Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Reducing Chlorothalonil Use in Fungicide Spray Programs for Powdery Mildew, Anthracnose, and Gummy Stem Blight in Melons.

Fungicides are applied to nearly 80% of U.S. melon acreage to manage the numerous foliar and fruit diseases that threaten yield. Chlorothalonil is the most widely used fungicide but has been associated with negative effects on human and bee health. We designed alternative fungicide programs to examine the impact of reducing chlorothalonil use (Bravo Weather Stik) on watermelon, cantaloupe, and honeydew melon in 2016, 2017, and 2018 in Maryland. Chlorothalonil was replaced in the tank mix of weekly sprays of targeted fungicides with either polyoxin D zinc salt (Oso) or an extract of Reynoutria sachalinensis (Regalia). Powdery mildew (PM; Podosphaera xanthii), gummy stem blight (GSB; Stagonosporopsis spp.), and anthracnose (Colletotrichum orbiculare) were the most prevalent diseases to occur in the 3 years. Replacing chlorothalonil with the biopesticides as the tank-mix component of the fungicide spray program was successful in reducing GSB and PM severity in cantaloupe, honeydew melon, and watermelon compared with the untreated control, with the exception of GSB in 2017 in cantaloupe, and similar to the program including chlorothalonil in all cases, except anthracnose in watermelon. Anthracnose disease severity was not significantly reduced compared with the untreated control when chlorothalonil was replaced with the biopesticides and yields were not improved over the chlorothalonil-alone treatment in any of the trials. Therefore, replacement of chlorothalonil may not fully address its loss as a fungicide resistance management tool but efficacy can be maintained when polyoxin D is alternated with R. sachalinensis as a tank mix with targeted fungicides to manage PM and GSB.

Interactions Between Salmonella enterica Newport, Fusarium spp., and Melon Cultivars.

Melons are perishable fruit of high food safety risk, grown in contact with soil and soil-borne organisms. To assess whether food safety risk could be augmented by the presence of soil-borne fungi, this study investigated the relationship between Fusarium spp. that were isolated from the surface of melon and the foodborne pathogen Salmonella enterica. In four repeated trials, rind discs from cultivars, Arava, Athena, Dulce Nectar, Jaune de Canaries, and Sivan fruit, grown in the field and in high tunnels in Maryland were inoculated separately with Fusarium isolates, F. oxysporum, F. fujikuroi, F. armeniacum, and F. proliferatum, with no Fusarium inoculation serving as a control and incubated at 25°C. Salmonella Newport was inoculated onto melon discs 4 d post-Fusarium inoculation and recovered 24 h later. Melon cultivar impacted the retrieval of Salmonella Newport. In all four replicated experiments, one or more of the netted varieties, Arava, Athena, and Sivan, yielded higher Salmonella Newport counts than one or both smooth-rind melons, Jaune de Canaries and Dulce Nectar (p < 0.05). Fusarium inoculation did not have a marked impact on Salmonella retrieval. The average Salmonella count recovered was 5.0 log colony-forming unit (CFU)/mL for both Fusarium-inoculated and uninoculated melons. However, in one trial, Salmonella Newport counts recovered from F. fujikuroi-inoculated melons were higher than all other treatments (8.6 log CFU/mL; p < 0.001), due to high levels of Salmonella recovered from Jaune de Canaries compared with other experiments. The food safety risk of melon did not appear to be enhanced by postharvest colonization with saprophytic Fusarium spp. However, melons with netted rinds appeared to favor Salmonella colonization compared with smooth melons. Choice of melon cultivar may be an important consideration in reducing Salmonella colonization risk in areas where Salmonella may be endemic in the environment.

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology.

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

Drip Line Flushing with Chlorine May Not Be Effective in Reducing Bacterial Loads in Irrigation Water Distribution Systems.

Irrigation water distribution systems are used to supply water to produce crops, but the system may also provide a protected environment for the growth of human pathogens present in irrigation water. In this study, the effects of drip tape installation depth and sanitization on the microbial quality of irrigation groundwater were evaluated. Drip tape lines were installed on the soil surface or 5 or 10 cm below the soil surface. Water samples were collected from the irrigation source and the end of each drip line every 2 weeks over an 11-week period, and the levels of Escherichia coli, total coliforms, aerobic mesophilic bacteria, and enterococci were quantified. Half of the lines installed at each depth were flushed with sodium hypochlorite for 1 h during week 6 to achieve a residual of 10 ppm at the end of the line. There was a statistically significant (P = 0.01) effect of drip tape installation depth and sanitizer application on the recovery of E. coli, with increased levels measured at the 5-cm depth and in nonsanitized lines, although the levels were at the limit of detection, potentially confounding the results. There was no significant effect of drip tape depth on total coliforms, aerobic mesophiles, or enterococci. In contrast, a statistically significant increase (P < 0.01) in the recovery of total coliforms was recorded from the ends of lines that received chlorine. This may be indicative of shedding of cells owing to degradation of biofilms that formed on the inner walls of the lines. These findings emphasize the need to better understand conditions that may lead to corrosion and increases in bacterial loads inside drip lines during flushing. Recommendations to growers should suggest collecting groundwater samples for testing at the end of drip lines rather than at the source. Guidelines on flushing drip lines with chlorine may need to include water pH monitoring, a parameter that influences the corrosive properties of chlorine.

Assessment of DAPG-producing Pseudomonas fluorescens for Management of Meloidogyne incognita and Fusarium oxysporum on Watermelon.

Pseudomonas fluorescens isolates Clinto 1R, Wayne 1R, and Wood 1R, which produce the antibiotic 2,4-diacetylphloroglucinol (DAPG), can suppress soilborne diseases and promote plant growth. Consequently, these beneficial bacterial isolates were tested on watermelon plants for suppression of Meloidogyne incognita (root-knot nematode: RKN) and Fusarium oxysporum f. sp. niveum (Fon). In a greenhouse trial, Wayne 1R root dip suppressed numbers of RKN eggs per gram root on 'Charleston Gray' watermelon by 28.9%. However, in studies focused on 'Sugar Baby' watermelon, which is commercially grown in Maryland, a Wayne 1R root dip did not inhibit RKN reproduction or plant death caused by Fon. When all three isolates were applied as seed coats, plant stand in the greenhouse was reduced up to 60% in treatments that included Fon ± P. fluorescens, and eggs per gram root did not differ among treatments. In a microplot trial with Clinto 1R and Wayne 1R root dips, inoculation with P. fluorescens and/or Fon resulted in shorter vine lengths than treatment with either P. fluorescens isolate plus RKN. Root weights, galling indices, eggs per gram root, and second-stage juvenile (J2) numbers in soil were similar among all RKN-inoculated treatments, and fruit production was not affected by treatment. Plant death was high in all treatments. These studies demonstrated that the tested P. fluorescens isolates resulted in some inhibition of vine growth in the field, and were not effective for enhancing plant vigor or suppressing RKN or Fon on watermelon.

Effects of Cover Crop Species and Season on Population Dynamics of Escherichia coli and Listeria innocua in Soil.

Cover crops provide several ecosystem services, but their impact on enteric bacterial survival remains unexplored. The influence of cover cropping on foodborne pathogen indicator bacteria was assessed in five cover crop/green manure systems: cereal rye, hairy vetch, crimson clover, hairy vetch-rye and crimson clover-rye mixtures, and bare ground. Cover crop plots were inoculated with Escherichia coli and Listeria innocua in the fall of 2013 and 2014 and tilled into the soil in the spring to form green manure. Soil samples were collected and the bacteria enumerated. Time was a factor for all bacterial populations studied in all fields (P < 0.001). E. coli levels declined when soil temperatures dipped to <5°C and were detected only sporadically the following spring. L. innocua diminished somewhat but persisted, independently of season. In an organic field, the cover crop was a factor for E. coli in year 1 (P = 0.004) and for L. innocua in year 2 (P = 0.011). In year 1, E. coli levels were highest in the rye and hairy vetch-rye plots. In year 2, L. innocua levels were higher in hairy vetch-rye (P = 0.01) and hairy vetch (P = 0.03) plots than in the rye plot. Bacterial populations grew (P < 0.05) or remained the same 4 weeks after green manure incorporation, although initial reductions in L. innocua numbers were observed after tilling (P < 0.05). Green manure type was a factor only for L. innocua abundance in a transitional field (P < 0.05). Overall, the impacts of cover crops/green manures on bacterial population dynamics in soil varied, being influenced by bacterial species, time from inoculation, soil temperature, rainfall, and tillage; this reveals the need for long-term studies.

Evaluation of Rotational Biopesticide Programs for Disease Management in Organic Cucurbit Production.

Downy mildew (Pseudoperonospora cubensis) and powdery mildew (Podosphaera xanthii) are two of the most economically important and widespread cucurbit diseases. Disease management relies primarily on fungicide use, but frequent fungicide applications can lead to the development of resistant pathogen populations. In addition, more vegetables are being produced with organic practices, which prohibit the use of many fungicides. Incorporating biorational products into a disease management program may help mitigate the risk of fungicide resistance development while being compatible with organic production. Field trials were conducted for two years on organically managed land in Maryland with cucumber, muskmelon, pumpkin, and butternut squash to evaluate the efficacy of four biorational products (i.e., Actinovate AG, OxiDate, Regalia, and Serenade Soil) when applied in a rotational program with copper against foliar cucurbit diseases. Generally, all biorational treatments resulted in significantly lower downy and powdery mildew severity compared with the nontreated plants, but the level of disease management was not significantly different than that provided by copper alone. However, Actinovate AG, OxiDate, and Serenade Soil each improved disease management on at least one crop, as compared with copper alone. Rotational programs with biopesticides are a viable disease management option for organic production of field-grown cucurbits in Maryland.

The growing season, but not the farming system, is a food safety risk determinant for leafy greens in the mid-Atlantic region of the United States.

Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.

Assessment of region, farming system, irrigation source and sampling time as food safety risk factors for tomatoes.

In the mid-Atlantic region of the United States, small- and medium-sized farmers use varied farm management methods and water sources to produce tomatoes. It is unclear whether these practices affect the food safety risk for tomatoes. This study was conducted to determine the prevalence, and assess risk factors for Salmonella enterica, Shiga toxin-producing Escherichia coli (STEC) and bacterial indicators in pre-harvest tomatoes and their production areas. A total of 24 organic and conventional, small- to medium-sized farms were sampled for six weeks in Maryland (MD), Delaware (DE) and New Jersey (NJ) between July and September 2012, and analyzed for indicator bacteria, Salmonella and STEC. A total of 422 samples--tomato fruit, irrigation water, compost, field soil and pond sediment samples--were collected, 259 of which were tomato samples. A low level of Salmonella-specific invA and Shiga toxin genes (stx1 or stx2) were detected, but no Salmonella or STEC isolates were recovered. Of the 422 samples analyzed, 9.5% were positive for generic E. coli, found in 5.4% (n=259) of tomato fruits, 22.5% (n=102) of irrigation water, 8.9% (n=45) of soil, 3/9 of pond sediment and 0/7 of compost samples. For tomato fruit, farming system (organic versus conventional) was not a significant factor for levels of indicator bacteria. However, the total number of organic tomato samples positive for generic E. coli (1.6%; 2/129) was significantly lower than for conventional tomatoes (6.9% (9/130); (χ(2) (1)=4.60, p=0.032)). Region was a significant factor for levels of Total Coliforms (TC) (p=0.046), although differences were marginal, with western MD having the highest TC counts (2.6 log CFU/g) and NJ having the lowest (2.0 log CFU/g). Tomatoes touching the ground or plastic mulch harbored significantly higher levels of TC compared to vine tomatoes, signaling a potential risk factor. Source of irrigation water was a significant factor for all indicator bacteria (p<0.0001), and groundwater had lower bacterial levels than surface water. End of line surface water samples were not significantly different from source water samples, but end of line groundwater samples had significantly higher bacterial counts than source (p<0.0001), suggesting that Good Agricultural Practices that focus on irrigation line maintenance might be beneficial. In general, local effects other than cropping practices, including topography, land use and adjacent industries, might be important factors contributing to microbiological inputs on small- and medium-sized farms in the mid-Atlantic region.

Reduced Fungicide Applications and Host Resistance for Managing Three Diseases in Pumpkin Grown on a No-Till Cover Crop.

Two recent changes in Maryland pumpkin production are (i) planting no-till into a cover crop with soil surface residue (70% of acreage) and (ii) adoption of cultivars with moderate resistance to powdery mildew. Pumpkin cultivar resistance to powdery mildew, planting method (no-till cover crop or conventional tillage bare ground), and fungicide schedules were examined for development of powdery mildew (caused primarily by Podosphaera xanthii), Plectosporium blight (Plectosporium tabacinum), and black rot (Didymella bryoniae), and pumpkin yield and quality. Fungicide application intervals were (i) nontreated, (ii) 7 days, (iii) 14 days, or (iv) 7 days early and 14 days late season. Pumpkin grown no-till on hairy vetch and hairy vetch plus rye cover crops had an average 36% less Plectosporium blight and 50% less black rot than those grown conventional tillage on bare ground. Powdery mildew was less severe on cv. Magic Lantern, which is moderately resistant to this disease, than on susceptible cv. Wizard. Regression equations to describe the impact of disease and treatment on pumpkin fruit number, weight, and peduncle quality (healthy, intact peduncles) were developed using three-stage least squares procedure. Powdery mildew caused the greatest reduction on fruit number, weight, and peduncle quality compared with other diseases. Plectosporium blight reduced fruit number in 1999 and 2000, and fruit weight and peduncle quality in 2000. Hairy vetch and hairy vetch plus rye cover crops resulted in greater fruit number (1,033 and 858 more marketable fruit/ha, respectively) than bare ground in 2000. Powdery mildew resistance (Magic Lantern) combined with pumpkin production on a cover crop resulted in lower levels of powdery mildew (average areas under the disease progress curve 1,474 versus 2,379), Plectosporium blight (average 5 versus 16% severity), and black rot (average 153 versus 217 symptomatic fruit/ha) compared with conventional production (Wizard on bare ground). A reduced fungicide schedule resulted in acceptable disease management, yield, and peduncle quality of Magic Lantern grown on a cover crop; Magic Lantern grown on a cover crop and sprayed every 14 days yielded the same as or more than Wizard grown on bare ground and sprayed weekly.

Foliar Disease in Fresh-Market Tomato Grown in Differing Bed Strategies and Fungicide Spray Programs.

A 3-year field study in central Maryland evaluated foliar disease in fresh-market tomato grown using combinations of four bed strategies and three fungicide programs. Bed strategies included uncovered beds with or without a composted dairy manure amendment or beds covered with black polyethylene or hairy vetch mulch. Fungicide programs included no fungicide, weekly fungicide, or fungicide applications scheduled according to the TOMCAST disease predictor. In plots with hairy vetch-covered beds, early blight caused by Alternaria solani, Septoria leaf spot caused by Septoria lycopersici, and defoliation were lower versus uncovered beds each year. Early blight and defoliation were lower in beds covered with vetch versus polyethylene mulch in 2 of 3 years. Disease severity, defoliation, and marketable yield were similar for the weekly and TOMCAST fungicide programs, with 40 to 50% fewer sprays using TOMCAST. Marketable yield was similar among bed strategies except for higher yields in covered versus uncovered and unamended beds in a relatively wet year and lower yields in vetch versus polyethylene beds in a dry year.

Factors Associated with Foliar Disease of Staked Fresh Market Tomatoes Grown Under Differing Bed Strategies.

The use of mulch or compost to reduce foliar disease in fresh market tomato could reduce fungicide use. Between 1997 and 1999, foliar disease was monitored in tomatoes grown in beds with bare soil, black polyethylene, composted dairy manure, or hairy vetch. Early blight was reduced in plots with vetch compared with bare soil or compost in all years and compared with polyethylene cover in 1 year. Early blight was reduced in plots with polyethylene versus compost and bare soil in 1 and 2 years, respectively. Septoria leaf spot was reduced in plots with vetch versus other bed strategies in 1998 and bare soil or compost in 1999. This disease was reduced in plots with polyethylene versus bare soil or compost in 1998 and 1999. Soil coverage of tomato leaflets and soil particle dispersal were reduced in plots with polyethylene or vetch versus bare soil or compost in both years that these variables were assessed. Sensor wetness duration was reduced in plots with polyethylene or vetch versus bare soil or compost in one of two years. These findings suggest that foliar disease reduction in mulch was associated with reduced splash dispersal and, in one year, reduced leaf wetness.

Impact of Powdery Mildew and Leaf Rust on Milling and Baking Quality of Soft Red Winter Wheat.

Changes in milling and baking quality (especially flour yield) of soft red winter wheat can have a large economic impact on flour mills. To determine the relationship between early-season powdery mildew and late-season leaf rust on flour yield, flour protein, alkaline water retention capacity, and kernel texture (softness equivalent), a study was conducted over 2 years at Kinston and Plymouth, NC. Different levels of powdery mildew and leaf rust developed on three winter wheat cultivars that varied in levels of disease resistance, the presence of seed treatment, and the presence and timing of foliar fungicide application. In Kinston and Plymouth in 1989-90, where leaf rust occurred early, the softness equivalent score was lower in wheat grown from seed treated with triadimenol. The following year, when the leaf rust epidemic increased later, foliar fungicide application reduced disease and resulted in lower softness equivalent scores in both Plymouth and Kinston for cv. Saluda and in Kinston for cv. Coker 983. A regression model was developed to describe the relationship between the log of the area under the disease progress curves and adjusted flour yield (AFY). The AFY of Saluda was reduced in the presence of powdery mildew such that %AFY = 103.96 - 0.92 (log AUMPC).