Ralstonia solanacearum (Rs), the causal agent of bacterial wilt of tomato, can cause severe economic losses to tomato growers in Louisiana (LA). Traditional management tactics are ineffective. Resistant cultivars lack durability and have undesirable horticultural traits. In addition, disease resistance is strain specific. For these reasons, many producers in LA have abandoned their fields for tomato production. Although R. solanacearum is endemic in LA, pathogen populations have never been characterized. Tomato samples with bacterial wilt symptoms were collected from Livingston, Tangipahoa, East Baton Rouge and St. Helena parishes in 2015. Bacterial wilt was confirmed using Agdia Inc. Rs-specific immunoflow strips and bacterial streaming test. Fifteen isolates were recovered from five fields and one greenhouse. Strains from LA were characterized and belong to phylotype I and II. Thirty-three percent of the strains were characterized as biovar 1 and 20% as biovar 3. Forty-seven percent of strains were unable to utilize dulcitol, and thus belong to a new biovar 6 classification. None of the isolates belong to a select agent Race 3 biovar 2. A worldwide collection of genetically diverse eggplant, tomato and pepper varieties was screened for resistance to seven Rs strains from LA. One pepper variety (PM702) and one eggplant variety (cv. MM15) were identified as being highly resistant to all seven strains from LA. All of the tomato varieties tested were susceptible to the LA strains. Grafting with resistant rootstock varieties was explored as a sustainable management strategy for bacterial wilt in LA. Three susceptible tomato cultivars (Celebrity, Florida 91 and BHN602) that are commonly produced in LA were grafted onto cvs. MM152 and PM702 resistant rootstocks and onto tomato cv. Hawaii 7996, which is considered a model resistant variety. None of the tomato plants that were grafted to pepper cv. PM702 survived the grafting process. Tomato plants (all varieties) grafted to eggplant (cv. MM152) rootstocks were the most resistant to Rs strains from LA compared to those grafted to Hawaii 7996. This study shows that Rs strains from LA were capable of infecting putatively resistant tomato rootstocks but may be managed by using resistant eggplant rootstocks.

Cercospora leaf blight (CLB) caused in soybean by Cercospora cf. flagellaris is an important disease in Louisiana. It was thought that CLB starts with leaf purpling, and then, as the disease progresses, leaves become blighted. Moreover, it was assumed that accumulation of cercosporin, a red/purple pigment, was the cause of the purple pigmentation in diseased leaves. However, our observations in Louisiana suggested that these two symptoms were not correlated. The first objective of this work was to examine the relationship between purple and blight symptoms as well as their relationships with endophytic colonization of leaves by the fungus and accumulation of cercosporin in soybean leaves. The second objective was to document biochemical changes in purple, blighted and asymptomatic leaves. We demonstrated that purple and blighted leaves were not necessarily correlated, and that cercosporin concentrations in purple leaves were one third that in blighted leaves. Location and cultivar also determined the type of symptoms shown by soybean leaves. This work provides the first report of accumulation of coumestrol (COU) in purple leaves of soybean affected by CLB and demonstrated that COU may be associated with resistance to C. cf. flagellaris via its antioxidant activity. Production of pterocarpin derivatives, a common reaction to biotic and abiotic stresses, may be the cause of purple discoloration of soybean leaves affected by CLB. Results from this work showed that purple CLB leaf symptoms probably are a plant response to low levels of cercosporin produced by the pathogen in its endophytic stage, and blight symptoms are produced when cercosporin production by the pathogen exceeds the plants antioxidant capability.

The AVR4 effector, secreted by Cladosporium fulvum, has been demonstrated to be involved in pathogen virulence. Recent studies further demonstrated that Avr4 is highly conserved among several Cercospora species, indicating a potential important role of this gene in fungal virulence. Therefore, investigation to determine whether this fungal effector gene is present in Cercospora cf. flagellaris (previously known as C. kikuchii), the causal agent of soybean cercospora leaf blight (CLB) disease, and whether it plays any role in CLB disease development, is of great interest. In the present study, the Avr4 gene from C. cf. flagellaris was cloned and mutants lacking the expression of Avr4 were created by homologous recombination to investigate its role in fungal virulence, cercosporin production as well as CLB disease development. The ∆avr4 mutants produced little or no cercosporin in vitro and the mutants also had significantly reduced cercosporin toxin biosynthesis genes expression. Furthermore, ∆avr4 mutants grew faster and were more sensitive to chitinase in vitro than the wild type. Surprisingly, cercosporin could also directly suppress chitinolytic activity in vitro. When inoculated onto detached soybean leaves, these mutants exhibited reduced virulence compared to the wild type and no cercosporin was detected in mutant inoculated soybean leaves. Taken all together, the results suggest that AVR4 may contribute to the virulence of C. cf. flagellaris on soybean through protecting fungal hyphae and regulating cercosporin biosynthesis. Considering the importance of AVR4 in C. cf. flagellaris virulence, we selected a region of this gene for targeted gene suppression through host induced gene silencing (HIGS) to determine whether this can reduce CLB disease in soybean. It was found that HIGS plants carrying the BPMV-Avr4 construct showed less disease symptoms compared to control plants, and the reduction of symptoms was positively correlated with reduction in Avr4 transcript levels and fungal growth. To the best of our knowledge, this is the first study demonstrating the involvement of AVR4 in CLB disease development possibly through regulating cercosporin production as well as protecting fungal hyphae. In addition, this study also showed the potential of using HIGS as a tool to control this important disease of soybean

Glyphosate (Roundup®, Monsanto, Inc., St. Louis, MO) is the most widely used herbicide in the world because of its broad spectrum and its efficacy in controlling annual broadleaf weeds and undesired grasses. The effect of glyphosate on mineral nutrition and plant diseases has been an important topic during the past decade, because of its controversial effects on plant mineral nutrition. In order to test the hypothesis that glyphosate affects soybean mineral nutrition, and therefore predisposes soybean to Cercospora leaf blight, six glyphosate-resistant (GR) soybean varieties were either not treated or treated with glyphosate in field experiments. Plants were then evaluated for leaf concentrations of 13 nutrients, foliar disease symptoms, and biomass of Cercospora cf. flagellaris as assessed with real-time PCR assays. Experiments were conducted at three locations in Louisiana. Three sets of soybean GR varieties were used in the experiments, six each year at each location, for a total of 18 varieties. These varieties corresponded to maturity groups III, early and late IV, and V. Control plots received no glyphosate application on the soybean foliage. Disease assessments were performed at R6 growth stage at one location using predefined scales for purple symptoms in 2014 and 2016. No symptoms of CLB were observed at any other location during the three-year period of the study. Differences were detected in nutrient uptake among research stations and years, and there were variations in fungal biomass across varieties. The effects of glyphosate on leaf concentrations of Al, Fe, Mn, N, Na, and K were location-dependent. Glyphosate enhanced uptake of Zn in all experiments. Real-time PCR analyses of the CTB6 gene of C. cf. flagellaris consistently detected higher fungal biomass in glyphosate-treated samples compared to controls, indicating that glyphosate affected colonization of host plants during the latent period of infection. Enhanced uptake of of Zn, which is a key part of a transcriptional activator (CTB8) in the cercosporin biosynthetic pathway, could be associated with higher concentrations of C. cf. flagellaris DNA in glyphosate-treated leaves. However, CLB purple leaf symptom severity was significantly lower in glyphosate-treated plots compared to controls in 2014 and 2016. Therefore, colonization by C. cf. flagellaris was not associated with disease severity. These results suggest glyphosate may affect colonization by C. cf. flagellaris only while the pathogen is in its endophytic stage of development.

Agrobacterium tumefaciens-mediated transformation is generally used for genetic transformation of higher plants. Several experimental factors important for the increase of beta-glucuronidase (GUS) reporter gene activity were evaluated in this study using leaf disks of tobacco (Nicotiana tabacum L. cv. Xanthi). We found that co-cultivation temperature at 20C is the most critical factor to obtain the reproducible enhancement of GUS activity. pCAMBIA 1305.01 resulted in higher GUS activity than the other two pCAMBIA vectors 1301 and 1305.02. The highest GUS activity and transformation efficiency were achieved under the following experimental conditions: Agrobacterium tumefaciens strain LBA4404 containing pCAMBIA1305.01 was grown overnight at 28oC in liquid Agrobacterium media, and the concentration was adjusted to 3x107 cells/mL (0.3 A600 units/mL). Tobacco leaf disks were inoculated with bacteria under 50 mm Hg vacuum infiltration for 20 min in the presence of 0.001% (w/v) Silwet L-77. Leaf disks were co-cultivated for four days under constant light at 20C in MS shoot media containing 200 uM acetosyringone without antibiotics. Leaf disks were then transferred to MS shoot selection media containing 50 mg/L hygromycin and 500 mg/L carbenicillin, and grown for an additional 14 days under constant light at 25C. Beta-Glucuronidase (GUS) activity was measured at the end of the growth period by quantitative GUS assay and GUS histochemical staining.

Sweetpotato production in the southern United States is being threatened by a soft rot that develops in storage roots when fields are flooded and by an important post-harvest disease caused mainly by the fungi Fusarium solani and Macrophomina phaseolina. To identify the pathogens responsible for development of the soft rot, samples were collected from storage roots with soft rot from intentionally flooded fields and decayed tissue was streaked on plates of nutrient dextrose agar plus 0.05% cysteine and incubated anaerobically. Two distinct groups of Gram positive strict anaerobic bacteria were re-isolated from rotting storage roots. Endospores were observed in all isolates by differential staining. Genomic DNA was extracted from representative isolates of each group, LSU-B1 and LSU-B7, and the 16s ribosomal RNA region was amplified and sequenced. BLASTn analysis of the 1425 bp sequence of LSU-B1 resulted in 99% homology with Clostridium puniceum strain BL 70/20 from rotting Irish potatoes. Isolate LSU-B7 generated a sequence 1376 bp long, which resulted in 99% homology with C. saccharobutylicum strain P262. To determine how and when end rot pathogens enter sweetpotato storage roots, two greenhouse experiments were designed using tissue culture-derived plants free of F. solani and M. phaseolina. In one experiment, plants were grown in autoclaved soil and one month after transplanting, plants were inoculated at the soil line with either non-infested toothpicks or with toothpicks infested with each fungus alone or combined. In the other experiment, plants were grown in non-infested soil or in soil infested with each fungus alone or combined. Isolations were attempted from different parts of the plants. F. solani and M. phaseolina were recovered from roots, storage roots, and plant stems below the soil line, at the soil line, and five centimeters above the soil line in both experiments. This suggests that these fungi are capable of invading the sweetpotato plants and storage roots from infested soil, and systemically colonize the plant from infected plant propagation material, eventually reaching the storage roots. These findings indicate that infection with F. solani and M. phaseolina can occur prior to harvest adding crucial information to end rot disease control.

There are more than 7800 species of Pucciniales (rust fungi) described. Aeciospores and urediniospores of rust fungi are a food source for the larval stage of members of the fly genus Mycodiplosis, hence these could be of interest as potential biological control agents. Currently, Mycodiplosis contains 49 described species based on adult male morphology. A survey of 1,350 rust-infected plants from 44 countries was recently conducted to assess the occurrence of Mycodiplosis fly larvae across a broad spectrum of Pucciniales. Larvae were found on 261 collections from 25 countries. Statistical analyses explored the distribution of larvae in relation to host species. Five of 127 rust species in the survey data were identified as infested at a greater frequency than expected using binomial probability analysis of presence/absence. DNA was extracted from individual larvae and 28S nuclear ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 (COI) genes were amplified and sequences were concatenated for maximum likelihood analyses. Test analyses were done using identical sequence regions of 28S and COI from Bradysia species and Asteromyia species respectively, to verify the ability of individual loci to resolve species. The 206 larval specimens analyzed were resolved into approximately 33 clades, 17 of which received significant support. One clade has a global distribution in the survey. Twelve clades occurred in the United States. There are currently seven species described from the United States, the finding of 12 clades in this study represents potential additional species in the Mycodiplosis. The distribution of larvae within clades could be explained partially by geographic origin but not by rust host at any taxonomic level; thus even though there is evidence for preferential feeding by larvae on some rust species, there is no evidence of host-specificity between clades of larvae and their hosts.

Greenhouse and microplot studies were conducted to evaluate the influence of soil texture on reproduction and pathogenicity of Rotylenchulus reniformis (reniform nematode) on cotton. A 45 day duration greenhouse experiment confirmed the pathogenicity of an isolate of R. reniformis from Avoyelles Parish on Stoneville LA887 cotton. A series of greenhouse experiments were conducted with three geographic isolates of R. reniformis (identified as Avoyelles, Evangeline, and Rapides to indicate the Parish of origin) on Stoneville LA887, Stoneville 5288B2F, and Phytogen 375WF cotton growing in soils with varying textures for 60 days. Soil types with sand, silt, and clay contents ranging from 74.4 to 7.8, 20.7 to 66.3, and 4.9 to 25.9, respectively, were employed in this research. Two experiments were conducted with Stoneville LA887 cotton, three soil types and Avoyelles isolate of reniform nematode for 150-152 days in a microplot environment. In the greenhouse, variations in soil texture significantly affected plant height and dry weights in both Stoneville 5288B2F and Phytogen 375WF, but did not have any significant effect on plant growth of Stoneville LA887, except in the 45-day duration experiment. Stoneville 5288B2F plants were significantly taller throughout the experiment in soil with 31.4% sand and 13.3% clay. Phytogen 375WF cultivar showed the same pattern, but the difference in plant height was not observed at harvest. Stoneville 5288B2F and Phytogen 375WF had significantly reduced dry root and shoot weights in sandier soils. Soil type had a significant effect on nematode reproduction on all three cotton cultivars. The interaction between soil type and reniform isolate significantly affected population densities of all reniform isolates tested among the multiple soil types in the cultivars Stoneville 5288B2F and Phytogen 375WF, but no effect was observed in Stoneville LA887 cultivar. In the microplot, plants growing in soil with more clay content had significantly greater root and shoot weights than the others. The number of bolls open and seed cotton weight were significantly reduced by R. reniformis in the microplots. Population densities of R. reniformis in the microplot followed the same pattern observed in greenhouse experiments.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice, which is becoming a major threat to global rice production. The genome of a highly virulent B. glumae strain, 336gr-1 that was isolated from rice in Louisiana, was sequenced to better understand the genome-scale characteristics, particularly that of its pathogenicity. Comparative genomic analyses with another strain, BGR1 that was isolated from Korea, revealed several unique regions present in the genomes of these two geographically separated phytopathogenic bacteria. Genome plasticity, primarily caused by a horizontal gene transfer, was observed in these closely related strains of Burkholderia that are capable of infecting the same host plant. The highly conserved nature of chromosome 2, along with the presence of important virulence determinant gene coding regions in it, such as those involved in type III secretion and toxoflavin production, indicates its importance in pathogenesis. The presence of multiple genomic islands, detectable pseudo genes, insertions, deletions, and paralogous genes indicates recent adaptation to diverse ecological niches and reduced selection pressures in specific regions of the B. glumae genome. These findings would help explain the genotype and host range diversity of B. glumae and augment characterization of its ecology and pathogenesis. Characterization of ECF σ70 gene and σ54 dependent response regulator gene in B. glumae has revealed that they are not directly involved in pathogenicity in this phytopathogenic bacterium. Global transcriptome analysis of B. glumae strain 336gr-1 has revealed that the expression of 87 genes is influenced by the quorum sensing genes tofI/tofR and their intergenic region, orf1. Especially, the genes for the type II secretion system and diguanylatecyclase activity, which play important roles in the pathogenesis of B. glumae, were found to be regulated by quorum sensing mechanism.

Sweetpotato potyviruses [Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2)] commonly infect sweetpotato and weedy morning glories in the USA. These viruses are transmitted in a non-persistent manner by various aphid species and cause up to 15% yield loss. Sweetpotato is vegetatively propagated, and in the USA growers are supplied with virus tested propagation material to minimize impact of viruses. However the rapid re-infection of these materials with viruses warranted further studies to determine factors that influence the epidemiology of these viruses. The objectives of this study were: (i) to determine if differences in acquisition hosts, aphid species and infection status influenced transmission of SPFMV; (ii) to determine how aphid abundance, aphid species diversity and virus titers relate to the spread of potyviruses in Louisiana sweetpotato fields; (iii) to determine the effects of virus infection on the population dynamics of aphids on sweetpotato and morning glories; and (iv) to determine the effects of virus infection on stylet penetration behaviors of aphids. SPFMV was transmitted at a greater rate from morning glories which also had greater virus titers compared with sweetpotato and from mixed infection sources than from singly infected sources, and Aphis gossypii was the most efficient vector. Aphids were captured in fields during the entire crop cycle, and A. gossypii and Rhopalosiphum padi, were the most abundant species occurring throughout the growing season. Virus infection of sentinel plants occurred mainly during the months of June to August when virus titers were high in sweetpotato plants. SPFMV was more commonly detected than SPVG or SPV2 in sentinel plants. Myzus persicae had a significantly greater reproduction on sweetpotato cvs. Beauregard and Evangeline with mixed virus infection compared with non-infected plants. Stylet penetration behaviors were variable depending on host and virus infection status. Differences in virus transmission rates depending on host plant, aphid species, virus species and virus titers, and pattern of spread in sweetpotato fields suggest the dissemination of sweetpotato potyviruses is influenced by the source of inoculum, the quantity of inoculum, virus species and aphid species vectors.