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Fusarium graminearum is an important fungus causing a variety of maize diseases, including stalk rot, ear rot, and sheath rot. However, conidia of F. graminearum are not easily obtained under normal culture conditions, which seriously affects the identification and pathogenicity assessment of the isolates and screening of resistance sources. This study was undertaken to develop and utilize a rapid sporulation technique of F. graminearum using liquid cultivation, which could meet the needs of various tests. The results show that the optimum conditions for sporulation of F. graminearum were as follows: culture medium, 0.154 mol/liter of saline; temperature, 28 to 30°C; incubation time, 96 h; initial pH, 9 to 10; illumination, continuous ultraviolet light; and shaking speed, 150 rpm. Using this culture method, conidial concentration of tested F. graminearum strains can reach >1.5 × 105 conidia/ml. Compared with the existing methods using mung bean and carboxylmethyl cellulose as matrix, saline is relatively inexpensive, and the culture process, relatively quick. Overall, this study provided a systematic, rapid, and simple method to obtain a large number of conidia of F. graminearum.

Phytophthora blight, caused by Phytophthora capsici, is one of the most destructive diseases in the production of solanaceous and cucurbitaceous vegetable crops. Fluopicolide has been used to control the disease; however, reduced efficacy of the fungicide was observed in Georgia. P. capsici isolates were collected from commercial vegetable fields in Georgia in 2018 and 2019 to determine sensitivity to fluopicolide, which were phenotyped to have 43.1% of the isolates as resistant. The fitness of resistant (R) and sensitive (S) isolates was assessed through mycelial growth and sporulation assays exposed to the fungicide (0 or 50 µg/ml). Fluopicolide did not reduce mycelial growth, sporangial production, and zoospore germination of the R isolates. In the absence of fluopicolide, there was no significant difference between the R and S isolates in sporangial production but mycelial growth and zoospore germination of the R isolates was greater than the S isolates (P = 0.01 and 0.001, respectively). The R isolates had an ability similar to that of the S isolates to induce disease on Aristotle bell pepper, and most of the R and S isolates caused the same level of disease on Paladin. Inoculating squash fruit using different R:S ratios and recovering R and S isolates after five cycles of inoculation resulted in similar trends in changes of R versus S isolate ratios. Overall, it appeared that fitness and competitive ability of the R isolates were not reduced compared with the S isolates. This is the first report of the occurrence of field isolates of P. capsici resistant to fluopicolide in the world. The results have significant implications in providing guidance for growers to avoid or limit use of this fungicide in vegetable production.

Diseases caused by oomycete plant pathogens result in devastating losses to agriculture and native forests, despite the significant research efforts that have advanced our understanding of these organisms. Limiting these pathogens has been challenging to plant pathologists and plant health practitioners. In this first focus issue , titled Managing Stubborn Oomycete Plant Pathogens, Plant Health Progress has assembled an array of manuscripts on the biology and management of Phytophthora, Pythium, Pseudoperonospora, Peronospora, and Aphanomyces spp. This focus issue has 28 peer-reviewed papers including three diagnostic guides, three mini-reviews, three briefs, two surveys, and 17 research papers. Of the 28 papers, 20 are on diseases caused by Phytophthora, four on Pythium, three on downy mildews, and one on Aphanomyces. All advance our understanding of these stubborn oomycete pathogens.

Phytophthora capsici, the causal agent of Phytophthora blight, is a prominent and economically damaging oomycete pathogen in South Georgia. P. capsici causes crown, root, leaf, stem, and fruit infections on a wide range of vegetable crops. Oomycete pathogens such as P. capsici are dispersed in water, as their zoospores are flagellated and can move through runoff. Irrigation ponds are often reservoirs for different pathogens, and reusing the captured runoff is increasing in popularity to decrease irrigation costs. This combination allows for unintended outbreaks of diseases by pumping the contaminated runoff onto susceptible crops. Detection and identification of these pathogens are crucial steps in disease management, and rapid detection can ensure timely application of disease control measures. In this study, 42 irrigation ponds in nine counties from South Georgia were surveyed for the presence of P. capsici using a novel filtration method in conjunction with a LAMP assay specific for P. capsici. Ten ponds in five counties were found to have P. capsici as detected from the assay, suggesting that testing of irrigation ponds for P. capsici and other pathogens should be conducted to assist in preventing disease outbreaks.

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers’ and researchers’ ability to detect and manage fungicide-resistant phytopathogens.

Alfalfa long-term continuous cropping (CC) can pose a serious threat to alfalfa production. However, the mechanism of alfalfa CC obstacle is unclear as of today. In this study, we determined physic-chemical property, microbial population structure, and metabolite differences of alfalfa rhizosphere soils with CC for 1, 7, and 14-years based on analysis of metabolomics and microbiomics. Shifts of functional microorganisms in rhizosphere soil were analyzed, key metabolites and their effects on alfalfa seeds, seedlings and root rot pathogens were assessed. Based on anlysis, p-coumaric acid and ferulic acid on alfalfa seed and seedling growth and root rot pathogens were basically consistent with the influence of CC obstacles in the field. With the increase of CC years, the microbial community in soils changed from fungal to bacterial, and beneficial microorganisms decreased with the increase of CC years, which echoed the performance of alfalfa CC obstacles. The autotoxicity of p-coumaric acid was the strongest.This study fully proved that the continuous accumulation of autotoxic substances in alfalfa rhizosphere was the key factor causing alfalfa CC obstacles.

Early detection of soilborne disease is essential for prediction of disease development and successful disease management. Traditional methods of monitoring soilborne diseases based on plant growth and symptoms did not take into account the presence and inoculum potential of pathogens in the soil, which generally delay the prevention of diseases. To detect and quantify soil populations of Sclerotium rolfsii, the causal agent of southern blight of tomatoes, quantitative polymerase chain reaction (Q-PCR) was used to measure the amount of soil general fungal and specific S. rolfsii DNA. Significantly higher amounts of total fungal and S. rolfsii DNA were detected in inoculated than in uninoculated soil in a greenhouse inoculation experiment. Fluorescent in situ hybridization (FISH) was successfully developed to detect the abundance of S. rolfsii through whole cell hybridization and was further applied for visual detection of the abundance of S. rolfsii in the soil samples of greenhouse inoculation experiment. Natural abundance of S. rolfsii in soil with a DNA amount of 0.06 pg µl−1 DNA extraction, which was equivalent to 8 pg g−1 soil, was successfully detected. The hybridization signal detected in soil for S. rolfsii had a great correlation with the amount of infested DNA levels of S. rolfsii. Molecular detection and quantification of soilborne pathogens in plant roots and in the soil could provide valuable information to predict disease development and apply management practices for soilborne diseases.

Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum is a serious, widespread disease of watermelon throughout the southern United States. To investigate whether soil temperature affects disease development, three cultivars of triploid watermelon were transplanted March 17 to 21, April 7 to 11, and April 26 to May 2 in 2015 and 2016 at Charleston, SC, and Tifton, GA into fields naturally infested with F. oxysporum f. sp. niveum. Incidence of Fusarium wilt was lower with late-season than with early and midseason transplanting in all four experiments (P ≤ 0.01). Cultivar Citation had more wilted plants than the cultivars Fascination and Melody in three of four experiments (P ≤ 0.05). In South Carolina, planting date did not affect weight and number of marketable fruit ≥4.5 kg apiece. In Georgia in 2016, weight and number of marketable fruit were greater with late transplanting than with early and midseason transplanting. In both states, yield and value for Fascination and Melody were higher than for Citation. Soil temperature averaged over the 4-week period after transplanting was negatively correlated with disease incidence for all four experiments (r = –0.737, P = 0.006). Transplanting after mid-April and choosing a cultivar with resistance to F. oxysporum f. sp. niveum race 1, like Fascination, or tolerance, like Melody, can help manage Fusarium wilt of watermelon and increase marketable yields in the southern United States.

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is pathogenic only to watermelon and has become one of the main limiting factors in watermelon production internationally. Detection methods for this pathogen are limited, with few published molecular assays available to differentiate FON from other formae speciales of F. oxysporum. FON has four known races that vary in virulence but are difficult and costly to differentiate using traditional inoculation methods and only race 2 can be differentiated molecularly. In this study, genomic and chromosomal comparisons facilitated the development of a conventional polymerase chain reaction (PCR) assay that could differentiate race 3 from races 1 and 2, and by using two other published PCR markers in unison with the new marker, the three races could be differentiated. The new PCR marker, FNR3-F/FNR3-R, amplified a 511 bp region on the &ldquo, pathogenicity chromosome&rdquo, of the FON genome that is absent in race 3. FNR3-F/FNR3-R detected genomic DNA down to 2.0 pg/&micro, L. This marker, along with two previously published FON markers, was successfully applied to test over 160 pathogenic FON isolates from Florida, Georgia, and South Carolina. Together, these three FON primer sets worked well for differentiating races 1, 2, and 3 of FON. For each marker, a greater proportion (60 to 90%) of molecular results agreed with the traditional bioassay method of race differentiation compared to those that did not. The new PCR marker should be useful to differentiate FON races and improve Fusarium wilt research.

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is pathogenic only to watermelon and has become one of the main limiting factors in watermelon production internationally. Detection methods for this pathogen are limited, with few published molecular assays available to differentiate FON from other formae speciales of F. oxysporum. FON has four known races that vary in virulence but are difficult and costly to differentiate using traditional inoculation methods and only race 2 can be differentiated molecularly. In this study, genomic and chromosomal comparisons facilitated the development of a conventional polymerase chain reaction (PCR) assay that could differentiate race 3 from races 1 and 2, and by using two other published PCR markers in unison with the new marker, the three races could be differentiated. The new PCR marker, FNR3-F/FNR3-R, amplified a 511 bp region on the “pathogenicity chromosome” of the FON genome that is absent in race 3. FNR3-F/FNR3-R detected genomic DNA down to 2.0 pg/µL. This marker, along with two previously published FON markers, was successfully applied to test over 160 pathogenic FON isolates from Florida, Georgia, and South Carolina. Together, these three FON primer sets worked well for differentiating races 1, 2, and 3 of FON. For each marker, a greater proportion (60 to 90%) of molecular results agreed with the traditional bioassay method of race differentiation compared to those that did not. The new PCR marker should be useful to differentiate FON races and improve Fusarium wilt research.

Phytophthora blight, caused by

Fusarium wilt of watermelon (Citrullus lanatus) caused by Fusarium oxysporum f. sp. niveum (Fon), has become an increasing concern of farmers in the southeastern USA, especially in Florida. Management of this disease, most often through the use of resistant cultivars and crop rotation, requires an accurate understanding of an area’s pathogen population structure and phenotypic characteristics. This study improved the understanding of the state’s pathogen population by completing multilocus sequence analysis (MLSA) of two housekeeping genes (BT and TEF) and two loci (ITS and IGS), aggressiveness and race-determining bioassays on 72 isolates collected between 2011 and 2015 from major watermelon production areas in North, Central, and South Florida. Multilocus sequence analysis (MLSA) failed to group race 3 isolates into a single large clade; moreover, clade membership was not apparently correlated with aggressiveness (which varied both within and between clades), and only slightly with sampling location. The failure of multilocus sequence analysis using four highly conserved housekeeping genes and loci to clearly group and delineate known Fon races provides justification for future whole genome sequencing efforts whose more robust genomic comparisons will provide higher resolution of intra-species genetic distinctions. Consequently, these results suggest that identification of Fon isolates by race determination alone may fail to detect economically important phenotypic characteristics such as aggressiveness leading to inaccurate risk assessment.

Here, we report the draft genome sequences of three Fusarium oxysporum f. sp. niveum isolates that were used to design markers for molecular race differentiation. The isolates were collected from watermelon fields in Georgia (USA) and were determined to be different races of F. oxysporum f. sp. niveum using a traditional bioassay.

Background: Rice blast disease, caused by Magnaporthe oryzae, is a major constraint for rice production in the world. Introgression of blast-durable resistance genes into high-yielding rice cultivars has been considered an agricultural priority in an effort to control the disease. The blast resistance Pik locus, located on chromosome 11, contains at least six important resistance genes, but these genes have not been widely employed in resistance breeding since existing markers hardly satisfy current breeding needs owing to their limited scope of application.Results: In the present study, two PCR-based markers, Pikp-Del and Pi1-In, were developed to target the specific InDel (insertion/deletion) of the Pik-p and Pi-1 genes, respectively. The two markers precisely distinguished Pik-p, Pi-1, and the K-type alleles at the Pik locus, which is a necessary element for functional genes from rice varieties. Conclusions:Two gene-specific markers of Pi-kp and Pi1 identified that only several old varieties contain the two genes, nearly half these varieties yet carry the K-type alleles. Therefore, these identified varieties can be new gene sources for developing blast resistant rice. The two newly developed markers should be highly useful for using Pi-kp, Pi1 and other resistance genes at the Pik locus in marker-assisted selection (MAS) breeding programs.

Summary Drought stress conditions decrease maize growth and yield, and aggravate preharvest aflatoxin contamination. While several studies have been performed on mature kernels responding to drought stress, the metabolic profiles of developing kernels are not as well characterized, particularly in germplasm with contrasting resistance to both drought and mycotoxin contamination. Here, following screening for drought tolerance, a drought‐sensitive line, B73, and a drought‐tolerant line, Lo964, were selected and stressed beginning at 14 days after pollination. Developing kernels were sampled 7 and 14 days after drought induction (DAI) from both stressed and irrigated plants. Comparative biochemical and metabolomic analyses profiled 409 differentially accumulated metabolites. Multivariate statistics and pathway analyses showed that drought stress induced an accumulation of simple sugars and polyunsaturated fatty acids and a decrease in amines, polyamines and dipeptides in B73. Conversely, sphingolipid, sterol, phenylpropanoid and dipeptide metabolites accumulated in Lo964 under drought stress. Drought stress also resulted in the greater accumulation of reactive oxygen species (ROS) and aflatoxin in kernels of B73 in comparison with Lo964 implying a correlation in their production. Overall, field drought treatments disordered a cascade of normal metabolic programming during development of maize kernels and subsequently caused oxidative stress. The glutathione and urea cycles along with the metabolism of carbohydrates and lipids for osmoprotection, membrane maintenance and antioxidant protection were central among the drought stress responses observed in developing kernels. These results also provide novel targets to enhance host drought tolerance and disease resistance through the use of biotechnologies such as transgenics and genome editing.

Fusarium wilt, incited by the fungus Fusarium oxysporum f. sp. niveum, is a soilborne disease that affects watermelon production worldwide. Approaches for effective management of Fusarium wilt in watermelon are limited. Studies conducted in recent years indicated that prothioconazole and thiophanate-methyl reduced the disease significantly under field conditions. However, effects of the fungicides on different life stages of F. oxysporum f. sp. niveum and potential existence of fungicide resistance in F. oxysporum f. sp. niveum populations are unknown. In the present study, effects of prothioconazole and thiophanate-methyl on mycelium growth and spore germination of F. oxysporum f. sp. niveum isolates collected in watermelon fields in Georgia were determined. In vitro mycelium growth studies indicated that all 100 isolates evaluated were sensitive to prothioconazole; the effective concentration that suppressed mycelium growth by 50% ranged from 0.75 to 5.69 μg/ml (averaged 1.62 μg/ml). In contrast, 33 and 4% of the isolates were resistant to thiophanate-methyl at 10 and 100 μg/ml, respectively. Microconidial germination assays showed that 36 and 64% of the isolates tested were sensitive or intermediately sensitive to prothioconazole at 100 μg/ml but the fungicide did not inhibit spore germination at 10 μg/ml. Sequencing a portion of the β-tubulin gene of eight isolates resistant or sensitive to thiophanate-methyl indicated that fungicide resistance was associated with a point mutation at nucleotide position 200, resulting in a substitution of phenylalanine by tyrosine. This is the first report of isolates of F. oxysporum resistant to thiophanate-methyl. Results of the research suggest that prothioconazole may be a viable option for management of Fusarium wilt of watermelon whereas thiophanate-methyl should be used judiciously due to the existence of isolates resistant to the fungicide.

About 50% of the watermelons in the United States are produced in the southeastern states, where optimal conditions for development of Phytophthora fruit rot prevail. Phytophthora fruit rot significantly limits watermelon production by causing serious yield losses before and after fruit harvest. Efficacy of fungicide rotation programs and Melcast-scheduled sprays for managing Phytophthora fruit rot was determined by conducting experiments in Phytophthora capsici-infested fields at three locations in southeastern United States (North Carolina, South Carolina, and Georgia). The mini seedless cultivar Wonder and seeded cultivar Mickey Lee (pollenizer) were used. Five weekly applications of fungicides were made at all locations. Significant fruit rot (53 to 91%, mean 68%) was observed in the nontreated control plots in all three years (2013 to 2015) and across locations. All fungicide rotation programs significantly reduced Phytophthora fruit rot compared with nontreated controls. Overall, the rotation of Zampro alternated with Orondis was highly effective across three locations and two years. Rotations of Actigard followed by Ranman+Ridomil Gold, Presidio, V-10208, and Orondis, or rotation of Revus alternated with Presidio were similarly effective. Use of Melcast, a melon disease-forecasting tool, may occasionally enable savings of one spray application without significantly impacting control. Although many fungicides are available for use in rotations, under very heavy rain and pathogen pressure, the fungicides alone may not offer adequate protection; therefore, an integrated approach should be used with other management options including well-drained fields.

Aims The objective of this study was to determine the effects of sophorolipids on several fungal and oomycete plant pathogens and the relationship between sophorolipids at different pH and antimicrobial activities. Methods and results Sophorolipids had different solubility at different pH with a dramatic increase in solubility when pH was 6 or higher. Inhibition of mycelial growth of Phytophthora infestans by sophorolipids was affected by pH values, showing that when the pH value was higher, the inhibition rate was lower. Sophorolipids inhibited spore germination and mycelial growth of several fungal and oomycete pathogens in vitro including Fusarium sp., F. oxysporum, F. concentricum, Pythium ultimum, Pyricularia oryzae, Rhizoctorzia solani, Alternaria kikuchiana, Gaeumannomyces graminis var. tritici and P. infestans and caused morphological changes in hyphae by microscope observation. Sophorolipids reduced β-1,3-glucanase activity in mycelia of P. infestans. In greenhouse studies, foliar application of sophorolipids at 3 mg ml-1 reduced severity of late blight of potato caused by P. infestans significantly. Conclusion Sophorolipids influenced spore germination and hyphal tip growth of several plant pathogens and pH solubility of sophorolipids had an effect on their efficacy. Application of sophorolipids reduced late blight disease on potato under greenhouse conditions. Significance and impact of the study The findings indicated that sophorolipids have the potential to be developed as a convenient and easy-to-use formulation for managing plant diseases.

Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). Endospores of P. penetrans attach to the cuticle of second-stage juveniles (J2) and complete their life cycle within the nematode female body. Infected females will be filled with spores and will be sterilized. Studies with Daphnia magna and its parasite Pasteuria ramosa showed that a poor maternal environment can lead to offspring resistant to P. ramosa. Therefore, we hypothesized that Meloidogyne arenaria females raised under a stressed environment would produce offspring that were more resistant to P. penetrans. Females were exposed to a stressed environment created by crowding and low-food supply, or a non-stressed environment and their offspring evaluated for endospore attachment and infection by P. penetrans. No difference in spore attachment was observed between the two treatments. However, infection rate of P. penetrans in the stressed treatment was significantly lower than that in the non-stressed treatment (8 vs 18%). Mothers raised under stressed conditions appeared to produce more resistant offspring than did mothers raised under favorable conditions. Under stressful conditions, M. arenaria mothers may provide their progeny with enhanced survival traits. In the field, when nematode populations are not managed, they often reach the carrying capacity of their host plant by the end of the season. This study suggests that the next generation of inoculum may be more resistant to infection by P. penetrans.

Black shank, caused by Phytophthora nicotianae, occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide mefenoxam. Races of P. nicotianae currently prevalent in tobacco production in Georgia, their sensitivity to mefenoxam, and genetic diversity of the pathogen are largely unknown. To determine population structure and genetic diversity of the pathogen, simple sequence repeat (SSR) markers were used. Three races of P. nicotianae (races 0, 1, and 3) were isolated from infected tobacco plants, with race 3 identified in Georgia for the first time. The majority of isolates were identified as A2 mating type and all isolates were sensitive or intermediately sensitive to mefenoxam at 1 or 10 μg/ml, with effective concentration of mefenoxam for 50% mycelial growth reduction values ranging from

Production of bell pepper is seriously affected by Phytophthora capsici, the causal agent of Phytophthora blight. Limited approaches are available for effective management of the disease. Oxathiapiprolin is a fungicide recently registered in the United States that suppressed P. capsici and reduced Phytophthora blight on bell pepper significantly in our previous studies. It is unknown whether oxathiapiprolin translocates in bell pepper plants systemically after application. Experiments were conducted to determine uptake of oxathiapiprolin by bell pepper plants and its systemic movement in the plant. Quantification of oxathiapiprolin in plant tissues was conducted by high-performance liquid chromatography (HPLC) that detected the compound sensitively and selectively. Percentage of recovery of oxathiapiprolin from plant tissues was calculated by comparing the quantities in plant tissues determined by HPLC with known quantities of the compound added to the plant tissues. Recovery rates of oxathiapiprolin from pepper plant tissues ranged from 87.0 to 119.3%. When oxathiapiprolin was applied to roots of bell pepper plants grown in hydroculture, the compound was detected in the root within 4 h and in the cotyledon, first true leaf, and second true leaf within 8 h. It was detectable in the top new leaf 48 h after application to the root. In greenhouse studies with bell pepper plants grown in pots, oxathiapiprolin was applied as a soil drench at 100 and 400 μg/ml. The compound was detected in the root within 3 days and in the stem and first true leaf within 6 days when applied at 100 μg/ml. It was detected in the root, stem, first true leaf, and top new leaf within 3 days when applied at 400 μg/ml. Phytophthora blight on pepper foliage was significantly reduced when oxathiapiprolin was applied as a soil drench at 100 or 400 μg/ml under greenhouse conditions. This is the first report indicating systemic movement of oxathiapiprolin in pepper plants that provides useful information for designing fungicide application programs for effective management of the disease.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (FON) is a destructive soilborne disease commonly found in watermelon producing areas throughout the world. At present, the control of Fusarium wilt depends heavily on host resistance and chemical fungicide application. In this study, we isolated fluorescent pseudomonads from the rhizosphere of healthy watermelon and evaluated their biocontrol capacity against FON race 2. Biochemical assays indicated that all 14 fluorescent Pseudomonas strains were able to produce indole-3-acetic acid and at least one type of biosurfactants. Both sulphur oxidising and proteolytic activities were exhibited in 2 of the 14 strains. However, none of them could synthesise phenazine antibiotics. Seven strains with different morphological and biochemical characteristics were identified by sequencing the rpoB gene. Among those, one was Pseudomonas resinovorans (WMT16-1-1), two were P. putida (WMC16-1-1 and WMC16-2-5), one was P. fluorescens (WMC16-1-8) and others were Pseudomonas sp. In vitro studies indicated that WMC16-1-1, WMC16-1-8 and WMC16-2-5 inhibited mycelial growth of FON significantly. Detection of biosynthetic loci of 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin and pyoluteorin using specific primers indicated that the three strains could not produce the antibiotics. In greenhouse studies, WMC16-1-1, WMC16-1-8 and WMC16-2-5 reduced the severity of Fusarium wilt significantly with WMC16-1-1 being the most effective. Inoculation with WMC16-2-5 resulted in the significantly greater weight of both stems and roots compared to the nontreated control, while WMC16-1-1 resulted in greater stem weight. Pseudomonas strains WMC16-1-1, WMC16-1-8 and WMC16-2-5 are potential candidates for controlling Fusarium wilt and promoting the growth of watermelon.

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is a devastating soil-borne disease in watermelon production. Race 1 and race 2 of the pathogen are widely distributed in different watermelon producing regions. To investigate whether the root-knot nematodes break resistance of watermelon genotypes with resistance to FON race 1 and race 2, four greenhouse experiments were conducted on two watermelon cultivars ‘Calhoun Gray’ and ‘Fascination’ that are resistant to race 1, and an accession PI 296 341-FR that is resistant to race 2. The treatments included seedlings of ‘Calhoun Gray’ and ‘Fascination’ inoculated with Meloidogyne incognita (1000 J2/plant) or FON race 1 alone, nematodes 5 days before FON, FON 5 days before nematodes, nematodes and FON simultaneously, and non-inoculated control. PI 296 341-FR was inoculated with FON race 2, with or without M. incognita inoculation. The presence of M. incognita enhanced the susceptibility of all watermelon genotypes to Fusarium wilt. Co-inoculation with M. incognita led to an early development of wilt symptoms and increased disease severity. Galls were observed on roots of all nematode-inoculated plants, and sequential inoculation of FON followed by nematodes resulted in numerically lower galling indices compared with other inoculation methods. Whereas inoculation of the nematodes alone did not reduce plant growth, growth suppression was evident when seedlings were inoculated with both pathogens. The results indicated that M. incognita could enhance susceptibility of resistant watermelon genotypes to respective FON races, and host resistance alone is not sufficient for managing Fusarium wilt on watermelon in soils infested with root-knot nematodes.

Black shank incited by Phytophthora nicotianae is a devastating disease in the production of tobacco. Fungicides have been commonly used for managing the disease; however, there is only a narrow pool of effective fungicides. A few new fungicides became available in recent years, including fluopicolide, mandipropamid, and oxathiapiprolin, which reduced diseases incited by oomycetes under field conditions. Limited information is available regarding sensitivity of P. nicotianae isolates to these new fungicides. Research was conducted to determine effects of the three new fungicides on P. nicotianae isolates from tobacco in Georgia. Studies with 106 isolates indicated that they did not grow when agar medium was amended with the fungicides at the rate of 1 μg/ml. Twenty isolates were used for in vitro studies to determine sensitivity to the fungicides. Fluopicolide, mandipropamid, and oxathiapiprolin inhibited mycelial growth of the isolates with mean EC50 values (effective concentrations that provide 50% growth reduction) of 0.09, 0.04, and 0.001 μg/ml, respectively. EC50 values of fluopicolide, mandipropamid, and oxathiapiprolin for inhibiting sporangial formation were 0.15, 0.03, and 0.0002 μg/ml, respectively. EC50 values for suppressing zoospore germination averaged 0.16, 0.04, and 0.002 μg/ml for fluopicolide, mandipropamid, and oxathiapiprolin, respectively. Results from the study indicated that P. nicotianae isolates from tobacco in Georgia were sensitive to the fungicides, with lower EC50 for oxathiapiprolin than for fluopicolide and mandipropamid. The information on effectiveness and baseline sensitivity of fungicides on P. nicotianae will facilitate monitoring of resistance development in the pathogen population.

Phomopsis longicolla T. W. Hobbs (syn. Diaporthe longicolla) is the primary cause of Phomopsis seed decay (PSD) in soybean, Glycine max (L.) Merrill. This disease results in poor seed quality and is one of the most economically important seed diseases in soybean. The objectives of this study were to infer protein-protein interactions (PPI) and to identify conserved global networks and pathogenicity subnetworks in P. longicolla including orthologous pathways for cell signaling and pathogenesis. The interlog method used in the study identified 215,255 unique PPIs among 3,868 proteins. There were 1,414 pathogenicity related genes in P. longicolla identified using the pathogen host interaction (PHI) database. Additionally, 149 plant cell wall degrading enzymes (PCWDE) were detected. The network captured five different classes of carbohydrate degrading enzymes, including the auxiliary activities, carbohydrate esterases, glycoside hydrolases, glycosyl transferases, and carbohydrate binding molecules. From the PPI analysis, novel interacting partners were determined for each of the PCWDE classes. The most predominant class of PCWDE was a group of 60 glycoside hydrolases proteins. The glycoside hydrolase subnetwork was found to be interacting with 1,442 proteins within the network and was among the largest clusters. The orthologous proteins FUS3, HOG, CYP1, SGE1, and the g5566t.1 gene identified in this study could play an important role in pathogenicity. Therefore, the P. longicolla protein interactome (PiPhom) generated in this study can lead to a better understanding of PPIs in soybean pathogens. Furthermore, the PPI may aid in targeting of genes and proteins for further studies of the pathogenicity mechanisms.

Soil microbial communities and their relationship with major soil factors and disease development under tillage, no-tillage and organic farming practices were studied. Real-time quantitative-PCR (Q-PCR) was performed to quantify soil fungal and bacterial abundance. Length heterogeneity polymerase chain reaction (LH-PCR) combined with cloning and sequencing was used to assess microbial communities. Soil chemical characteristics were significantly different under different management practices. Organic agricultural practice significantly increased soil bacterial and fungal abundances, but not the diversity when compared to tillage and no-tillage management practices. Tillage farming agricultural practice yielded a significant higher bacterial abundance than no-tillage agricultural practice. No difference in fungal abundance was observed between tillage and no-tillage. Bacterial communities in no-tillage and organic cultivation were relatively similar, but significantly different from those in tillage cultivation. Differences of bacterial communities were correlated to soil organic matter (R2 = 0.558), cation exchange capacity (R2 = 0.583) and percentage of Mg (R2 = 0.590). Fungal communities in soil under organic cultivation were significantly different from relatively similar fungal community structure in no-tillage and tillage cultivation treatments. Differences of fungal communities were correlated to soil pH (R2 = 0.421) and soil organic matter (R2 = 0.401). In greenhouse assays, severity of southern blight on tomato plants grown in soils collected from the no-tillage was significantly lower than in soils from the tillage field. Different soil management practices have played an important role in altering soil microbial communities and potentially reducing disease incidence of southern blight on tomato.

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

Fusarium wilt incited by Fusarium oxysporum f. sp. niveum is a seed-transmitted disease that causes significant yield loss in watermelon production. The pathogen may infect watermelon seeds latently, which can be an important inoculum source and contribute to severe disease outbreak. However, information regarding infection courts of F. oxysporum f. sp. niveum leading to infestation of watermelon seeds is limited. To determine how seeds in watermelon fruit can be infested by F. oxysporum f. sp. niveum during the watermelon growing season, greenhouse and field experiments were conducted in 2014 and 2015 where watermelon flowers and immature fruit were inoculated with F. oxysporum f. sp. niveum. Seeds were extracted from mature watermelon fruit, and infestation of watermelon seeds was determined by isolation of F. oxysporum f. sp. niveum and further confirmed by real-time polymerase chain reaction (PCR) analysis. Inoculation of the pericarp of immature fruit resulted in 17.8 to 54.4% of infested seeds under field conditions and 0.6 to 12.8% of infested seeds under greenhouse conditions when seeds were not surface disinfested prior to isolation. Seed infestation was also detected in 0 to 4.5% of the seeds when seeds were surface disinfested prior to isolation. Inoculation of pistil resulted in 0 to 7.2% and 0 to 18.3% of infested seeds under greenhouse and field conditions when seeds were surface disinfested or not disinfested before isolation, respectively. Inoculation of peduncle resulted in 0.6 to 6.1% and 0 to 10.0% of infested seeds in the greenhouse and field experiments when seeds were surface disinfested or not disinfested before isolation, respectively. Seed infestation was also detected in all the experiments using real-time PCR assay when pericarp or pistil was inoculated, and in three of four experiments when peduncle was inoculated, regardless of whether seeds were surface disinfested or not disinfested. Pericarp and peduncle of immature watermelon fruit and pistil of watermelon flowers could be potential infection courts for F. oxysporum f. sp. niveum leading to infestation of seeds in asymptomatic watermelon fruit.

Black shank, caused by Phytophthora nicotianae, is responsible for serious yield and quality reduction in tobacco production. Application of effective fungicides continues to be a viable component in developing integrated disease management programs. Experiments were conducted in 2011 to 2013 to determine the efficacy and application methods of a new fungicide, Zorvec (a.i. oxathiapiprolin), for management of black shank under field conditions. Oxathiapiprolin is the first member of a new class of isoxazoline fungicide. Application of Zorvec (0.35 liter/ha) onto tobacco seedlings 1 week prior to transplanting in conjunction with directed applications of the product at 0.7 liter/ha at first cultivation and lay-by (last cultivation) reduced black shank significantly compared to the nontreated control in the experiments conducted in 2011 and 2012. Application of Zorvec at 1.4 liter/ha through transplant water followed by directed sprays at first cultivation and lay-by at 0.7 liter/ha reduced black shank significantly compared with the nontreated control in 2012 and 2013 studies. These treatments were not significantly different (P = 0.05) in disease reduction compared to mefenoxam. All treatments involving Zorvec increased tobacco yield significantly (P = 0.05) or showed a tendency to increase tobacco yield over the nontreated control in all experiments conducted in 2011 to 2013. The results indicated that the new fungicide oxathiapiprolin was effective in reduction of P. nicotianae on tobacco.

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), occurs worldwide and is responsible for substantial yield losses in watermelon-producing areas of the southeastern United States. Management of this disease largely relies on the use of integrated pest management (i.e., fungicides, resistant cultivars, crop rotation, etc.). Knowledge about race structure and genetic diversity of FON in the southeastern US is limited. To determine genetic diversity of the pathogen, FON isolates were collected from symptomatic watermelon plants in commercial fields in Georgia and Florida, USA, and identified based on morphological characteristics and PCR analysis using FON-specific primers. Discriminant analysis of principal components (DAPC) of 99 isolates genotyped with 15 simple sequence repeat (SSR) markers grouped the isolates in eight distinct clusters with two prominent clusters (clusters 1 and 8). Cluster 1 consisted of a total of 14 isolates, out of which 85.7% of the isolates were collected in Florida. However, most of the isolates (92.4%) in cluster 8 were collected in Georgia. Both DAPC and pairwise population differentiation analysis (ФPT) revealed that the genetic groups were closely associated with geographical locations of pathogen collection. Three races of FON (races 0, 2 and 3) were identified in the phenotypic analysis; with race 3 identified for the first time in Georgia. Overall, 5.1%, 38.9% and 55.9% of the isolates were identified as race 0, race 2 and race 3, respectively. The majority of the isolates in cluster 1 and cluster 8 belonged to either race 2 (35.6%) or race 3 (45.8%). Additionally, no relationship between genetic cluster assignment and races of the isolates was observed. The information obtained on genotypic and phenotypic diversity of FON in the southeastern US will help in development of effective disease management programs to combat Fusarium wilt.

Greenhouse studies were performed to determine the reactions of 10 “California Wonder” (Capsicum annuum) accessions to the three forms of Phytophthora blight (root rot, stem blight and foliar blight) caused by Phytophthora capsici. Differences in root rot, stem blight and foliar blight severities among accessions were significant. The accessions consistently differentiated into two groups across the three disease syndromes. Simple sequence repeat (SSR) markers showed variability both within and between accessions of California Wonder. The variability in the responses to the three forms of Phytophthora blight does not warrant its usefulness as a standard susceptible control in studies involving the Capsicum-P. capsici patho-system.

A mixture of six Georgia isolates of Phytophthora capsici (Leon.), the causal agent of phytophthora blight, were used for greenhouse mass screening of over 700 accessions of Capsicum annuum for both stem blight and foliar blight. From this screening, it was determined that resistance to both forms of the disease were relatively common in the germplasm, but resistance to one form of the disease was not strongly correlated to resistance to the other form. Ten accessions previously shown to possess root rot resistance were tested for resistance to stem rot and leaf blight, and were found to also be highly resistant to these forms of the disease. It appears that single accessions have resistance to foliar, stem and root rot caused by P. capsici, which may simplify breeding for resistance to all three forms of the disease.