Transmission of Spinach Downy Mildew via Seed and Infested Leaf Debris.

Spinach downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is a worldwide constraint on spinach production. The role of airborne sporangia in the disease cycle of P. effusa is well established, but the role of the sexual oospores in the epidemiology of P. effusa is less clear and has been a major challenge to examine experimentally. To evaluate seed transmission of spinach downy mildew via oospores in this study, isolated glass chambers were employed in two independent experiments to grow out oospore-infested spinach seed and noninfested seeds mixed with oospore-infested crop debris. Downy mildew diseased spinach plants were observed 37 and 34 days after planting in the two isolator experiments, respectively, in the chambers that contained one of two oospore-infested seed lots or seeds coated with oospore-infested leaves. Spinach plants in isolated glass chambers initiated from seeds without oospores did not show downy mildew symptoms. Similar findings were obtained using the same seed lot samples in a third experiment conducted in a growth chamber. In direct grow out tests to examine oospore infection on seedlings performed in a containment greenhouse with oospore-infested seed of two different cultivars, characteristic Peronospora sporangiophores were observed growing from a seedling of each cultivar. The frequency of seedlings developing symptoms from 82 of these oospore-infested seed indicated that approximately 2.4% of seedlings from infested seed developed symptoms, and 0.55% of seedlings from total seeds assayed developed symptoms. The results provide evidence that oospores can serve as a source of inoculum for downy mildew and provide further evidence of direct seed transmission of the downy mildew pathogen to seedlings in spinach via seedborne oospores.

Early Detection of the Spinach Downy Mildew Pathogen in Leaves by Recombinase Polymerase Amplification.

Downy mildew of spinach, caused by Peronospora effusa, is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for P. effusa could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the P. effusa specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, P. schachtii. TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect P. effusa in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.

Changes in Bacterial Endophyte Community Following Aspergillus flavus Infection in Resistant and Susceptible Maize Kernels.

Aspergillus flavus (A. flavus)-mediated aflatoxin contamination in maize is a major global economic and health concern. As A. flavus is an opportunistic seed pathogen, the identification of factors contributing to kernel resistance will be of great importance in the development of novel mitigation strategies. Using V3-V4 bacterial rRNA sequencing and seeds of A. flavus-resistant maize breeding lines TZAR102 and MI82 and a susceptible line, SC212, we investigated kernel-specific changes in bacterial endophytes during infection. A total of 81 bacterial genera belonging to 10 phyla were detected. Bacteria belonging to the phylum Tenericutes comprised 86-99% of the detected phyla, followed by Proteobacteria (14%) and others (<5%) that changed with treatments and/or genotypes. Higher basal levels (without infection) of Streptomyces and Microbacterium in TZAR102 and increases in the abundance of Stenotrophomonas and Sphingomonas in MI82 following infection may suggest their role in resistance. Functional profiling of bacteria using 16S rRNA sequencing data revealed the presence of bacteria associated with the production of putative type II polyketides and sesquiterpenoids in the resistant vs. susceptible lines. Future characterization of endophytes predicted to possess antifungal/ anti-aflatoxigenic properties will aid in their development as effective biocontrol agents or microbiome markers for maize aflatoxin resistance.

Transcriptional analyses of differential cultivars during resistant and susceptible interactions with Peronospora effusa, the causal agent of spinach downy mildew.

Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.

Spinach Downy Mildew: Advances in Our Understanding of the Disease Cycle and Prospects for Disease Management.

Downy mildew on spinach is caused by Peronospora effusa, an oomycete pathogen that poses a challenge to spinach production worldwide, especially in organic production. Following infection, P. effusa produces abundant amounts of asexual sporangia. Sporangia become windborne and initiate new infections locally or distantly, leading to widespread epidemics. Oospores produced from the union of opposite mating types have been observed within infected leaves and seeds and may remain viable for many years. Sexual reproduction increases the genetic diversity of P. effusa through sexual recombination, and thus, the movement of oospores on seed has likely fueled the rapid explosion of new pathotypes in different regions of the world over the past 20 years. This review summarizes recent advances in spinach downy mildew research, especially in light of the findings of oospores in contemporary commercial spinach seed lots as well as their germination. Knowledge of the role of the oospores and other aspects of the disease cycle can directly translate into new and effective disease management strategies.

Corrigendum: An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

[This corrects the article DOI: 10.3389/fmicb.2017.00386.].

Do Endophytes Promote Growth of Host Plants Under Stress? A Meta-Analysis on Plant Stress Mitigation by Endophytes.

Endophytes are microbial symbionts living inside plants and have been extensively researched in recent decades for their functions associated with plant responses to environmental stress. We conducted a meta-analysis of endophyte effects on host plants' growth and fitness in response to three abiotic stress factors: drought, nitrogen deficiency, and excessive salinity. Ninety-four endophyte strains and 42 host plant species from the literature were evaluated in the analysis. Endophytes increased biomass accumulation of host plants under all three stress conditions. The stress mitigation effects by endophytes were similar among different plant taxa or functional groups with few exceptions; eudicots and C4 species gained more biomass than monocots and C3 species with endophytes, respectively, under drought conditions. Our analysis supports the effectiveness of endophytes in mitigating drought, nitrogen deficiency, and salinity stress in a wide range of host species with little evidence of plant-endophyte specificity.

An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

Microbial communities in the endosphere of Salicaceae plants, poplar (Populus trichocarpa) and willow (Salix sitchensis), have been demonstrated to be important for plant growth promotion, protection from biotic and abiotic stresses, and degradation of toxic compounds. Our study aimed to investigate bio-control activities of Salicaceae endophytes against various soil borne plant pathogens including Rhizoctonia solani AG-8, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Pythium ultimum. Additionally, different plant growth promoting traits such as biological nitrogen fixation (BNF), indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, and siderophore production were assessed in all bio-control positive strains. Burkholderia, Rahnella, Pseudomonas, and Curtobacterium were major endophyte genera that showed bio-control activities in the in-vitro assays. The bio-control activities of Burkholderia strains were stronger across all tested plant pathogens as compared to other stains. Genomes of sequenced Burkholderia strains WP40 and WP42 were surveyed to identify the putative genes involved in the bio-control activities. The ocf and hcnABC gene clusters responsible for biosynthesis of the anti-fungal metabolites, occidiofungin and hydrogen cyanide, are present in the genomes of WP40 and WP42. Nearly all endophyte strains showing the bio-control activities produced IAA, solubilized tricalcium phosphate, and synthesized siderophores in the culture medium. Moreover, some strains reduced acetylene into ethylene in the acetylene reduction assay, a common assay used for BNF. Salicaceae endophytes could be useful for bio-control of various plant pathogens, and plant growth promotion possibly through the mechanisms of BNF, IAA production, and nutrient acquisition.

Relationship Between Climatic Factors and Distribution of Pratylenchus spp. in the Dryland Wheat-Production Areas of Eastern Washington.

Field surveys were conducted by collecting soil samples to estimate nematode densities in soil from winter wheat, spring wheat, spring barley, and spring legumes (lentil, chickpea, and pea) fields during 2010 and 2011. Pratylenchus spp. were observed in 60% of sampled fields. However, nematodes were detected in nearly all of the survey fields in high numbers where crops were grown every year. To identify climatic variables associated with density of Pratylenchus spp. in soil, correlation and regression analyses were performed using climate data of survey sites from 1979 to 2010. Fifty-seven climate variables were significantly correlated with densities of Pratylenchus spp. All precipitation variables were significantly positively correlated with nematode abundance. Summer maximum air temperature was negatively correlated and winter minimum air temperature was positively correlated with nematode densities. In addition, both years' nematode densities were significantly correlated with cropping intensity. Five multivariate regression models for 2010 and seven models for 2011 nematode abundance levels were developed. The majority of the climate variables selected in the models were related to precipitation. Knowledge of root-lesion nematode distribution in the dryland region of eastern Washington and associated climate variables may be helpful to determine risk and apply management practices to minimize crop damage.

Spring Wheat Tolerance and Resistance to Heterodera avenae in the Pacific Northwest.

The cereal cyst nematode Heterodera avenae reduces wheat yields in the Pacific Northwest. Previous evaluations of cultivar resistance had been in controlled environments. Cultivar tolerance had not been evaluated. Seven spring wheat trials were conducted in naturally infested fields in three states over 2 years. A split-plot design was used for all trials. Five trials evaluated both tolerance and resistance in 1.8-by-9-m plots treated or not treated with nematicides. Two trials evaluated resistance in 1-m head rows where each wheat entry was paired with an adjacent row of a susceptible cultivar. Cultivars with the Cre1 resistance gene ('Ouyen' and 'Chara') reduced the postharvest density of H. avenae under field conditions, confirming Cre1 parents as useful for germplasm development. Ouyen was resistant but it was also intolerant, producing significantly lower grain yield in controls than in plots treated with nematicides. Susceptible cultivars varied in tolerance. Undefined resistance was identified in one commercial cultivar ('WB-Rockland) and four breeding lines (UC1711, SO900163, SY-B041418, and SY-97621-05). This research was the first systematic field demonstration of potential benefits to be derived through development and deployment of cultivars with resistance plus tolerance to cereal cyst nematode in North America.