescent-, migration, and population genetic analysis of sequence and microsatellite data, to determine the pathways of spread and the demographic history of this pathogen. We found P. plurivora populations to be moderately diverse but not geographically structured. High levels of gene flow were observed within Europe and unidirectional from Europe to the US. Coalescent analyses revealed a signal of a recent expansion of the global P. plurivora population. Our study shows that P. plurivora has most likely been spread around the world by nursery trade of diseased plant material. In particular, P. plurivora was introduced into the US from Europe. International trade has allowed the pathogen to colonize new environments and/or hosts, resulting in population growth.     

Effects of Propagule Density and Survival Strategies on Establishment and Growth: Further Investigations in the Phylloplane Fungal Model System

This work builds on an earlier culture study where we determined that species diversity of competing saprotrophic phyllpolane fungi had only a negligible effect on the establishment and coexistence of a target fungus, Pestalotia vaccinii. Here, we explore preliminary evidence suggesting that spore density is a more important contributing factor to colonization and coexistence. We examine the influence of propagule density in vitro on establishment and growth of select members of the phylloplane of Vaccinium macrocarpon (American cranberry). To evaluate the response of the weak pathogen P. vaccinii to changes in competitors spore density, we chose saprotrophs from the previous investigation that had the greatest inhibitory effect on the establishment of P. vaccinii (Curvularia lunata), an intermediate inhibitory effect (Alternaria alternata) and the least inhibitory effect (Penicillium sp.). A constant target spore concentration of 50 viable spores of P. vaccinii was pit against densities of the three individual competitors ranging between 12 and 200 spores. As viable propagule density increased, establishment and coexistence of P. vaccinii significantly decreased, with C. lunata and A. alternata decreasing the growth of P. vaccinii more than Penicillium sp. Concomitantly, both C. lunata and Penicillium sp. were not significantly affected by overall spore density but were significantly affected by the presence of P. vaccinii. A. alternata, on the other hand, was not significantly influenced by the presence of P. vaccinii but was significantly affected by overall spore density. An in vitro investigation into the effect of interspecific competition on mycelial growth suggests how different survival strategies and community assembly rules might influence both growth and development. Growth of P. vaccinii was significantly less when interacting with C. lunata than when interacting with either A. alternata or Penicillium sp. Conversely, P. vaccinii had the greatest effect on the growth of C. lunata, less of an effect on the growth of A. alternata, and the least effect on Penicillium sp.     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

Soil pathogen communities associated with native and non‐native Phragmites australis populations in freshwater wetlands

Soil pathogens are believed to be major contributors to negative plant–soil feedbacks that regulate plant community dynamics and plant invasions. While the theoretical basis for pathogen regulation of plant communities is well established within the plant–soil feedback framework, direct experimental evidence for pathogen community responses to plants has been limited, often relying largely on indirect evidence based on above‐ground plant responses. As a result, specific soil pathogen responses accompanying above‐ground plant community dynamics are largely unknown. Here, we examine the oomycete pathogens in soils conditioned by established populations of native noninvasive and non‐native invasive haplotypes of Phragmites australis (European common reed). Our aim was to assess whether populations of invasive plants harbor unique communities of pathogens that differ from those associated with noninvasive populations and whether the distribution of taxa within these communities may help to explain invasive success. We compared the composition and abundance of pathogenic and saprobic oomycete species over a 2‐year period. Despite a diversity of oomycete taxa detected in soils from both native and non‐native populations, pathogen communities from both invaded and noninvaded soils were dominated by species of Pythium. Pathogen species that contributed the most to the differences observed between invaded and noninvaded soils were distributed between invaded and noninvaded soils. However, the specific taxa in invaded soils responsible for community differences were distinct from those in noninvaded soils that contributed to community differences. Our results indicate that, despite the phylogenetic relatedness of native and non‐native P. australis haplotypes, pathogen communities associated with the dominant non‐native haplotype are distinct from those of the rare native haplotype. Pathogen taxa that dominate either noninvaded or invaded soils suggest different potential mechanisms of invasion facilitation. These findings are consistent with the hypothesis that non‐native plant species that dominate landscapes may “cultivate” a different soil pathogen community to their rhizosphere than those of rarer native species.     

Fitness consequences of infection of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with its natural bacterial pathogen <Emphasis Type="Italic">Pseudomonas viridiflava</Emphasis>

Variation in plant resistance to pathogen infection is commonly observed in interactions between wild plants and their foliar pathogens. Models of host–pathogen interactions indicate that a large cost of infection is generally necessary to maintain this variation, yet there is limited evidence that foliar pathogens cause detectable fitness reductions in wild host plants. Most published work has focused on fungal pathogens. Pseudomonas viridiflava, a common bacterial pathogen of the annual weed Arabidopsis thaliana across its range, comprises two distinct genetic clades that cause disease symptoms of different severity. Here we measured the extent of infection of wild A. thaliana populations in the Midwest, USA, and examined the effect on seed production, in field and growth-chamber experiments, of experimental inoculation with isolates from the two clades. We found infection with P. viridiflava varied from 0 to 56% in Midwest A. thaliana populations, with the possibility of several leaves per plant infected later in the growing season. In the growth chambers, experimental inoculation reduced seed set by averages of 15 and 11% for clades A and B, respectively. In the field experiment, only clade A affected plant fitness significantly, reducing seed set by an average of 38%. Underlying these average effects we observed both negative and positive effects of infection, and variation in both fitness among plant genotypes and sensitivity to environmental conditions.     

Phylogenetic and environmental DNA insights into emerging aquatic parasites: implications for risk management

Species translocation leads to disease emergence in native species of considerable economic importance. Generalist parasites are more likely to be transported, become established and infect new hosts, thus their risk needs to be evaluated. Freshwater systems are particularly at risk from parasite introductions due to the frequency of fish movements, lack of international legislative controls for non-listed pathogens and inherent difficulties with monitoring disease introductions in wild fish populations. Here we used one of the world’s most invasive freshwater fish, the topmouth gudgeon, Pseudorasbora parva, to demonstrate the risk posed by an emergent generalist parasite, Sphaerothecum destruens. Pseudorasbora parva has spread to 32 countries from its native range in China through the aquaculture trade and has introduced S. destruens to at least five of these. We systematically investigated the spread of S. destruens through Great Britain and its establishment in native fish communities through a combination of phylogenetic studies of the host and parasite and a novel environmental DNA detection assay. Molecular approaches confirmed that S. destruens is present in 50% of the P. parva communities tested and was also detected in resident native fish communities but in the absence of notable histopathological changes. We identified specific P. parva haplotypes associated with S. destruens and evaluated the risk of disease emergence from this cryptic fish parasite. We provide a framework that can be applied to any aquatic pathogen to enhance detection and help mitigate future disease risks in wild fish populations.     

Animal movements and the spread of infectious diseases

Domestic and wild animal population movements are important in the spread of disease. There are many recent examples of disease spread that have occurred as a result of intentional movements of livestock or wildlife. Understanding the volume of these movements and the risks associated with them is fundamental in elucidating the epidemiology of these diseases, some of which might entail zoonotic risks. The importance of the worldwide animal trade is reviewed and the role of the unregulated trade in animals is highlighted. A range of key examples are discussed in which animal movements have resulted in the introduction of pathogens to previously disease-free areas. Measures based on heightened surveillance are proposed that mitigate the risks of new pathogen introductions.     

First record of the freshwater snail Pseudosuccinea columella (Gastropoda: Lymnaeidae) in southern Pampas (Argentina) and assessment of future spread

ABSTRACT

The freshwater snail Pseudosuccinea columella was recorded for the first time in Argentina more than 60 years ago. Since then its distribution in the wild has been restricted to the northeastern provinces. Here we record the presence of P. columella in southern Pampas for the first time, extending its distribution more than 500?km southwards. The climatic suitability of this and other areas of South America for its establishment and spread was analysed using habitat modelling software. Hitherto its spread within and between watercourses in southern Pampas has been very limited, probably through a combination of low climatic suitability, recent introduction and low connectivity of the drainage basins. The suitability of other areas where it has been recently recorded indicates a moderate risk of further spread in central and northwestern Argentina and in coastal areas of the Pacific rim of South America (southern Perú and northern Chile). The recent spread of P. columella in the wild in Argentina may be the result of an increase in the trade in aquarium plants or of the evolution of a new lineage with different ecological capabilities.     

A hyperparasite affects the population dynamics of a wild plant pathogen

Assessing the impact of natural enemies of plant and animal pathogens on their host's population dynamics is needed to determine the role of hyperparasites in affecting disease dynamics, and their potential for use in efficient control strategies of pathogens. Here, we focus on the long‐term study describing metapopulation dynamics of an obligate pathogen, the powdery mildew (Podosphaera plantaginis) naturally infecting its wild host plant (Plantago lanceolata) in the fragmented landscape of the Åland archipelago (southwest Finland). Regionally, the pathogen persists through a balance of extinctions and colonizations, yet factors affecting extinction rates remain poorly understood. Mycoparasites of the genus Ampelomyces appear as good candidates for testing the role of a hyperparasite, i.e. a parasite of other parasites, in the regulation of their fungal hosts' population dynamics. For this purpose, we first designed a quantitative PCR assay for detection of Ampelomyces spp. in field‐collected samples. This newly developed molecular test was then applied to a large‐scale sampling within the Åland archipelago, revealing that Ampelomyces is a widespread hyperparasite in this system, with high variability in prevalence among populations. We found that the hyperparasite was more common on leaves where multiple powdery mildew strains coexist, a pattern that may be attributed to differential exposure. Moreover, the prevalence of Ampelomyces at the plant level negatively affected the overwinter survival of its fungal host. We conclude that this hyperparasite may likely impact on its host population dynamics and argue for increased focus on the role of hyperparasites in disease dynamics.     

Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages

The spread of aggressive fungal pathogens into previously non‐endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil‐borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well‐differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.     

Molecular Inversion Probe: A New Tool for Highly Specific Detection of Plant Pathogens

Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.     

Population structure of Erysiphe necator on domesticated and wild vines in the Middle East raises questions on the origin of the grapevine powdery mildew pathogen

Plant pathogens usually originate and diversify in geographical regions where hosts and pathogens co-evolve. Erysiphe necator, the causal agent of grape powdery mildew, is a destructive pathogen of grapevines worldwide. Although Eastern US is considered the centre of origin and diversity of E. necator, previous reports on resistant native wild and domesticated Asian grapevines suggest Asia as another possible origin of the pathogen. By using multi-locus sequencing, microsatellites and a novel application of amplicon sequencing (AmpSeq), we show that the population of E. necator in Israel is composed of three genetic groups: Groups A and B that are common worldwide, and a new group IL, which is genetically differentiated from any known group in Europe and Eastern US. Group IL showed distinguished ecological characteristics: it was dominant on wild and traditional vines (95%); its abundance increased along the season; and was more aggressive than A and B isolates on both wild and domesticated vines. The low genetic diversity within group IL suggests that it has invaded Israel from another origin. Therefore, we suggest that the Israeli E. necator population was founded by at least two invasions, of which one could be from a non-East American source, possibly from Asian origin.     

Genetic evidence for a high diversity and wide distribution of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphibians

Population declines and extinctions of amphibians have been attributed to the chytrid fungus Batrachochytrium dendrobatidis (Bd), especially one globally emerging recombinant lineage (‘Bd‐GPL’). We used PCR assays that target the ribosomal internal transcribed spacer region (ITS) of Bd to determine the prevalence and genetic diversity of Bd in South Korea, where Bd is widely distributed but is not known to cause morbidity or mortality in wild populations. We isolated Korean Bd strains from native amphibians with low infection loads and compared them to known worldwide Bd strains using 19 polymorphic SNP and microsatellite loci. Bd prevalence ranged between 12.5 and 48.0%, in 11 of 17 native Korean species, and 24.7% in the introduced bullfrog Lithobates catesbeianus. Based on ITS sequence variation, 47 of the 50 identified Korean haplotypes formed a group closely associated with a native Brazilian Bd lineage, separated from the Bd‐GPL lineage. However, multilocus genotyping of three Korean Bd isolates revealed strong divergence from both Bd‐GPL and the native Brazilian Bd lineages. Thus, the ITS region resolves genotypes that diverge from Bd‐GPL but otherwise generates ambiguous phylogenies. Our results point to the presence of highly diversified endemic strains of Bd across Asian amphibian species. The rarity of Bd‐GPL‐associated haplotypes suggests that either this lineage was introduced into Korea only recently or Bd‐GPL has been outcompeted by native Bd strains. Our results highlight the need to consider possible complex interactions among native Bd lineages, Bd‐GPL and their associated amphibian hosts when assessing the spread and impact of Bd‐GPL on worldwide amphibian populations.     

Multiple guests in a single host: interactions across symbiotic and phytopathogenic bacteria in phloem‐feeding vectors – a review

Some pathogenic phloem‐limited bacteria are a major threat for worldwide agriculture due to the heavy economic losses caused to many high‐value crops. These disease agents – phytoplasmas, spiroplasmas, liberibacters, and Arsenophonus‐like bacteria – are transmitted from plant to plant by phloem‐feeding Hemiptera vectors. The associations established among pathogens and vectors result in a complex network of interactions involving also the whole microbial community harboured by the insect host. Interactions among bacteria may be beneficial, competitive, or detrimental for the involved microorganisms, and can dramatically affect the insect vector competence and consequently the spread of diseases. Interference is observed among pathogen strains competing to invade the same vector specimen, causing selective acquisition or transmission. Insect bacterial endosymbionts are another pivotal element of interactions between vectors and phytopathogens, because of their central role in insect life cycles. Some symbionts, either obligate or facultative, were shown to have antagonistic effects on the colonization by plant pathogens, by producing antimicrobial substances, by stimulating the production of antimicrobial substances by insects, or by competing for host infection. In other cases, the mutual exclusion between symbiont and pathogen suggests a possible detrimental influence on phytopathogens displayed by symbiotic bacteria; conversely, examples of microbes enhancing pathogen load are available as well. Whether and how bacterial exchanges occurring in vectors affect the relationship between insects, plants, and phytopathogens is still unresolved, leaving room for many open questions concerning the significance of particular traits of these multitrophic interactions. Such complex interplays may have a serious impact on pathogen spread and control, potentially driving new strategies for the containment of important diseases.     

First Record of Batrachochytrium dendrobatidis Infecting Four Frog Families from Peninsular Malaysia

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects amphibians on every continent where they occur and is linked to the decline of over 200 amphibian species worldwide. At present, only three published Bd surveys exist for mainland Asia, and Bd has been detected in South Korea alone. In this article, we report the first survey for Bd in Peninsular Malaysia. We swabbed 127 individuals from the six amphibian families that occur on Peninsular Malaysia, including two orders, 27 genera, and 47 species. We detected Bd on 10 out of 127 individuals from four of five states and five of 11 localities, placing the 95% confidence interval for overall prevalence at 4–14%. We detected no variation in Bd prevalence among regions, elevations, or taxonomic groups. The infection intensity ranged from 1 to 157,000 genome equivalents. The presence of Bd infections in native species without clinical signs of disease suggests that Bd may be endemic to the region. Alternately, Bd may have been introduced from non-native amphibians because of the substantial amphibian food trade in Peninsular Malaysia. Under both scenarios, management efforts should be implemented to limit the spread of non-native Bd and protect the tremendous amphibian diversity in Peninsular Malaysia.     

Soil calcium and plant disease in serpentine ecosystems: a test of the pathogen refuge hypothesis

Ecologists have long sought mechanistic explanations for the patterns of plant distribution and endemism associated with serpentine soils. We conducted the first empirical test of the serpentine pathogen refuge hypothesis, which posits that the low levels of calcium found in serpentine soils provide associated plants with a refuge from attack by pathogens. We measured the range of soil calcium concentrations experienced by 16 wild population of California dwarf flax (Hesperolinon californicum) and experimentally recreated part of this range in the greenhouse by soaking serpentine soils in calcium chloride solutions of varying molarity. When flax plants grown in these soils were inoculated with spores of the rust fungus Melampsora lini we found a significant negative relationship between infection rates and soil calcium concentrations. This result refutes the pathogen refuge hypothesis and suggests that serpentine plants, by virtue of their association with low calcium soils, may be highly vulnerable to attack by pathogens. This interaction between plant nutrition and disease may in part explain demographic patterns associated with serpentine plant populations and suggests scenarios for the evolution of life history traits and the distribution of genetic resistance to infection in serpentine plant communities.     

Climate change accelerates local disease extinction rates in a long‐term wild host–pathogen association

Pathogens are a significant component of all plant communities. In recent years, the potential for existing and emerging pathogens of agricultural crops to cause increased yield losses as a consequence of changing climatic patterns has raised considerable concern. In contrast, the response of naturally occurring, endemic pathogens to a warming climate has received little attention. Here, we report on the impact of a signature variable of global climate change – increasing temperature – on the long‐term epidemiology of a natural host–pathogen association involving the rust pathogen Triphragmium ulmariae and its host plant Filipendula ulmaria. In a host–pathogen metapopulation involving approximately 230 host populations growing on an archipelago of islands in the Gulf of Bothnia we assessed changes in host population size and pathogen epidemiological measures over a 25‐year period. We show how the incidence of disease and its severity declines over that period and most importantly demonstrate a positive association between a long‐term trend of increasing extinction rates in individual pathogen populations of the metapopulation and increasing temperature. Our results are highly suggestive that changing climatic patterns, particularly mean monthly growing season (April‐November) temperature, are markedly influencing the epidemiology of plant disease in this host–pathogen association. Given the important role plant pathogens have in shaping the structure of communities, changes in the epidemiology of pathogens have potentially far‐reaching impacts on ecological and evolutionary processes. For these reasons, it is essential to increase understanding of pathogen epidemiology, its response to warming, and to invoke these responses in forecasts for the future.     

Life cycle and life strategy features of Puccinia glechomatis (Uredinales) favorable for extending the natural range of distribution

The objective of this study was to find features in microcyclic rust fungi (Uredinales) on wild host plants favorable for extension of the natural range of distribution. Puccinia glechomatis, a leptosporic rust fungus and its herbal host Glechoma hederacea (Lamiaceae), both natives to Eurasia and introduced in North America, were used for this study. Although the host has been known from North America since the beginning of the nineteenth century, the rust fungus was first observed there only in recent years. Favorable features were identified by studying the life cycle of the rust, including nuclear conditions and seasonal characteristics as well as its spread in North America. The life cycle was studied macroscopically by inoculation experiments, by various light microscope techniques, and by scanning electron microscopy. The spread of the pathogen and its host were reconstructed by evaluating host plant herbarium specimens and databases, literature, and field study data. The studies on P. glechomatis show that, generally for microcyclic rust fungi, establishment and potential for spread are based on several favorable features of both the host (e.g., synanthropic occurence and dispersal, genetic stability, regeneration of vegetative plant parts) and the rust fungus (asexual reproduction/genetic stability, homothallism, propagation with host plant, formation of both leptospores and thick-walled teliospores).     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.