Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival.

Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have evolved independently in distinct kingdoms of life. How these microorganisms interact and to what extent those interactions affect plant health are poorly understood. We examined root-associated microbial communities from three Arabidopsis thaliana populations and detected mostly negative correlations between bacteria and filamentous microbial eukaryotes. We established microbial culture collections for reconstitution experiments using germ-free A. thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, we observed a profound impact of the bacterial root microbiota on fungal and oomycetal community structure and diversity. We demonstrate that the bacterial microbiota is essential for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation experiments in planta, indicate that biocontrol activity of bacterial root commensals is a redundant trait that maintains microbial interkingdom balance for plant health.

Modulation of host cell biology by plant pathogenic microbes.

Plant-pathogen interactions can result in dramatic visual changes in the host, such as galls, phyllody, pseudoflowers, and altered root-system architecture, indicating that the invading microbe has perturbed normal plant growth and development. These effects occur on a cellular level but range up to the organ scale, and they commonly involve attenuation of hormone homeostasis and deployment of effector proteins with varying activities to modify host cell processes. This review focuses on the cellular-reprogramming mechanisms of filamentous and bacterial plant pathogens that exhibit a biotrophic lifestyle for part, if not all, of their lifecycle in association with the host. We also highlight strategies for exploiting our growing knowledge of microbial host reprogramming to study plant processes other than immunity and to explore alternative strategies for durable plant resistance.

Fatal attraction: How Phytophthora zoospores find their host.

Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.

Recent developments in plant-downy mildew interactions.

Downy mildews are obligate oomycete pathogens that attack a wide range of plants and can cause significant economic impacts on commercial crops and ornamental plants. Traditionally, downy mildew disease control relied on an integrated strategies, that incorporate cultural practices, deployment of resistant cultivars, crop rotation, application of contact and systemic pesticides, and biopesticides. Recent advances in genomics provided data that significantly advanced understanding of downy mildew evolution, taxonomy and classification. In addition, downy mildew genomics also revealed that these obligate oomycetes have reduced numbers of virulence factor genes in comparison to hemibiotrophic and necrotrophic oomycetes. However, downy mildews do deploy significant arrays of virulence proteins, including so-called RXLR proteins that promote virulence or are recognized as avirulence factors. Pathogenomics are being applied to downy mildew population studies to determine the genetic diversity within the downy mildew populations and manage disease by selection of appropriate varieties and management strategies. Genome editing technologies have been used to manipulate host disease susceptibility genes in different plants including grapevine and sweet basil and thereby provide new soucres of resistance genes against downy mildews. Previously, it has proved difficult to transform and manipulate downy mildews because of their obligate lifestyle. However, recent exploitation of RNA interference machinery through Host-Induced Gene Silencing (HIGS) and Spray-Induced Gene Silencing (SIGS) indicate that functional genomics in downy mildews is now possible. Altogether, these breakthrough technologies and attendant fundamental understanding will advance our ability to mitigate downy mildew diseases.

Oomycete Metabolism Is Highly Dynamic and Reflects Lifestyle Adaptations.

The selective pressure of pathogen-host symbiosis drives adaptations. How these interactions shape the metabolism of pathogens is largely unknown. Here, we use comparative genomics to systematically analyze the metabolic networks of oomycetes, a diverse group of eukaryotes that includes saprotrophs as well as animal and plant pathogens, with the latter causing devastating diseases with significant economic and/or ecological impacts. In our analyses of 44 oomycete species, we uncover considerable variation in metabolism that can be linked to lifestyle differences. Comparisons of metabolic gene content reveal that plant pathogenic oomycetes have a bipartite metabolism consisting of a conserved core and an accessory set. The accessory set can be associated with the degradation of defense compounds produced by plants when challenged by pathogens. Obligate biotrophic oomycetes have smaller metabolic networks, and taxonomically distantly related biotrophic lineages display convergent evolution by repeated gene losses in both the conserved as well as the accessory set of metabolisms. When investigating to what extent the metabolic networks in obligate biotrophs differ from those in hemibiotrophic plant pathogens, we observe that the losses of metabolic enzymes in obligate biotrophs are not random and that gene losses predominantly influence the terminal branches of the metabolic networks. Our analyses represent the first metabolism-focused comparison of oomycetes at this scale and will contribute to a better understanding of the evolution of oomycete metabolism in relation to lifestyle adaptation. Numerous oomycete species are devastating plant pathogens that cause major damage in crops and natural ecosystems. Their interactions with hosts are shaped by strong selection, but how selection affects adaptation of the primary metabolism to a pathogenic lifestyle is not yet well established. By pan-genome and metabolic network analyses of distantly related oomycete pathogens and their nonpathogenic relatives, we reveal considerable lifestyle- and lineage-specific adaptations. This study contributes to a better understanding of metabolic adaptations in pathogenic oomycetes in relation to lifestyle, host, and environment, and the findings will help in pinpointing potential targets for disease control. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The pathogenicity of Plasmopara viticola: a review of evolutionary dynamics, infection strategies and effector molecules.

Oomycetes are filamentous organisms that resemble fungi in terms of morphology and life cycle, primarily due to convergent evolution. The success of pathogenic oomycetes lies in their ability to adapt and overcome host resistance, occasionally transitioning to new hosts. During plant infection, these organisms secrete effector proteins and other compounds during plant infection, as a molecular arsenal that contributes to their pathogenic success. Genomic sequencing, transcriptomic analysis, and proteomic studies have revealed highly diverse effector repertoires among different oomycete pathogens, highlighting their adaptability and evolution potential.The obligate biotrophic oomycete Plasmopara viticola affects grapevine plants (Vitis vinifera L.) causing the downy mildew disease, with significant economic impact. This disease is devastating in Europe, leading to substantial production losses. Even though Plasmopara viticola is a well-known pathogen, to date there are scarce reviews summarising pathogenicity, virulence, the genetics and molecular mechanisms of interaction with grapevine.This review aims to explore the current knowledge of the infection strategy, lifecycle, effector molecules, and pathogenicity of Plasmopara viticola. The recent sequencing of the Plasmopara viticola genome has provided new insights into understanding the infection strategies employed by this pathogen. Additionally, we will highlight the contributions of omics technologies in unravelling the ongoing evolution of this oomycete, including the first in-plant proteome analysis of the pathogen.

From glycans to green biotechnology: exploring cell wall dynamics and phytobiota impact in plant glycopathology.

Filamentous plant pathogens, including fungi and oomycetes, pose significant threats to cultivated crops, impacting agricultural productivity, quality and sustainability. Traditionally, disease control heavily relied on fungicides, but concerns about their negative impacts motivated stakeholders and government agencies to seek alternative solutions. Biocontrol agents (BCAs) have been developed as promising alternatives to minimize fungicide use. However, BCAs often exhibit inconsistent performances, undermining their efficacy as plant protection alternatives. The eukaryotic cell wall of plants and filamentous pathogens contributes significantly to their interaction with the environment and competitors. This highly adaptable and modular carbohydrate armor serves as the primary interface for communication, and the intricate interplay within this compartment is often mediated by carbohydrate-active enzymes (CAZymes) responsible for cell wall degradation and remodeling. These processes play a crucial role in the pathogenesis of plant diseases and contribute significantly to establishing both beneficial and detrimental microbiota. This review explores the interplay between cell wall dynamics and glycan interactions in the phytobiome scenario, providing holistic insights for efficiently exploiting microbial traits potentially involved in plant disease mitigation. Within this framework, the incorporation of glycobiology-related functional traits into the resident phytobiome can significantly enhance the plant's resilience to biotic stresses. Therefore, in the rational engineering of future beneficial consortia, it is imperative to recognize and leverage the understanding of cell wall interactions and the role of the glycome as an essential tool for the effective management of plant diseases.

Diversity and distribution of Phytophthora species across different types of riparian vegetation in Italy with the description of Phytophthora heteromorpha sp. nov.

Riparian formations encompass a diverse suite of transitional zones between terrestrial and aquatic ecosystems. During the last decades, these formations have been impacted by several emerging diseases. The first outbreaks were detected on alder formations, but have progressively also been observed on other plant species such as Betula pubescens, Nerium oleander, Populus alba, Salix alpina, Salix purpurea and Tamarix gallica. Declining plants showed a plethora of symptoms (leaf spot, shoot blight, bleeding cankers and root rot) indicative of Phytophthora infections. Since there is little information about the aetiology of these pathosystems, from November 2019 to March 2023, an in-depth study was conducted in 46 riparian ecosystems spanning from the Mediterranean to Alpine regions. Overall, 744 symptomatic samples (stem bleeding cankers and root with rhizosphere) from 27 host species were collected for Phytophthora isolation. Based on morphology and DNA sequence data, 20 known Phytophthora species belonging to seven phylogenetic clades have been identified: P. plurivora (202 isolates), P. gonapodyides (156), P. pseudosyringae (84), P. lacustris (57), P. acerina (31), P. idaei (30), P. alpina (20), P. pseudocryptogea (19), P. cambivora (13), P. pseudotsugae (13), P. cactorum (9), P. honggalleglyana (6), P. pseudogregata (6), P. debattistii (4), P. multivora (4), P. cinnamomi (3), P. bilorbang (2) P. crassamura (2), P. ilicis (2) and P. inundata (2). In addition, 26 isolates of a new putative species obtained from Alnus incana and Pinus sylvestris are described here as Phytophthora heteromorpha sp. nov. The new species proved to be pathogenic on grey alder causing symptoms congruent with field observations. This study represents the most comprehensive investigation on the Phytophthora species associated with declining riparian vegetation in Italy and highlights that the polyphagous pathogen P. plurivora represents a growing threat to Mediterranean, temperate and alpine ecosystems.

Rapid Detection of Viral, Bacterial, Fungal, and Oomycete Pathogens on Tomatoes with Microneedles, LAMP on a Microfluidic Chip, and Smartphone Device.

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.

Assessing the Capacity of High-resolution Commercial Satellite Imagery for Grapevine Downy Mildew Detection and Surveillance in New York state.

Grapevine downy mildew (GDM), caused by the oomycete Plasmopara viticola, can cause 100% yield loss and vine death under conducive conditions. High resolution multispectral satellite platforms offer the opportunity to track rapidly spreading diseases like GDM over large, heterogeneous fields. Here, we investigate the capacity of PlanetScope (3 m) and SkySat (50 cm) imagery for season-long GDM detection and surveillance. A team of trained scouts rated GDM severity and incidence at a research vineyard in Geneva, NY, USA from June to August of 2020, 2021, and 2022. Satellite imagery acquired within 72 hours of scouting was processed to extract single-band reflectance and vegetation indices (VIs). Random forest models trained on spectral bands and VIs from both image datasets could classify areas of high and low GDM incidence and severity with maximum accuracies of 0.85 (SkySat) and 0.92 (PlanetScope). However, we did not observe significant differences between VIs of high and low damage classes until late July-early August. We identified cloud cover, image co-registration, and low spectral resolution as key challenges to operationalizing satellite-based GDM surveillance. This work establishes the capacity of spaceborne multispectral sensors to detect late-stage GDM and outlines steps towards incorporating satellite remote sensing in grapevine disease surveillance systems.

Combining single-gene-resistant and pyramided cultivars of perennial crops in agricultural landscapes compromises pyramiding benefits in most production situations.

While resistant cultivars are valuable in safeguarding crops against diseases, they can be rapidly overcome by pathogens. Numerous strategies have been proposed to delay pathogen adaptation (evolutionary control), while still ensuring effective protection (epidemiological control). For perennial crops, multiple resistance genes can be deployed 1) in the same cultivar (pyramiding strategy), in single-gene-resistant cultivars grown 2) in the same field (mixture strategy) or 3) in different fields (mosaic strategy), or 4) in hybrid strategies that combine the three previous options. In addition, the spatial scale at which resistant cultivars are deployed can affect the plant-pathogens interaction: small fields are thought to reduce pest density and disease transmission. Here we used the spatially-explicit stochastic model landsepi to compare the evolutionary and epidemiological control across spatial scales and deployment strategies relying on two major resistance genes. Our results, broadly focused on resistance to downy mildew of grapevine, show that the evolutionary control provided by the pyramiding strategy is at risk when single-gene-resistant cultivars are concurrently planted in the landscape (hybrid strategies), especially at low mutation probability. Moreover, the effectiveness of pyramiding compared to hybrid strategies is influenced by whether the adapted pathogen pays a fitness cost across all hosts or only for unnecessary virulence, particularly when the fitness cost is high rather than intermediate. Finally, field size did not affect model outputs for a wide range of mutation probabilities and associated fitness costs. The socio-economic policies favoring the adoption of optimal resistant management strategies are discussed.

New Genotypes of Phytophthora nicotianae Found on Strawberry in Florida.

Phytophthora cactorum is the most common causal agent of Phytophthora crown rot and leather rot of strawberry, but P. nicotianae is also responsible for the disease in Florida. Studies of P. nicotianae populations have suggested that different groups of genotypes are associated with different hosts; however, it is not yet clear how many lineages exist globally and how they are related to different production systems. The aim of this study was to determine the genetic relationships of P. nicotianae isolates from Florida strawberry with genotypes reported from other hosts, quantify the genetic variation on strawberry, and test for an association with nursery source. A total of 49 isolates of P. nicotianae were collected from strawberry plants originating from multiple nursery sources during six seasons of commercial fruit production in Florida. Microsatellite genotyping identified 28 multilocus genotypes on strawberry that were distinct among 208 isolates originating from various hosts and locations. Based on STRUCTURE analysis, two genetic groups were identified: one consisting of isolates from strawberry, and the other comprising samples from different hosts. Multilocus genotypes were shared among nursery sources, and populations defined by nursery were not differentiated. Both mating types were found among the isolates from North Carolina- and California-origin plants and in most strawberry seasons; however, a predominance of A1 was observed, and regular sexual reproduction was not supported by the data. This study reveals a unique genetic population of P. nicotianae associated with strawberry and emphasizes the vital role of nursery monitoring in mitigating disease spread.

Untargeted metabolomic analysis reveals a potential role of saponins in the partial resistance of pea (Pisum sativum) against a root rot pathogen, Aphanomyces euteiches.

In soil-borne diseases, the plant-pathogen interaction begins as soon as the seed germinates and develops into a seedling. Aphanomyces euteiches, an oomycete, stays dormant in soil and gets activated by sensing the host through chemical signals present in the root exudates. The composition of plant exudates may, thus, play an important role during the early phase of infection. To better understand the role of root exudates in plant resistance, we investigated the interaction between partially resistant lines (PI660736 and PI557500) and susceptible pea cultivars (CDC Meadow and AAC Chrome) against Aphanomyces euteiches during the pre-invasion phase. The root exudates of two sets of cultivars clearly distinguished from each other in inducing oospore germination. PI557500 root exudate not only had diminished induction but also inhibited the oospore germination. The contrast between the root exudates of resistance and susceptible cultivars was reflected in their metabolic profiles. Data from fractionation and oospore germination inhibitory experiments identified a group of saponins that accumulated differentially in susceptible and resistant cultivars. We detected 56 saponins and quantified 44 of them in pea root and 30 from root exudate; the majority of them, especially Soyasaponin I and dehydrosoyasaponin I with potent in vitro inhibitory activities, were present in significantly higher amounts in both roots and root exudates of PI660736 and PI557500 as compared to Meadow and Chrome. Our results provide evidence for saponins as deterrents against Aphanomyces euteiches, which might have contributed to the resistance against root rot in the studied pea cultivars.

The functional role of Daphnia in the host-pathogen interaction of crayfish and the crayfish plague disease agent (Aphanomyces astaci).

Pathogen spores have been recognized as prey with implications for resource dynamics, energy transfer and disease transmission. In aquatic ecosystems, filter-feeders are able to consume such motile forms of pathogens that can cause severe disease in susceptible hosts. The interactions between European crayfish and the crayfish plague pathogen Aphanomyces astaci are of particular conservation interest. In this study, we aim to evaluate the ecological interactions between Ap. astaci, its host Astacus astacus and individuals of the genus Daphnia, filter-feeding planktonic crustaceans. Our focus was on the consumption of the motile zoospores by Daphnia individuals, but we also considered the potential of Daphnia as non-target hosts. We conducted a series of infection and life-history experiments with Ap. astaci, three Daphnia species (D. magna, D. galeata, and D. pulex) and the noble crayfish As. astacus. We did not observe any lethal effects in the infection experiments involving Ap. astaci and Daphnia. Only D. pulex showed differences in some life-history traits. The feeding experiment using the motile zoospores of Ap. astaci as alternative food source or as supplement to different amounts of algal food revealed their nutritional value: D. magna individuals survived, grew, and reproduced on a zoospore diet alone. When zoospores were supplemented to the regular algal diet, all life-history parameters have been significantly improved. However, this successful consumption of zoospores did not result in a reduced mortality of the susceptible crayfish As. astacus during the infection experiment. Nevertheless, the pathogen load of Ap. astaci in the tissues of As. astacus was significantly reduced as a consequence of the feeding activity of Daphnia. Our results indicate that an abundant filter-feeding community can reduce the amount of infective zoospores in the water body and thus be beneficial to susceptible crayfish hosts, potentially acting as a general buffer against zoospore-transmitted diseases in lentic waters.

Evaluation of a Formulation of Bacillus subtilis for Control of Phytophthora Blight of Bell Pepper.

Phytophthora blight, caused by Phytophthora capsici, is one of the most economically significant diseases of bell pepper in the United States. Over the past several decades, isolates of P. capsici exhibiting resistance to mefenoxam and other fungicides have been reported. Fungicide resistance coupled with an increased market for organically grown crops has led to interest in biological control as a disease management option. In this work, an isolate of Bacillus subtilis (AFS032321) was evaluated for control of Phytophthora blight of bell pepper in the greenhouse and field. A 28% active ingredient wettable powder formulation of the strain was applied as a soil drench at transplanting prior to inoculation. Treatment with this formulation of B. subtilis significantly reduced the area under the disease progress curve (AUDPC) by up to 52% compared to untreated control plants in greenhouse tests. Comparisons between applying the biocontrol weekly after seeding for 5 weeks versus a single application at transplanting (5 weeks) indicated no significant benefits of additional applications. The formulation of B. subtilis reduced disease caused by a mefenoxam-resistant isolate of P. capsici, while mefenoxam failed. The biocontrol efficacy of formulated strains was not affected in different soil types or potting media. However, disease was more severe in sandy soils. In field experiments that were conducted with a mefenoxam-sensitive isolate, disease incidence and severity of Phytophthora blight were significantly reduced at all rates of B. subtilis in 2019 except the 16.8 kg ha-1 rate. In both years, mefenoxam was more effective than B. subtilis in controlling disease in the field. B. subtilis did not affect the spatial dynamics of pathogen spread within rows. While the precise mechanism(s) of action is unclear, in vitro dual-culture tests suggest direct antagonism, as B. subtilis significantly inhibited colony growth of P. capsici. AgBiome has recently released a new formulation of the AFS032321 strain named Theia, with higher active ingredients for commercial applications and biocontrol of P. capsici.

Effect of Cucurbit Host, Production Region, and Season on the Population Structure of Pseudoperonospora cubensis in Florida.

Pseudoperonospora cubensis, the causal agent of Cucurbit downy mildew (CDM), is one of the most important diseases affecting cucurbit production in the United States. This disease is especially damaging to Florida production areas, as the state is a top producer of many cucurbit species. In addition, winter production in central and south Florida likely serves as a likely source of P. cubensis inoculum for spring and summer cucurbit production throughout the eastern United States, where CDM is unable to overwinter in the absence of a living host. Over 2 years (2017 and 2018) and four seasons (spring 2017, spring 2018, fall 2017, and fall 2018), 274 P. cubensis isolates were collected from cucurbit hosts at production sites in south, central, and north Florida. The isolates were analyzed with 10 simple sequence repeat (SSR) markers to establish population structure and genetic diversity and further assigned to a clade based on a qPCR assay. Results of population structure and genetic diversity analyses differentiated isolates based on cucurbit host and clade (1 or 2). Of the isolates assigned to clade by qPCR, butternut squash, watermelon, and zucchini were dominated by clade 1 isolates, whereas cucumber isolates were split 34 and 59% between clades 1 and 2, respectively. Clade assignments agreed with isolate clustering observed within discriminant analysis of principal components (DAPC) based on SSR markers, although watermelon isolates formed a group distinct from the other clade 1 isolates. For seasonal collections from cucumber at each location, isolates were typically skewed to one clade or the other and varied across locations and seasons within each year of the study. This variable population structure of cucumber isolates could have consequences for regional disease management. This is the first study to characterize P. cubensis populations in Florida and evaluate the effect of cucurbit host and clade-type on isolate diversity and population structure, with implications for CDM management in Florida and other United States cucurbit production areas.

Characterizing the Dynamics of Virulence and Fungicide Resistance of Phytophthora capsici in Michigan Vegetable Fields Reveals Loci Associated with Virulence.

The oomycete Phytophthora capsici is a destructive pathogen infecting more than 50 plant species and is one of the most serious threats to cucurbit production. Phytophthora blight caused by Phytophthora capsici can affect all plant growth stages, and fungicides and cultural controls are used to limit losses. Dissecting pathogen virulence and fungicide resistance can provide insights into pathogenic mechanisms and inform effective management practices to control P. capsici. In this study, we assessed virulence, mefenoxam sensitivity, and genetic diversity of nine P. capsici populations collected from Cucurbitaceae, Solanaceae, and Fabaceae host families in Michigan from 2002 to 2016. We developed 992 simple sequence repeats (SSRs) in the P. capsici genome and identified 60 SSRs located within or close to RXLR-class (Arginine-any amino acid-Leucine-Arginine) effectors and 29 SSRs within or close to effector CRN (CRinkling and Necrosis) family protein, which represent 62 RXLR and 34 putative CRNs. Population structure analysis shows that mefenoxam resistance was not associated with the year of collection, host type, or location, but there were significant differences in virulence among the populations. Using the general linear model and mixed linear model-based association analyses with all effector-related SSR markers, we identified four SSR markers significantly associated with at least one of the virulence-related parameters. Of these, one (Pce_SC18) was in a predicted CRN effector and had high identity with the putative PhCRN37 effector in the pathogen Plasmopara halstedii, which can be further verified for virulence identification in P. capsici.

First global report of Hyaloperonospora brassicae in commercial broccoli and cabbage microgreens.

Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover and distribution to population centers. Eleven of the 25 most broadly grown microgreens are brassicas (Choe et al. 2018). In November 2023, kale, broccoli (H009B), and cabbage (H009C) microgreen crops in Michigan were observed with downy mildew, at disease severities of 3%, 40%, and 20% foliage on 10 x 16 cm seeded blocks of plants, respectively. These crops shared a germination chamber for at least three days, which was maintained at approximately 22℃ in very humid, dark conditions. Chlorosis and grayish, sunken necrosis characterized symptoms on cotyledon surfaces (Fig. 1). In humid conditions, thick, white-light gray sporulation was present on adaxial cotyledon surfaces, accompanied by sparse sporulation on abaxial surfaces and hypocotyls. Severely diseased plants were stunted and approximately 50% gradually succumbed to downy mildew. On microscopic examination, a Hyaloperonospora spp. was tentatively identified, with long sporangiophores that dichotomously branched 3 to 6 times and hyaline sporangia borne singly on flexuous terminal sterigmata (Fig. 2). Sporangia were round to oval, with average length of 23.1 (range 16.0 to 28.3) µm; width of 20.0 (15.0 to 25.6) µm; and average length:width of 1.2 (1.0 to 1.4); (n = 97 for all). Sporangia dislodged rapidly if disturbed or as humidity decreased. Two pathogenicity tests were initiated on two sequential days. Two cotyledons from originally infected broccoli and cabbage were suspended, abaxial-side down, on coarse mesh over an open 60-mm plate of pregerminated brassica seeds on a water-saturated filter, inside a sealed, clear plastic box. Boxes contained only one type of originally diseased host, with 15 to 20 seeds of transfer varieties in unique dishes. Boxes were incubated in the dark for 2 days at 19°C with a wet paper towel atop the cotyledons. Before removal, cotyledons were lightly brushed across the surfaces of the seedlings they were just suspended above. Seedlings were grown in boxes in the presence of indirect, ambient light for 9.5 hr/day for an additional 5 days before pathogen sporulation was apparent. Filter paper was resaturated as needed. Noninoculated control plants, maintained separate from inoculated plants, were asymptomatic throughout the experiments. Total disease incidence in transfer varieties was 43.5% of 'Graffiti' cauliflower, 18.7% and 15.7% of 'Nixon' and 'Blue Vantage' cabbage; 11.8% of 'Red Russian' kale, and 6.0% of 'Ironman' broccoli, combined from two experiments. All varieties listed had at least one plant successfully infected in both pathogenicity tests. Sporulation on transfer hosts was morphologically identical to originally affected crops. Sporangiophores and sporangia were removed from H009B broccoli and H009C cabbage plants using surface sterilized forceps, placed directly into DNA extraction tubes containing buffer CD1 (Qiagen PowerSoil Pro), then kit instructions were followed. Extracts were utilized as template for ITS and cox1 PCR amplification, using DreamTaq Mastermix and ITS4/6 (45 cycles; White et al. 1990) and Levup/Levlo primers (30 cycles; Robideau et al. 2011). Cycling conditions were as published, with the number of cycles indicated by primer set. Each reaction yielded a single amplicon of approximately 1000 and 700 bp, for ITS and cox1, respectively,. Amplicons were cleaned using ExoSap-IT and submitted for Sanger sequencing, using ITS6 and Levup as sequencing primers (Robideau et al. 2011; White et al. 1990). After quality trimming, amplicons shared >98.5% identity with H. brassicae (NCBI Genbank accession MG757792 or reference genome CANTFL010000892.1). Sequences were submitted to Genbank (PP093830, PP093831, PP776812, PP776813). This is the first report of downy mildew, caused by H. brassicae, in commercial brassica microgreens, crops with vast nutritional value and expanding production.

First Report of Phytophthora cinnamomi causing root rot of avocado trees in Greece.

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees ('Hass' grafted on 'Zutano' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 µm (avg 29±0.8 µm) with characteristic thick walls (avg 1.2±0.2 µm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 µm (avg 56±4.8 µm) long and 20 to 42 µm (avg 31±3.2 µm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado 'Zutano' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

Picarbutrazox Effectiveness Added to a Seed Treatment Mixture for Management of Oomycetes that Impact Soybean in Ohio.

None of the current oomycota fungicides are effective towards all species of Phytophthora, Phytopythium, Globisporangium, and Pythium that affect soybean seed and seedlings in Ohio. Picarbutrazox is a new oomyceticide with a novel mode of action towards oomycete pathogens. Our objectives were to evaluate picarbutrazox to determine (i) baseline sensitivity (EC50) to 189 isolates of 29 species, (ii) the efficacy with a base seed treatment with three cultivars with different levels of resistance in 14 field environments; and (iii) if the rhizosphere microbiome was affected by the addition of the seed treatment on a moderately susceptible cultivar. The mycelial growth of all isolates was inhibited beginning at 0.001 µg, and the EC50 ranged from 0.0013 to 0.0483 µg of active ingredient (a.i.)/ml. The effect of seed treatment was significantly different for plant population and yield in eight of 14 and six of 12 environments, respectively. The addition of picarbutrazox at 1 and 2.5 g of a.i./100 kg seed to the base seed treatment compared to the base alone was associated with higher plant populations and yield in three and one environments, respectively. There was limited impact of the seed treatment mefenoxam 7.5 g of a.i. plus picarbutrazox 1 g of a.i./100 kg seed on the oomycetes detected in the rhizosphere of soybean seedlings collected at the V1 growth stage. Picarbutrazox has efficacy towards a wider range of oomycetes that cause disease on soybean, and this will be another oomyceticide tool to combat early season damping-off in areas where environmental conditions highly favor disease development.