Development of resistant cultivars is the main control measure against sugarcane brown rust caused by Puccinia melanocephala. Durability is uncertain, since the pathogen possesses adaptive ability to overcome host plant resistance. A differential gene expression study utilizing suppressive subtraction hybridization was conducted to improve understanding of brown rust resistance mechanisms in sugarcane. The expression patterns of 11 unigenes representing biosynthetic pathways, defense-related genes, and signaling genes were analyzed in L 99-233, a cultivar exhibiting quantitative resistance, L 01-299, a resistant cultivar with the major resistance gene Bru1, and two susceptible cultivars, Ho 95-988 and L 09-125, at 24 h, 48 h, 72 h, and 1 week after inoculation with P. melanocephala using (semi)quantitative RT-PCR. All genes analyzed for their expression showed message accumulation upon infection in susceptible and resistant cultivars, but the maintenance of high amounts of mRNAs of the genes for a prolonged time period appeared to be the most important factor contributing to brown rust resistance. Differences in the time-course of gene expression were detected between L 01-299 and L 99-233 suggesting variable mechanisms for resistance between the cultivars. Molecular markers were used to screen the World Collection of Sugarcane and Related Grasses (WCSRG) for Bru1 to determine its distribution and frequency in Saccharum species and related genera. A total of 1,282 clones were screened. Bru1 was distributed across the Saccharum complex, but the frequency varied among species. Bru1was more prevalent in S. robustum clones (59.1%), whereas it occurred in low frequency and exhibited the highest level of variability in clones of S. spontaneum (18.8%). Bru1 frequency was highest in the two secondary cultivated species, S. barberi (79.3%) and S. sinense (71.8%). The frequency of Bru1 detection was 26.4% and 21.0% for S. officinarum and interspecific hybrid clones, respectively. The characterization of the WCSRG for Bru1 distribution and prevalence will complement efforts to characterize diversity in the Saccharum complex for the expected expanded use of marker-assisted selection in the future. Selection for quantitative resistance in combination with Bru1 could allow breeding programs to develop sugarcane cultivars with effective and durable resistance against brown rust.

Common bean (Phaseolus vulgaris) is the most important legume for direct human consumption. Common bean originated and was domesticated in the Americas but now is grown worldwide. As in the case of other crops, common bean can be infected with acute and persistent plant viruses. A modified dsRNA extraction method was developed and used in this study. The method was fast, economic, versatile, and required relatively small amounts of desiccated plant tissue. The method was successfully used to extract dsRNAs from plants infected with RNA plant viruses and to investigate the occurrence of two endornaviruses, Phaseolus vulgaris endornavirus 1 (PvEV1) and Phaseolus vulgaris endornavirus 2 (PvEV2), in breeding lines, cultivars, landraces, and wild genotypes of common bean from the two centers of common bean domestication: Mesoamerica and the Andes. The two endornaviruses were detected in many common bean genotypes of Mesoamerican origin but rarely in genotypes of Andean origin. A comparative study of morphological and physiological characteristics between two common bean lines of the cultivar Black Turtle Soup (BTS); one infected with PvEV1 and PvEV2 (BTS+) and the other endornavirus-free (BTS-) was conducted. Morphological differences between the two lines were not observed. However, the study revealed that common bean endornaviruses may promote seed germination, pod length, and carotenoid content. Nevertheless, endornaviruses were associated with lower chlorophyll content. When interactions studies were conducted between PvEV1 and PvEV2 and three acute viruses, synergistic effects were obtained. Quantitative RT-PCR results supported a synergism between PvEV1 and Sunn-hemp mosaic virus. More research should be conducted to determine the type of symbiotic interaction that exists between common bean and endornaviruses.

Brown rust, caused by Puccinia melanocephala, is an important disease of sugarcane. Breeding for host plant resistance is the primary control measure. Screening for resistance has relied on rating the severity of symptoms caused by natural infection; however, erratic results make this method problematic. A method accomplishing both infection and disease expression under controlled conditions could avoid the problems associated with resistance evaluations under natural infection. Inoculation of seedlings was evaluated to determine whether it could provide accurate resistance ratings in cross appraisal, and inoculation under controlled conditions was evaluated for the potential to accurately determine resistance reactions in clones with known and unknown reactions in comparison to field reactions. Seedlings from crosses between parents with different levels of resistance were inoculated with urediniospores at concentrations ranging from 1 x 103 to 1 x 106 spores per ml. Disease severity was visually assessed at 1 and 2 weeks after inoculation, and resistance ratings were assigned on a modified 1 to 9 scale. Inoculum concentration strongly affected severity and the frequency of resistant progeny in crosses. Brown rust resistance is a heritable trait; however, parental reaction was not a consistent determinant of progeny distribution across resistance rating categories. These results suggest that seedling inoculation may not be suitable for the evaluation of brown rust resistance. Clones were inoculated with 1 x106 spores per ml, and severity was determined as percentage of leaf area occupied by rust lesions by image analysis. Resistance reactions could not be reliably determined for susceptible clones in single inoculations. Controlled conditions inoculation and natural infection results were not correlated. Multiple inoculations under controlled conditions accurately identified resistant and susceptible clones with severe infection resulting from any single inoculation indicating susceptibility. Therefore, controlled conditions inoculation has the potential to be useful in limited studies to characterize parents in a recurrent selection program and for basic studies of resistance to brown rust.

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.