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.

Phyllosphere microbial diversity and specific taxa mediate within-cultivar resistance to Phytophthora palmivora in cacao.

The oomycete pathogen Phytophthora palmivora, which causes black pod rot (BPR) on cacao (Theobroma cacao L.), is responsible for devastating yield losses worldwide. Genetic variation in resistance to Phytophthora spp. is well documented among cacao cultivars, but variation has also been observed in the incidence of BPR even among trees of the same cultivar. In light of evidence that the naturally occurring phyllosphere microbiome can influence foliar disease resistance in other host-pathogen systems, it was hypothesized that differences in the phyllosphere microbiome between two field accessions of the cultivar Gainesville II 164 could be responsible for their contrasting resistance to P. palmivora. Bacterial alpha diversity was higher but fungal alpha diversity was lower in the more resistant accession MITC-331, and the accessions harbored phyllosphere microbiomes with distinct community compositions. Six bacterial and 82 fungal amplicon sequence variants (ASVs) differed in relative abundance between MITC-333 and MITC-331, including bacterial putative biocontrol agents and a high proportion of fungal pathogens, and nine fungal ASVs were correlated with increased lesion development. The roles of contrasting light availability and host mineral nutrition, particularly potassium, are also discussed. Results of this preliminary study can be used to guide research into microbiome-informed integrated pest management strategies effective against Phytophthora spp. in cacao. IMPORTANCE Up to 40% of the world's cacao is lost each year to diseases, the most devastating of which is black pod rot, caused by Phytophthora palmivora. Though disease resistance is often attributed to cacao genotypes (i.e., disease-resistant rootstocks), this study highlights the role of the microbiome in contributing to differences in resistance even among accessions of the same cacao cultivar. Future studies of plant-pathogen interactions may need to account for variation in the host microbiome, and optimizing the cacao phyllosphere microbiome could be a promising new direction for P. palmivora resistance research.

Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae.

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for more than a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high sensitivity (<0.08 µg/ml), moderate sensitivity (0.14 to 0.64 µg/ml), and low sensitivity (>1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50 group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1,161 nucleotides encoding 386 amino acids. The cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a polymerase chain reaction (PCR) assay was developed to screen for mutations. According to this assay, 80% (n = 99) of tested C. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n = 9) carried the G143A mutation and were highly resistant. A receiver operating characteristic curve analysis suggested the PCR assay was a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from that of other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly occurring cytb mutation.

Cytochrome b Mutations F129L and G143A Confer Resistance to Azoxystrobin in Cercospora nicotianae, the Frogeye Leaf Spot Pathogen of Tobacco.

Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (Nicotiana tabacum L.), caused by Cercospora nicotianae. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC50) for 47 (in 2016) and 53 (in 2017) C. nicotianae isolates. Distributions of C. nicotianae EC50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae of moderate resistance (azoxystrobin at 0.177 ≤ EC50 ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC50 > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (<4 weeks prior to harvest; 4 of 62) or organically produced samples (1 of 7), respectively. Together, these results suggest that resistance to azoxystrobin in C. nicotianae occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.

Environmental and Management Factors Associated with Bacterial Diseases of Onion in Pennsylvania.

Bacterial diseases of onion may result in over 60% yield loss in crops grown in the Mid-Atlantic region, even when managed with recommended chemical and cultural practices. To identify environmental and production factors associated with the high incidence of bacterial rots in Pennsylvania, data on 32 environmental and management variables ranging from soil temperature to foliar nutrients were recorded during three visits to each of 28 and 26 fields, surveyed in 2011 and 2012, respectively. Multiple linear regression indicated negative relationships between foliar nitrogen and carbon at midseason and total incidence of bacterial rots. Soil temperatures near the physiological onset of bulbing were positively related to bacterial rots in multiple datasets. These results suggest greater complexity may be necessary for N fertility recommendations: timing of inorganic N application should be considered in addition to the seasonal N rate applied. Lower soil temperatures, particularly near the physiological onset of bulbing, may also reduce the incidence of bacterial rots of onion.

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple.

RpoN is a σ(54) factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild-type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)-inducing liquid medium. A plasmid-borne copy of the wild-type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.

Orchard factors associated with resistance and cross resistance to sterol demethylation inhibitor fungicides in populations of Venturia inaequalis from Pennsylvania.

Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 µg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-µg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all three fungicides (odds ratio = 1.76; P = 0.048). Results identify management practices that can reduce the risk of V. inaequalis developing resistance to DMI fungicides.

2,4-diacetylphloroglucinol alters plant root development.

Pseudomonas fluorescens isolates containing the phlD gene can protect crops from root pathogens, at least in part through production of the antibiotic 2,4-diacetylphloroglucinol (DAPG). However, the action mechanisms of DAPG are not fully understood, and effects of this antibiotic on host root systems have not been characterized in detail. DAPG inhibited primary root growth and stimulated lateral root production in tomato seedlings. Roots of the auxin-resistant diageotropica mutant of tomato demonstrated reduced DAPG sensitivity with regards to inhibition of primary root growth and induction of root branching. Additionally, applications of exogenous DAPG, at concentrations previously found in the rhizosphere of plants inoculated with DAPG-producing pseudomonads, inhibited the activation of an auxin-inducible GH3 promoter::luciferase reporter gene construct in transgenic tobacco hypocotyls. In this model system, supernatants of 17 phlD+ P. fluorescens isolates had inhibitory effects on luciferase activity similar to synthetic DAPG. In addition, a phlD() mutant strain, unable to produce DAPG, demonstrated delayed inhibitory effects compared with the parent wild-type strain. These results indicate that DAPG can alter crop root architecture by interacting with an auxin-dependent signaling pathway.