Metabolomics and Microbiomics Reveal Impacts of Rhizosphere Metabolites on Alfalfa Continuous Cropping.

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. Our preliminary study showed that the main factors of CC obstacle were not the lack of nutrients or water in alfalfa rhizosphere soils. Further, we evaluated 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. Four phenolic acid metabolites, including p-coumaric acid, ferulic acid, vanillic acid, and p-hydroxybenzoic acid, were found to have significant differences among different CC years, which may be the key factors of CC obstacle. Among them, p-coumaric acid and ferulic acid could significantly decrease the germination rate of alfalfa seeds by 21.11 and 16.67% at the concentration of 100 µg/mL and the height (root length) of alfalfa seedlings by 21% (32.9%) and 13.72% (16.45%). Moreover, these metabolites could effectively promote the growth of some pathogenic fungi, causing alfalfa root rot. Among them, p-coumaric acid obviously and significantly aggravated the occurrence of alfalfa root rot. With the increase of CC years, soil microbial community changed from fungi to bacteria; fungi decreased by 10.83%, fungi increased by 8.08%, and beneficial microorganisms decreased with the increase of CC years. Field analysis and experimental verification showed that the above results were consistent with that of CC obstacle in the field. Among the key metabolites, 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.

Development of a Rapid Sporulation Method of Fusarium graminearum Using Liquid Cultivation.

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.

Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide.

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.

Phylogenetic and phenotypic characterization of Fusarium oxysporum f. sp. niveum isolates from Florida-grown watermelon.

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.

Marker Development for Differentiation of Fusarium Oxysporum f. sp. Niveum Race 3 from Races 1 and 2.

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.

Fitness and Competitive Ability of Field Isolates of Phytophthora capsici Resistant or Sensitive to Fluopicolide.

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.

Draft Genome Sequences of Three Fusarium oxysporum f. sp. niveum Isolates Used in Designing Markers for Race Differentiation.

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.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry.

Bacterial leaf scorch, caused by Xylella fastidiosa, is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x105 cfu/mL of Xylella fastidiosa. Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn't require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X. fastidiosa on blueberry.

Genetic and phenotypic diversity of Fusarium oxysporum f. sp. niveum populations from watermelon in the southeastern United States.

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.

Maternal Stress Reduces the Susceptibility of Root-Knot Nematodes to Pasteuria Penetrans.

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.

Managing Fusarium Wilt of Watermelon with Delayed Transplanting and Cultivar Resistance.

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.

The Interactomic Analysis Reveals Pathogenic Protein Networks in Phomopsis longicolla Underlying Seed Decay of Soybean.

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.

Deciphering drought-induced metabolic responses and regulation in developing maize kernels.

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.

Influence of Root Exudates and Soil on Attachment of Pasteuria penetrans to Meloidogyne arenaria.

The bacterium Pasteuria penetrans is a parasite of root-knot nematodes (Meloidogyne spp.). Endospores of P. penetrans attach to the cuticle of second-stage juveniles (J2) and subsequently sterilize infected females. When encumbered by large numbers of spores, juveniles are less mobile and their ability to infect roots is reduced. This study looked at different factors that influence spore attachment of P. penetrans to the root-knot nematode Meloidogyne arenaria. Pretreatment of J2 with root exudates of eggplant (Solanum melongena cv. Black beauty) reduced spore attachment compared with pretreatment with phosphate-buffered saline (PBS), suggesting that the nematode surface coat was altered or the spore recognition domains on the nematode surface were blocked. Spore attachment was equally reduced following exposure to root exudates from both host and nonhost plants for M. arenaria, indicating a common signal that affects spore attachment. Although phytohormones have been shown to influence the lipophilicity of the nematode surface coat, auxins and kinetins did not affect spore attachment compared with PBS. Root exudates reduced spore attachment more in sterilized soil than in natural soil. Sterilization may have eliminated microbes that consume root exudates, or altered the chemical components of the soil solution or root exudates. Root exudates caused a greater decrease in spore attachment in loamy sand than in a sandy loam soil. The sandy loam had higher clay content than the loamy sand, which may have resulted in more adsorption of compounds in the root exudates that affect spore attachment. The components of the root exudates could have also been modified by soil type. The results of this study demonstrate that root exudates can decrease the attachment of P. penetrans endospores to root-knot nematodes, indicating that when these nematodes enter the root zone their susceptibility to spore attachment may decrease.

Infection Courts in Watermelon Plants Leading to Seed Infestation by Fusarium oxysporum f. sp. niveum.

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.

Synthesis and antifungal activity of 2-allylphenol derivatives against fungal plant pathogens.

2-Allylphenol (2-AP) is an effective fungicide against a number of plant pathogens, which can be metabolized and bio-transformed to four chemical compounds by Rhizoctonia cerealis. To determine if its degradation affects antifungal activity, two major metabolites derived from 2-AP including 2-(2-hydroxypropyl) phenol and 2-(3-hydroxypropyl) phenol were synthesized. Inhibition of mycelial growth of several plant pathogens by the metabolites was evaluated, and structures of two metabolites were determined by hydrogen nuclear magnetic resonance (1H NMR). Among these metabolites, only 2-(2-hydroxypropyl) phenol inhibited test pathogens effectively. EC50 values of 2-(2-hydroxypropyl) phenol for inhibition of mycelial growth of R. cerealis, Pythium aphanidermatum, Valsa mali and Botrytis cinerea ranged from 1.0 to 23.5µg/ml, which were lower than the parental fungicide 2-AP that ranged from 8.2 to 48.8µg/ml. Hyphae of R. cerealis and P. aphanidermatum treated with 2-(2-hydroxypropyl) phenol were twisted. Newly developed hyphae were slender, twisted and swollen on the tip, while old hyphae were hollow and ruptured. This is the first report indicating the formation of 2-(2-hydroxypropyl) phenol may have contributed to toxicity of 2-allylphenol in control of plant pathogens.

Sensitivity of Fusarium oxysporum f. sp. niveum to Prothioconazole and Thiophanate-Methyl and Gene Mutation Conferring Resistance to Thiophanate-Methyl.

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 ß-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.

Population Structure and Genetic Diversity of Phytophthora nicotianae from Tobacco in Georgia.

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 <0.01 to 0.12 µg/ml. Bayesian and unweighted pair group method with arithmetic means analyses of 59 isolates using SSR markers grouped the isolates in two major groups. Group I contained 20 isolates, of which 19 isolates were collected from Berrien County. Group II contained 39 isolates collected from Bacon, Cook, Tift, and Toombs Counties as well as one sample from Berrien County. Genetic diversity of the isolates was associated with geographical location of collection, and isolates in group I were primarily (75%) race 1, whereas isolates in group II were primarily (69%) race 0. The presence of a single pathogen mating type at most of the locations implies low probability of sexual recombination that may have contributed to the low genetic diversity at a particular geographical location. Sensitivity of the isolates to mefenoxam indicates that the fungicide remains to be a potent tool for growers to combat the disease. Information generated in the study advances our knowledge about diversity and population structure of P. nicotianae, which facilitates development and implementation of effective disease management programs.

Translocation of Oxathiapiprolin in Bell Pepper Plants and Systemic Protection of Plants Against Phytophthora Blight.

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.