Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants.

BACKGROUND: Anti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars 'Vardar Valley' and 'Justin Brouwers' on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification. RESULTS: Bacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted. CONCLUSION: This study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Characterization of Boxwood Shoot Bacterial Communities and Potential Impact from Fungicide Treatments.

Phyllosphere bacterial communities play important roles in plant fitness and growth. The objective of this study was to characterize the epiphytic and endophytic bacterial communities of boxwood shoots and determine how they may respond to commonly used fungicides. In early summer and early fall, shoot samples were collected immediately before and 1, 7, and 14 days after three fungicides containing chlorothalonil and/or propiconazole were applied to the canopy. Total genomic DNA from shoot surface washings and surface-sterilized shoot tissues was used as the template for 16S rRNA metabarcoding, and the amplicons were sequenced on a Nanopore MinION sequencer to characterize the epiphytic and endophytic communities. The bacterial communities were phylogenetically more diverse on the boxwood shoot surface than in the internal tissue, although the two communities shared 12.7% of the total 1,649 identified genera. The most abundant epiphytes were Methylobacterium and Pantoea, while Stenotrophomonas and Brevundimonas were the dominant endophytes. Fungicide treatments had strong impacts on epiphytic bacterial community structure and composition. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) and analysis of variance (ANOVA)-like differential expression (ALDEx2) together identified 312 and 1,362 epiphytes changed in abundance due to fungicide treatments in early summer and early fall, respectively, and over 50% of these epiphytes were negatively impacted by fungicide. The two chlorothalonil-based contact fungicides demonstrated more marked effects than the propiconazole-based systemic fungicide. These results are foundational for exploring and utilizing the full potential of the microbiome and fungicide applications and developing a systems approach to boxwood health and production. IMPORTANCE Agrochemicals are important tools for safeguarding plants from invasive pathogens, insects, mites, and weeds. How they may affect the plant microbiome, a critical component of crop health and production, was poorly understood. Here, we used boxwood, an iconic low-maintenance landscape plant, to characterize shoot epiphytic and endophytic bacterial communities and their responses to contact and systemic fungicides. This study expanded our understanding of the above-ground microbiome in ornamental plants and is foundational for utilizing the full benefits of the microbiome in concert with different fungicide chemistries to improve boxwood health. This study also sets an example for a more thorough evaluation of these and other agrochemicals for their effects on boxwood microbiomes during production and offers an expanded systems approach that could be used with other crops for enhanced integrated pest management.

Differential Adaptation Has Resulted in Aggressiveness Variation of Calonectria pseudonaviculata on Hosts Buxus, Pachysandra, and Sarcococca.

Calonectria pseudonaviculata (Cps) infects Buxus (boxwood), Pachysandra (pachysandra), and Sarcococca spp. (sweet box); yet, how it adapts to its hosts has been unclear. Here, we performed serial passage experiments with the three hosts and measured Cps changes in three aggressiveness components: infectibility, lesion size, and conidial production. The detached leaves of individual hosts were inoculated with isolates (P0) from the originating host, followed by nine serial inoculations of new leaves of the same host with conidia from the infected leaves of the previous inoculation. All boxwood isolates maintained their capability of infection and lesion expansion through the 10 passages, whereas most non-boxwood isolates lost these abilities during the passages. Isolates from plants of origin (*-P0) and their descendants isolated from passages 5 (*-P5) and 10 (*-P10) were used to evaluate aggressiveness changes on all three hosts with cross-inoculation. While post-passage boxwood isolates gave enlarged lesions on pachysandra, sweet box P5 and pachysandra P10 isolates showed reduced aggressiveness on all hosts. Cps appears to be most adapted to boxwood and less adapted to sweet box and pachysandra. These results suggest speciation of Cps, with its coevolutionary pace with the hosts the fastest with boxwood, intermediate with sweet box, and the slowest with pachysandra.

Combating an Invasive Boxwood Pathogen - Calonectria pseudonaviculata - in the United States by Shifting Production to Less-Susceptible Cultivars.

Boxwood blight (BB) caused by Calonectria pseudonaviculata (Cps), an economically devastating disease affecting everyone in the supply chain from growers to gardeners, was first officially documented in the United States in 2011. This disease has taken a heavy toll on boxwood, an iconic landscape plant and the number one evergreen nursery crop. Instead of abandoning boxwood production and switching to other evergreens, growers in the United States, informed by the latest research, have elected to combat this disease. One of the strategies employed has been to shift boxwood production from highly susceptible to less-susceptible cultivars. The objective of this study was to investigate the ongoing shift by comparing boxwood sales of 17 selected nurseries from seven states across the country in 2011, 2016, and 2021. Results revealed an additional 21.24% (from 38.58 to 60.04%) in sales of less-susceptible boxwood cultivars in 2021 compared with 2016. The less-susceptible cultivars responsible for increased boxwood sales are 'Winter Gem', 'Wintergreen', 'SB 300' (Freedom), 'SB 108' (Independence), and 'Little Missy'. The potential for long-term positive impact on sustainable boxwood production and plantings in the United States through the use of less-susceptible cultivars is discussed. This shift in boxwood choices builds crop health into new plantings of this landmark plant and sustains growth in demand for boxwood. This sets a new example of sustainable protection for a crop that is under serious pressure from an invasive pathogen.

Multi-Year Field Plantings Evaluating Boxwood Cultivars for Susceptibility to the Blight Pathogens (Calonectria spp.) in Northern Germany.

Two multiyear field trials were conducted to evaluate boxwood cultivars for their susceptibility to the blight pathogens Calonectria pseudonaviculata and C. henricotiae in northern Germany. Fifteen cultivars were included in the first trial from 2007 to 2012, and 46 cultivars were included in the second trial from 2014 to 2017. Both trials were done in a naturally infested field that was supplemented with infected plant tissue added to the soil before planting. Each cultivar had three replicate hedge sections with 10 plants per section, and they were assessed annually for blight severity expressed as proportion of leaves blighted and fallen. Blight severity varied significantly among years (P < 0.0001) and cultivars (P < 0.05) within each trial. In the first trial, mean severity ranged from 0.03 to 0.11 for the most resistant cultivars and 0.35 to 0.96 for the most susceptible ones. Similarly, in the second trial, mean severity ranged from 0.06 to 0.27 and 0.71 to 0.97 for the most resistant and susceptible cultivars, respectively. 'Suffruticosa' was consistently the most susceptible cultivar, followed by 'Marianne', 'Myosotidifolia', 'Raket', and 'Morris Midget'. 'Herrenhausen' was the most resistant cultivar, followed by B. microphylla var. japonica, B. microphylla var. koreana, 'Green Mound', 'Faulkner', and 'Winter Beauty'. This study provides field data showing the performance of boxwood cultivars under different levels of disease pressure in an area where C. henricotiae was dominant. This knowledge will help boxwood growers and gardeners to choose less susceptible cultivars and help plant breeders to select for disease resistance.

Characterization of the Soil Bacterial Community from Selected Boxwood Gardens across the United States.

In a recent study, we observed a rapid decline of the boxwood blight pathogen Calonectria pseudonaviculata (Cps) soil population in all surveyed gardens across the United States, and we speculated that these garden soils might be suppressive to Cps. This study aimed to characterize the soil bacterial community in these boxwood gardens. Soil samples were taken from one garden in California, Illinois, South Carolina, and Virginia and two in New York in early summer and late fall of 2017 and 2018. Soil DNA was extracted and its 16S rRNA amplicons were sequenced using the Nanopore MinION® platform. These garden soils were consistently dominated by Rhizobiales and Burkholderiales, regardless of garden location and sampling time. These two orders contain many species or strains capable of pathogen suppression and plant fitness improvement. Overall, 66 bacterial taxa were identified in this study that are known to have strains with biological control activity (BCA) against plant pathogens. Among the most abundant were Pseudomonas spp. and Bacillus spp., which may have contributed to the Cps decline in these garden soils. This study highlights the importance of soil microorganisms in plant health and provides a new perspective on garden disease management using the soil microbiome.

Potential Distribution of Invasive Boxwood Blight Pathogen (Calonectriapseudonaviculata) as Predicted by Process-Based and Correlative Models.

Boxwood blight caused by Cps is an emerging disease that has had devastating impacts on Buxus spp. in the horticultural sector, landscapes, and native ecosystems. In this study, we produced a process-based climatic suitability model in the CLIMEX program and combined outputs of four different correlative modeling algorithms to generate an ensemble correlative model. All models were fit and validated using a presence record dataset comprised of Cps detections across its entire known invaded range. Evaluations of model performance provided validation of good model fit for all models. A consensus map of CLIMEX and ensemble correlative model predictions indicated that not-yet-invaded areas in eastern and southern Europe and in the southeastern, midwestern, and Pacific coast regions of North America are climatically suitable for Cps establishment. Most regions of the world where Buxus and its congeners are native are also at risk of establishment. These findings provide the first insights into Cps global invasion threat, suggesting that this invasive pathogen has the potential to significantly expand its range.

Rapid Decline of Calonectria pseudonaviculata Soil Population in Selected Gardens Across the United States.

Calonectria pseudonaviculata (Cps) poses a serious threat to boxwood, an iconic landscape plant in American and European gardens. Under the mild climatic conditions of the United Kingdom, Cps remained recoverable in infected leaf debris after being left on the soil surface or buried for 5 years. The primary objective of this study was to determine how this fungus may be affected by the warmer summers and colder winters in the United States by sampling and baiting soil with boxwood cuttings and by on-site testing with sentinel plants. Soil sampling started in a Virginia garden in January 2016 and was extended to California, Illinois, New York, and South Carolina in early summer of 2017 through late fall of 2018. The Cps soil population as measured by the percentage of infected bait leaves declined sharply within the first year of blighted boxwood removal and fell to an almost undetectable level at the end of this study. To validate these baiting results, the Virginia garden was tested on site four times with container-grown boxwood plants while the South Carolina garden and three New York gardens were tested once. Each test began with sentinel plants set out for field exposure, followed by evaluation on site and then in the laboratory after plants were retrieved from these gardens and incubated under conducive environments for 2 weeks. Cps was not observed on any sentinel boxwood plant on site or in the laboratory with one exception. These observations indicate that Cps did not survive in the United States garden soil over time as well as it did in the United Kingdom. These results have important practical implications while challenging the notion that fungi producing microsclerotia will always survive in the soil for many years.

cDNA Transcriptome of Arabidopsis Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol Agent Burkholderia sp. SSG.

Burkholderia sp. SSG is a potent biological control agent. Even though its survival on the leaf surface declined rapidly, SSG provided extended, moderate plant protection from a broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants, eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-treated controls to elucidate whether SSG triggers plant defense priming. Only eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential gene expression analysis identified 120 and 119 differentially expressed genes (DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing four regulation patterns. Functional categorization of these shared DEGs, and 44 very significantly upregulated DEGs revealed that SSG triggered various defense priming mechanisms, including responses to phosphate or iron deficiency, modulation of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-related gene regulation, and chromatin modification. These data support that SSG is an induced systemic resistance (ISR) trigger conferring plant protection upon pathogen encounter.

Differential Tolerance to Calonectria pseudonaviculata of English Boxwood Plants Associated with the Complexity of Culturable Fungal and Bacterial Endophyte Communities.

Isolated boxwood endophytes have been demonstrated to effectively protect boxwood plants from infection by Calonectria pseudonaviculata (Cps). However, the roles of endophytes as communities in plant defense are not clear. Here, we demonstrated differential tolerance to Cps of English boxwood (Buxus sempervirens 'Suffruticosa'), an iconic landscape plant and generally regarded as highly susceptible, and its link to endophyte complexity. Fifteen boxwood twig samples were collected in triplicates from three historic gardens-Colonial Williamsburg, George Washington's Mount Vernon and River Farm, and Virginia Tech's research farm in Virginia Beach in the summer and fall of 2019. A portion of individual samples was inoculated with Cps under controlled conditions. Significant differences in disease severity were observed among samples but not between the two seasons. Examining the endophyte cultures of the summer samples revealed that bacterial and fungal abundance was negatively and positively correlated with the disease severity. Nanopore metagenomics analysis on genomic DNA of the tolerant and susceptible group representatives confirmed the associations. Specifically, tolerant English boxwood plants had an endophyte community dominated by Bacilli and Betaproteobacteria, while susceptible ones had a distinct endophyte community dominated by Gammaproteobacteria, Actinobacteria, and diverse fungi. These findings may lead to boxwood health management innovations-devising and utilizing cultural practices to manipulate and increase the abundance and performance of beneficial endophytes for enhanced boxwood resistance to Cps.

Endophytic Burkholderia sp. SSG as a potential biofertilizer promoting boxwood growth.

BACKGROUND: Burkholderia sp. SSG is a bacterial endophyte isolated from boxwood leaves showing a resistant response to infection by the boxwood blight pathogen Calonectria pseudonaviculata. SSG acted as a protective and curative biocontrol agent for boxwood blight and as a bio-sanitizer of disease inoculum in the field. Many gene clusters involved in antibiotic production and plant growth promotion (PGP) were found in the genome, giving this endophyte great application potential as a treatment for plant protection. However, the PGP features have not been documented. This study investigated the plant growth promotion activity of SSG in boxwood. METHODS: To determine whether SSG is a plant growth promoting bacterium, four PGP traits, auxin and siderophore production, nitrogen fixation and phosphate solubilization, were examined in the laboratory with colorimetric or agar plate assays. The plant growth promoting activity of SSG was tested on three boxwood varieties characterized by slow, intermediate and fast growth rates, namely Justin Brouwers, Buddy and Winter Gem, respectively. These plants were drenched with an SSG cell suspension or water and washed plant weight was compared before and after treatment to determine growth changes after 10 months. RESULTS: The SSG culture was sustainable on nitrogen free media, suggesting that SSG may fix atmospheric nitrogen. It was also a strong phosphate solubilizer and a potent siderophore and indole-3-acetic acid (IAA) producer. Significant growth promotion was observed on boxwood cultivars Justin Brouwers, Buddy and Winter Gem 10 months after plant roots were drenched with SSG cells. The growth rate of treated plants was 76.1, 58.3, and 37.3% higher than that of the control, respectively. The degree of growth promotion was significantly different among plant varieties, notably more pronounced with the slow and intermediate growers. This study demonstrates that the SSG bacterium has multiple PGP traits and is a prospective plant biofertilizer.

Complete genome sequence of a boxwood endophyte Burkholderia sp. SSG with broad biotechnological application potential.

Burkholderia sp. strain SSG is a boxwood endophyte with potent antagonistic activities against a variety of plant pathogens. Here we present its complete genome sequence that is 8.6 Mb long with a GC content of 66.9%, 10,209 predicted protein-coding sequences, and 866 secondary metabolism gene clusters. Many of these genes and clusters involve antibiosis and other antagonistic activities against plant pathogens and insect pests as well as plant growth promoting traits but none for the Burkholderia cepacia epidemic strain marker. This genome sequence supports SSG as a potent biocontrol agent and source of other biotechnological applications.

A Potent Burkholderia Endophyte against Boxwood Blight Caused by Calonectria pseudonaviculata.

Calonectria pseudonaviculata (Cps) poses an increasing threat to boxwood, a major nursery crop and iconic landscape plant worldwide. Here, we report on a potent biocontrol agent that produces small sage green (SSG) colonies on potato dextrose agar. SSG is a bacterial strain recovered from Justin Brouwers boxwood leaves with unusual response to Cps inoculation. Water-soaked symptoms developed on leaves 2 days after inoculation then disappeared a few days later. This endophyte affected several major steps of the boxwood blight disease cycle. SSG at 107 cfu/mL lysed all conidia in mixed broth culture. SSG at 108 cfu/mL reduced blight incidence by >98% when applied one day before or 3 h after boxwood were inoculated with Cps. Its control efficacy decreased with decreasing bacterial concentration to 103 cfu/mL and increasing lead time up to 20 days. When applied on diseased leaf litter under boxwood plants, SSG reduced Cps sporulation and consequently mitigated blight incidence by 90%. SSG was identified as a new member of the Burkholderia cepacia complex with distinct characters from known clinical strains. With these protective, curative, and sanitizing properties, this Burkholderia endophyte offers great promise for sustainable blight management at production and in the landscape.

Indications of Susceptibility to Calonectria pseudonaviculata in Some Common Groundcovers and Boxwood Companion Plants.

Knowing the host range of a pathogen is critical to developing and implementing effective disease management programs. Calonectria pseudonaviculata (Cps) is known to attack a number of species, varieties, and cultivars in the genus Buxus as well as three Pachysandra species (Pachysandra terminalis, Pachysandra procumbens, and Pachysandra axillaris) and several Sarcococca species, all in the Buxaceae family. The objective of this study was to evaluate non-Buxaceae groundcovers and companion plants commonly associated with boxwood plantings for their susceptibility to Cps. Twenty-seven plant species belonging to 21 families were exposed to different levels of inoculum: 50 to 300 conidia per drop for detached leaf assays and 30,000 to 120,000 conidia per 1 ml for whole-plant assays. Inoculated plants were incubated in humid environments for at least 48 h to facilitate infection. Cps infection and sporulation were observed on 12 plant species: Alchemilla mollis, Arctostaphylos uva-ursi, Brunnera macrophylla, Epimedium × youngianum, Galium odoratum, Geranium sanguineum, Phlox subulata, Tiarella cordifolia, Callirhoe involucrata, Iberis sempervirens, Mazus reptans, and Vinca minor. These results suggest that there may be more hosts of Cps commonly grown in nurseries and landscapes. If corroborated by observations of natural infection, these findings have implications for the Boxwood Blight Cleanliness Program instituted by the National Plant Board and for planning disease mitigation at production and in the landscape.

Host responses and impact on the boxwood blight pathogen, Calonectria pseudonaviculata.

MAIN CONCLUSION: Boxwood leaves are more susceptible to Calonectria pseudonaviculata (Cps) and better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. Calonectria pseudonaviculata (Cps) infects boxwood and its two common companion plants-pachysandra and sweet box. This study investigated how boxwood, pachysandra, and sweet box respond to Cps isolates of different host origin. Detached leaves were inoculated with nine isolates, three from each host, and evaluated for colonization, infection rate, lesion size, and production of conidia and microsclerotia. Cps colonized boxwood leaf tissue within 12 h of inoculation, and 60 h ahead of pachysandra and sweet box. Cps also produced significantly larger lesions and more conidia on boxwood than on pachysandra and sweet box. Isolates originating from different host plants did not differ in all the components evaluated except for conidia production. Isolates from boxwood and sweet box produced significantly more conidia than those from pachysandra. Overall, boxwood leaves are more susceptible to the disease and are better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. These results advance the understanding of Cps biology and affirm the importance of taking pachysandra and sweet box into consideration in disease management planning.

Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species.

The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (ß-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and ß-tub. Resolution of these four markers also varied with (sub)clade. ß-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and ß-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade.

Microsclerotial Enumeration, Size, and Survival of Calonectria pseudonaviculata.

Boxwood blight caused by Calonectria pseudonaviculata (Cps) is an emerging disease in the United States. Understanding the biology and survival of microsclerotia, the resting propagules of Cps, is important to managing the disease and restoring Cps-infested sites. In this study, an image-analyzing software (OpenCFU) was adapted to enumerate microsclerotia produced in culture plates. Microsclerotial counts increased with time up to 42 days while their size continued to grow for 70 days. Additionally, younger microsclerotia were less sensitive to extreme temperatures of -10 and 40°C than older ones. For instance, 21-day-old microsclerotia survived at almost twice the rate of 70-day-old ones after a 24-h exposure to -10°C. Likewise, the 21-day-old microsclerotia survived at much greater rates than 70-day-old ones (50 to 70% versus <10%) after a 24-h exposure to 40°C. The practical implications of these results are discussed.

An expanded phylogeny for the genus Phytophthora.

A comprehensive phylogeny representing 142 described and 43 provisionally named Phytophthora species is reported here for this rapidly expanding genus. This phylogeny features signature sequences of 114 ex-types and numerous authentic isolates that were designated as representative isolates by the originators of the respective species. Multiple new subclades were assigned in clades 2, 6, 7, and 9. A single species P. lilii was placed basal to clades 1 to 5, and 7. Phytophthora stricta was placed basal to other clade 8 species, P. asparagi to clade 6 and P. intercalaris to clade 10. On the basis of this phylogeny and ancestral state reconstructions, new hypotheses were proposed for the evolutionary history of sporangial papillation of Phytophthora species. Non-papillate ancestral Phytophthora species were inferred to evolve through separate evolutionary paths to either papillate or semi-papillate species.

Zoospore exudates from Phytophthora nicotianae affect immune responses in Arabidopsis.

Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF) and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA) and jasmonic acid (JA): eds16 (enhanced disease susceptibility16), pad4 (phytoalexin deficient4), and npr1 (nonexpressor of pathogenesis-related genes1). Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora.