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 Report of Alternaria alternata Causing Decay in Exported Sweet Basil During Freight from Israel to Europe.

Sweet basil (Ocimum basilicum) is an important spice herb grown in Israel for local markets and export. The crop is used as a fresh culinary herb or spice, and the essential oils are used in cosmetics and food flavorings. Due to increased demand, the production area of basil has increased in Israel. Postharvest losses due to fungal disease are a major economic concern for growers. In the summer of 2019, a leaf spot was observed in postharvest shipments of sweet basil destined for Europe; in late winter of 2022, leaf spots were observed on greenhouse-grown sweet basil. Fungal isolates from infected leaves were characterized by morphology in culture as Alternaria spp. PCR amplification of the Alternaria major allergen Alt a1, ITS, and gdp gene regions of the recovered isolates confirmed the presence of A. alternata, a common pathogen of numerous herbs and spice plants. In vitro growth tests demonstrated that 25°C was the optimum temperature for growth of the isolates. The isolates were tested for pathogenicity and found to infect a commonly grown cultivar of basil, cultivar Eli (previously cultivar Perrie). Foliar symptoms in pathogenicity tests were identical to those observed in commercial shipments and in the field, which completed Koch's postulates. Control of the nascent disease by applying fungicides to the plants may be necessary to reduce postharvest losses.

First Report of Stem and Foliage Blight of Chrysanthemum Caused by Phytophthora drechsleri in the United States.

Chrysanthemum (Chrysanthemum × morifolium) plants exhibiting stem and foliage blight were observed in a commercial nursery in eastern Oklahoma in June 2019. Disease symptoms were observed on ~10% of plants during a period of frequent rain and high temperatures (26-36°C). Dark brown lesions girdled the stems of symptomatic plants and leaves were wilted and necrotic. The crown and roots were asymptomatic and not discolored. A species of Phytophthora was consistently isolated from the stems of diseased plants on selective V8 agar (Lamour and Hausbeck 2000). The Phytophthora sp. produced ellipsoid to obpyriform sporangia that were non-papillate and persistent on V8 agar plugs submerged in distilled water for 8 h. Sporangia formed on long sporangiophores and measured 50.5 (45-60) × 29.8 (25-35) µm. Oospores and chlamydospores were not formed by individual isolates. Mycelium growth was present at 35°C. Isolates were tentatively identified as P. drechsleri using morphological characteristics and growth at 35°C (Erwin and Ribeiro 1996). DNA was extracted from mycelium of four isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS 4 and ITS 6. The PCR product was sequenced and a BLASTn search showed 100% sequence similarity to P. drechsleri (GenBank Accession Nos. KJ755118 and GU111625), a common species of Phytophthora that has been observed on ornamental and vegetable crops in the U.S. (Erwin and Ribeiro 1996). The gene sequences for each isolate were deposited in GenBank (accession Nos. MW315961, MW315962, MW315963, and MW315964). These four isolates were paired with known A1 and A2 isolates on super clarified V8 agar (Jeffers 2015), and all four were mating type A1. They also were sensitive to the fungicide mefenoxam at 100 ppm (Olson et al. 2013). To confirm pathogenicity, 4-week-old 'Brandi Burgundy' chrysanthemum plants were grown in 10-cm pots containing a peat potting medium. Plants (n = 7) were atomized with 1 ml of zoospore suspension containing 5 × 103 zoospores of each isolate. Control plants received sterile water. Plants were maintained at 100% RH for 24 h and then placed in a protected shade-structure where temperatures ranged from 19-32°C. All plants displayed symptoms of stem and foliage blight in 2-3 days. Symptoms that developed on infected plants were similar to those observed in the nursery. Several inoculated plants died, but stem blight, dieback, and foliar wilt were primarily observed. Disease severity averaged 50-60% on inoculated plants 15 days after inoculation. Control plants did not develop symptoms. The pathogen was consistently isolated from stems of symptomatic plants and verified as P. drechsleri based on morphology. The pathogenicity test was repeated with similar results. P. drechsleri has a broad host range (Erwin and Ribeiro 1996; Farr et al. 2021), including green beans (Phaseolus vulgaris), which are susceptible to seedling blight and pod rot in eastern Oklahoma. Previously, P. drechsleri has been reported on chrysanthemums in Argentina (Frezzi 1950), Pennsylvania (Molnar et al. 2020), and South Carolina (Camacho 2009). Chrysanthemums are widely grown in nurseries in the Midwest and other regions of the USA for local and national markets. This is the first report of P. drechsleri causing stem and foliage blight on chrysanthemum species in the United States. Identifying sources of primary inoculum may be necessary to limit economic loss from P. drechsleri.

Characteristics of Resistance to Phytophthora Root and Crown Rot in Cucurbita pepo.

Root and crown rot incited by Phytophthora capsici causes considerable annual losses in squash-producing regions in the United States. 'Spineless Perfection' zucchini and 'Cougar' straightneck squash (Cucurbita pepo L.), partially resistant and susceptible to root and crown rot, respectively, were investigated for differences in root and crown physical factors and the histology of crown infection by P. capsici. The pH and titratable acidity of healthy root and crown tissue from tissue extracts were not significantly different between cultivars (P = 0.05). Crude fiber content (%) of blended and oven-dried root and crown tissue from healthy plants was similar between cultivars. However, dry matter (%) was highest for Cougar (P = 0.05). Colonies of P. capsici grown from mycelial plugs in root exudates collected from each cultivar were similar in diameter. Whole mounts and histological sections of healthy and infected crown tissue revealed that vascular bundles and metaxylem vessels were more abundant in crowns of Spineless Perfection than Cougar. Twelve to 48 h post inoculation (hpi), mycelia in the crown of each cultivar was limited to the cortex and hypodermal tissue. By 72 hpi, hyphae were observed in the cortex and endodermal tissue of Cougar and were concentrated in the phloem and parenchyma cells surrounding vascular bundles. Mycelia were limited to the outer cortex in Spineless Perfection. Mycelia and occluding material were present in the majority of metaxylem vessels of Cougar but not Spineless Perfection at 92 hpi; dissolution of parenchyma cells surrounding vascular bundles was apparent in Cougar. The vascular occlusions observed in Cougar may be responsible for plant wilting, a common disease symptom. Additional straightneck, crookneck, scallop, and acorn squash (C. pepo ssp. ovifera), and zucchini, marrow, and pumpkin (C. pepo ssp. pepo) cultivars were evaluated in a greenhouse study for resistance to root and crown rot. Cucurbita pepo ssp. ovifera cultivars were significantly more susceptible than C. pepo ssp. pepo to root and crown rot (P < 0.0001). Growing C. pepo ssp. pepo cultivars may be beneficial in an integrated Phytophthora management program.

Transport and Retention of Phytophthora capsici Zoospores in Saturated Porous Media.

Phytophthora capsici is an important plant pathogen capable of infecting several major vegetable crops. Water-induced P. capsici transport is considered to be a significant contributor to disease outbreaks and subsequent crop loss. However, little is known about factors controlling P. capsici zoospore transport in porous media, thus impeding our understanding of their environmental dispersal and development of filtration techniques for contaminated irrigation water. This study investigated the transport and retention of P. capsici zoospores in saturated columns packed with iron-oxide-coated sand (IOCS) or uncoated sand in Na(+) or Ca(2+) background solution at pH 7.7 ± 0.5 or 4.0 ± 0.3, in combination with XDLVO interaction energy calculations and microscopic visualizations. Significantly more encysted zoospores were retained in IOCS than in uncoated sand, and at pH 4.0 than at pH 7.7, which likely resulted from increased electrostatic attraction between zoospores and grain surface. At pH 7.7, up to 99% and 96% of the encysted zoospores were removed in IOCS and uncoated sand, respectively, due to a combination of strong surface attachment, pore straining, and adhesive interactions. Motile biflagellate zoospores were more readily transported than encysted zoospores, thus posing a greater dispersal and infection risk. This study has broad implications in environmental transport of Phytophthora zoospores in natural soils as well as in cost-effective engineered filtration systems.

Pathogenicity of Phytophthora capsici to Brassica Vegetable Crops and Biofumigation Cover Crops (Brassica spp.).

The soilborne oomycete Phytophthora capsici causes root, crown, and fruit rot of many vegetable crops in the Cucurbitaceae and Solanaceae families. P. capsici is a persistent problem in vegetable fields due to long-lived oospores that survive in soil and resist weathering and degradation. Vegetable crops in the Brassicaceae family have been considered nonhosts of P. capsici and are planted as rotational crops in infested fields. Brassica spp. are also grown as biofumigation cover crops to reduce inoculum levels of P. capsici and other soilborne pathogens, and this use has increased concurrent with restrictions on soil fumigation. Oriental mustard (Brassica juncea), oilseed rape (B. napus), and oilseed radish (Raphanus sativus var. oleiferus) contain high levels of glucosinolates and are widely recommended for biofumigation and as cover crops. The objective of this study was to evaluate vegetables and biofumigation cover crops in the Brassicaceae family for susceptibility to P. capsici. Brassica spp. used as vegetable crops and for biofumigation were grown in P. capsici-infested potting soil in the greenhouse and disease incidence and severity were recorded. In greenhouse trials, infection by the pathogen reduced the fresh weight of all Brassica spp. tested and resulted in plant death of 44% of plants of B. juncea 'Pacific Gold'. P. capsici isolates exhibited differences in virulence (P < 0.0001), and were reisolated from the roots of all Brassica spp. included in the study. The biofumigation cover crop Pacific Gold mustard may not reduce populations of P. capsici in soil and, instead, may sustain or increase pathogen levels. Further research is necessary to test this possibility under field conditions.