Leaf scald is an important disease of sugarcane caused by Xanthomonas albilineans (Xa). Leaf scald is controlled by the development and planting of resistant cultivars. However, erratic symptom expression makes conventional screening for resistance difficult. In addition, the mechanisms of resistance to leaf scald are not completely understood. Real-time, quantitative polymerase-chain-reaction (qPCR) assays were developed utilizing SYBR Green for a highly sensitive detection method or a TaqMan probe to quantify Xa populations in infected plants. Xa populations detected by qPCR followed similar trends to disease severity ratings and vascular infection results for two resistant and two susceptible cultivars under greenhouse and field conditions. Low bacterial populations were found in newly emerged, systemically infected leaves of resistant cultivars. The results suggest that Xa population quantification by qPCR has the potential to be used as an alternative method for leaf scald resistance screening. A comparative proteomic analysis identified differentially expressed proteins that suggested mechanisms for the sugarcane resistance response to Xa infection. Protein expression was compared for inoculated and mock-inoculated plants of two resistant and two susceptible cultivars during a time-course encompassing the responses to initial and systemic infection. Differential expression also was compared across cultivars with and without Xa infection. The number of up- and down-regulated proteins increased in the resistant cultivars during systemic infection. Identified, differentially expressed proteins were mostly in the chloroplast (67%), and 48% were involved in photosynthesis. Identified proteins were homologous to cyclophilin, translationally controlled tumor protein (TCTP), thylakoid ascorbate peroxidase (tAPOD), germin-like protein (GLP), and thioredoxins. All are proteins that have been associated with induced defense responses. Down-regulation of APOD and the thioredoxins and up-regulation of GLP could result in accumulation of reactive oxygen species, particularly H2O2, in the cytoplasm and the apoplast. In addition, proteins involved in ethylene biosynthesis, another key signaling molecule in induced systemic resistance (ISR), were differentially expressed in resistant cultivars. Differences found between the two resistant cultivars indicate that resistance mechanisms can vary between genotypes, but the suggested resistance mechanisms for both were inducible responses. Finally, some differentially expressed proteins were involved in primary metabolism that could represent a plant fitness mechanism to provide energy needed for ISR. The differences in Xa populations detected by qPCR and comparative proteomic analysis both suggest the existence of an induced mechanism of resistance against Xa during systemic infection. Multiple lines of evidence from the proteomic analysis suggest a triggering of ISR that would result in the limited Xa colonization of the new xylem developing above the apical meristem and lack of symptom development that is evident in leaf scald resistant sugarcane genotypes.

Orthodox and recalcitrant seeds exhibit differential tolerance to water loss. Recalcitrant seeds are not able to tolerate desiccation and die when dried, while the orthodox seeds can be stored dry without losing viability for years. Spartina is a good model to study recalcitrance, because unlike most other recalcitrance studies, which contain only a recalcitrant species, this system has both recalcitrant S. alterniflora and orthodox species, S. pectinata and S. spartinae, as close-related physiological comparators. Lack of protective proteins, e.g. late embryogenesis abundant proteins (LEAs), has been proposed to be the cause of recalcitrant seed death. A common feature of these protective proteins is their heat stability. In order to identify any heat-stable proteins that may be associated with a lack of desiccation tolerance in S. alterniflora, it is necessary to optimize the protocol of heat-stable protein extraction first. Heating the protein extracts at 95C for 40 minutes and centrifuging the heated protein extracts at 20,000g and 4C for 40 minutes yield a constant protein concentration of heat-stable fractions both in the S. alterniflora and S. pectinata. Comparisons of one-dimensional SDS-PAGE gels or total protein concentration provide little information about the minimum amount of protease inhibitor needed to stop the protease activity in Spartina seed protein extracts. Results of the Protease Determine Quick testTM protease assay indicated that 50 ul of protease inhibitor were sufficient to totally quench the protease activity in protein extracts in both S. pectinata and S. alterniflora. To investigate an association between heat-stable fraction percentage and desiccation tolerance, heat-stable fractions of S. alterniflora and S. pectinata were compared. There was no association between heat-stable fraction percentage and desiccation tolerance in Spartina. Comparative 1-DE profiles between dry S. alterniflora and dry S. pectinata did not reveal any differences. Therefore, two-dimensional gel electrophoresis, which has a much higher capability to resolve proteins, was used to investigate the differences in protein patterns between recalcitrant S. alterniflora and orthodox S. pectinata.

Burkholderia glumae is the major causal agent of an economically important rice disease, bacterial panicle blight (BPB). The known virulence factors of B. glumae share the TofI/TofR quorum sensing system as their regulator. tofI and tofR genes encode the N-acyl homoserine lactone (AHL) synthase for the B. glumae quorum sensing signals, N-octanoyl homoserine lactone (C8-HSL) and N-hexanoyl homoserine lactone (C6-HSL), and the receptor for AHL, respectively. To better understand the relationship between quorum sensing and known virulence factors (toxoflavin, flagella and lipase), as well as, putative virulence factors (i.e. extracellular polysaccharide), mutagenetic and phenotypic analyses were applied to this study. A technical breakthrough is the creation of a novel deletion mutation system-pBBSacB vector, which can effectively delete target genes from the genome and gives more reliable results. Quorum sensing gene deletion mutants were successfully created by using pBBSacB with a sucrose-sensitive counter selective marker, SacB. The parental strain 336gr-1 and its mutants have undergone a series of phenotypic observations and quantification tests for virulence changes. Toxoflavin and swarming motility were confirmed as the major virulence factors in 336gr-1, whereas lipase and EPS were not determined as critical for causing symptoms. The results confirmed the importance of quorum sensing system in expressing virulence, but also indicated that other regulators may be implicated in pathogenicity. Additionally, orf1, which is located between tofI and tofR, was postulated as a functional regulatory component.

Phakopsora pachyrhizi, the causal agent of soybean rust (SBR), is a serious disease on soybeans.The objectives of this project were to identify additional alternative and possible overwintering hosts of the SBR pathogen and to validate the current detection assays for SBR. For the first objective, we attempted to identify naturalized Louisiana legume(s) that can serve as hosts and overwintering sites for P. pachyrhizi. It was theorized that New Iberia and Iberville Parishes in south Louisiana are usually the first locations of soybean infection in the spring. This suggests that there may be alternative leguminous hosts located in these parishes that may serve as overwintering hosts for P. pachyrhizi. Over 100 species of naturalized legumes were tested in field experiments to identify susceptible alternative hosts of the pathogen from November to May. For the second project, samples of P. pachyrhizi were collected from different parishes across Louisiana and sequenced across the ITS locus.These sequences were analyzed for variability within the locus and compared to the standard used in the current assay for pathogen detection. The Frederick et al. (2002) assay discriminates between P. pachyrhizi and P. meibomiae, a closely related mildly virulent species on soybeans in the US. A second assay, Barnes et al. (2009), was designed to detect a single spore of P. pachyrhizi in rain wash material, to monitor spore deposition and predict the movement of SBR in major soybean-producing states. However, the Barnes et al. (2009) assay has been shown to produce false positive and false negative results when used to detect the presence of P. pachyrhizi in North America. We tested five other Phakopsora spp. by both assays and found that only the Barnes et al. (2009) assay was able to detect all other Phakopsora spp. that were tested. Furthermore, the DNA segments used in the Barnes et al. (2009) assay, the primers had base pairs similarities that were 100% with other Phakopsora spp. and more than 60% with the specific P. pachyrhizi probe. In addition, we had determine that neither assay was placed in variable regions of the ITS locus.

Cercospora leaf blight (CLB) and purple seed stain (PSS) are common soybean diseases in the Gulf South of the United States (USA). For nearly a century, <i>Cercospora kikuchii</i> has been considered as the only pathogen causing these diseases. However, previous reports of genetic diversity among isolates collected throughout Louisiana suggested the presence of multiple lineages or species. Recent systematic studies classified species of <i>Cercospora</i> using a taxonomic system based on phylogenetic analysis of five nuclear loci (legacy genes). Using a similar approach, cercosporoid fungi tentatively identified as <i>C. kikuchii</i> were evaluated along with 53 other species of <i>Cercospora</i>. No isolates from this study were nested within the clade including the ex-type strain of <i>C. kikuchii</i>. Five isolates grouped with <i>C.</i> cf. <i>sigesbeckiae</i> and all others were part of <i>C.</i> cf. <i>flagellaris</i>. Several isolates of <i>C.</i> cf. <i>flagellaris</i> were also obtained from <i>Gossypyium hirsutum</i> and <i>Phytolacca americana</i>. These results suggest that <i>C. kikuchii</i> is not the organism responsible for causing CLB or PSS in the Gulf South and other areas of the USA. Multiple haplotypes were observed at each locus and individual genes varied in their resolving power. Most species were monophyletic in concatenated analyses, but reciprocal monophyly was generally not observed within individual gene trees. Furthermore, node support values were generally low across all topologies, indicating that the phylogenetic markers most commonly used for systematic studies of <i>Cercospora</i> are limited to answering shallow-level taxonomic questions. However, existing genome sequence data provided an excellent opportunity to develop new markers with stronger phylogenetic signal to better understand the evolutionary history of <i>Cercospora</i>. Sixty-three exon-flanked intergenic regions, syntenic between the genomes of <i>C.</i> cf <i>sigesbeckiae</i> and <i>C. canescens</i>, were extracted and aligned, then ranked and filtered according to several metrics to assess their phylogenetic utility. Candidate markers were validated by PCR on 24 <i>Cercospora</i> species, including 16 type strains. Assessment of phylogenetic informativeness profiles and phylogenetic analyses showed that all of the new markers provide greater interspecific resolution than the legacy genes and offer new options for identifying cryptic species in complex clades like <i>C.</i> cf. <i>flagellaris</i>.

Among the variety of pathogens of cotton (Gossypium hirsutum), nematodes play a major role in reducing yield. Across the U.S. cotton belt, millions of dollars are lost annually due to nematode infestation. In the Mid-South and Southeast United States, root-knot nematode (Meloidogyne incognita) and reniform nematode (Rotylenchulus reniformis) are responsible for the highest percentage of damage. Crop rotation and nematicides are currently the most commonly used management strategies for nematode management. Soil fertility, which has a direct effect on plant growth, is also known to influence disease severity. Therefore, soil fertility would be an additional factor to consider for management of nematodes. The objectives of these studies were to evaluate the effects of soil nutrients on reniform nematode reproduction and pathogenicity on cotton. Four 60-day-duration greenhouse studies were conducted to evaluate the effect of different soil nutrients on reniform nematode pathogenicity and reproduction. Nutrients used in greenhouse studies were phosphorus (P), potassium (K) and sulfur (S). For the first study, P and K were used in high (112 kg ha-1) and low (0 kg ha-1) levels with a soil mixture of 70.1% sand, 25.4% silt, and 2.5% clay. Treatments for the second, third and fourth studies were five increasing levels of P (10, 20, 35, 60, and 73 mg kg-1), K (44, 70, 106, 123, and 153 mg kg-1), and S (3, 12, 20, 40, and 50 mg kg-1 ) mixed with soil comprised of 68% sand, 30% silt and 2% clay. Application of P produced a significant increase in plant shoot and root dry weights in studies one and two. Similarly, reproduction of reniform nematodes in these two studies were significantly influenced by levels of P. Studies three and four focused on K and S and did not show any effect on reproduction of reniform nematodes. Treatment with S had a significant negative influence on shoot height and dry weights. Under field conditions, nematicide application significantly reduced nematode population density at mid-season and at harvest in 2011 and at planting in 2012. In both 2011 and 2012, management of soil nutrients did not significantly influence nematode reproduction. In both years, seed cotton yield was significantly increased with nematicide, but not with nutrients.

Quorum sensing (QS) is a cell-to-cell communication mechanism that allows bacterial cells to collectively behave like a multicellular organism. It regulates the expression of toxoflavin, one of the major virulence factors of the rice pathogen, Burkholderia glumae. The QS system of B. glumae is mediated by the core genes, tofI and tofR. N-octanoyl-L-homoserine lactone, the primary QS signal molecule of B. glumae, is synthesized by tofI and binds to the cognate receptor tofR at the quorum point. However, tofI and tofR null mutants produce toxoflavin in certain growth conditions, indicating the presence of tofI- and tofR-independent pathways for toxoflavin production. The present study identified regulators required for the tofI- and tofR-independent pathways, including flagella transcriptional activator, diguanylate cyclase, O-antigen polymerase family protein, QsmR QS-dependent master regulator and one hypothetical protein with its encoding gene located upstream of toxJ (encoding toxoflavin production activator). A novel QS regulatory element, tofM, was identified as a positive regulator of pathogenicity and a putative modulator of tofR in B. glumae. RNA-sequencing was also performed to investigate the QS regulon and medium condition-dependent gene expression in B. glumae. A large collection of target genes and noncoding RNAs was detected by comparative transcriptome analysis. From a comparison of the transcriptional profile of the wild type (336gr-1) and quorum sensing mutants grown on solid and liquid media, it is postulated that an alternative global regulator is activated to compensate for the dysfunction of AHL QS on solid medium.

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), has the potential to cause severe yield losses as all United States commercial soybean varieties are susceptible. In this study, 10 soybean recombinant inbred line (RIL) derived sibling lines of two populations (RN06-32-2 and RN06-16-1) were evaluated for differences in response to infection by P. pachyrhizi. These lines, which had previously shown differential responses to Florida soybean rust isolates, were evaluated using Louisiana soybean rust isolates under both detached leaf assay and greenhouse in planta inoculation conditions. Sibling lines showed significant differences in response to P. pachyrhizi infection under both conditions. Lines 8-a, 8-b, 94-c of population RN06-32-2 and lines 15-b and 16-c of population RN06-16-1 showed a resistant response against Louisiana rust isolates in comparison with the immune response against Florida rust isolates. Whereas, lines 15-c and 16-b of population RN06-16-1 and lines 8-c, 94-a, and 94-b showed similar responses against Louisiana rust isolates as that of Florida rust isolates. Lines 15-c and 16-b showed moderately resistant response; lines 8-c, 94-a, and 94-b showed susceptible and resistant response, respectively. To understand the compatible and incompatible host-pathogen interactions at the molecular level, we conducted a time-course study (0 h, 10 h, 1 d, 2 d, 3 d, 4 d, 5 d, 8 d, 10 d, 12 d and 14 d) of P. pachyrhizi infection and compared protein profiles of 8-a (resistant) and 8-c (susceptible) lines in response to ASR inoculation, using DIGE proteomics. Based on the gel analysis, we observed approximately 100 differentially expressed spots between 8-a and 8-c lines. Among these, 37 proteins were identified using mass spectrometry. Most of the identified proteins are involved in photosynthesis and carbon metabolism, defense mechanism, seed storage and include some uncharacterized proteins.

Soybean (Glycine max) is one of the most widely grown crops in the world. Many pathogens attack soybeans, but of particular importance to tropical and subtropical areas such as Louisiana is Cercospora leaf blight (CLB). This disease is caused by the fungus Cercospora kikuchii and favored by high temperatures. This fungus utilizes a toxin, cercosporin, as its primary pathogenicity factor. Soybean rust (SBR), caused by Phakopsora pachyrhizi, is another common disease that occurs in Louisiana under cooler environmental conditions of spring and fall. Both diseases occur regularly in Louisiana and may result in severe yield losses. Cercospora kikuchii has recently developed fungicide resistance, and there are few if any resistant soybean cultivars. For these reasons, a search for new management strategies is underway. This study analyzed the efficacy of using plant-essential micronutrients applied foliarly to suppress CLB and SBR. This was tested in field experiments on these diseases as they occurred, as well as on isolates of C. kikuchii on amended agar media; both in randomized complete block design. Disease severity and yield were measured in field experiments, while fungal growth and toxin production were measured in vitro for C. kikuchii. Results showed that copper (150 300 g ha-1) and high rates of aluminum (160 190 g ha-1) and iron (200 400 g ha-1) applied foliarly to soybeans were effective in suppressing CLB severity. Iron; in the forms of iron nitrate and iron sulfate, as well as low rates of aluminum (20 100 g ha-1) were effective in reducing SBR severity. Agar media amended with iron (> 250 mg L-1), manganese (> 250 mg L-1) and zinc (> 125 mg L-1) inhibited fungal growth at high physiological concentrations found in soybean leaf tissue. Additions of aluminum (up to 150 mg L-1) and copper (up to 35 mg L-1) resulted in increased fungal growth. Aluminum, manganese, and zinc treatments caused higher toxin production by C. kikuchii than unamended controls. Iron at lower concentrations (up to 80 mg L-1) resulted in higher fungal growth rates than unamended controls, but this effect was reversed as concentrations exceeded this value. Conversely, toxin production was enhanced as fungal growth began to decline up to the point of complete fungal inhibition. Finally, some metals applied in vitro affected pigmentation of C. kikuchii. The normally purple pigmentation of cercosporin shifted to yellow when grown on aluminum, black when grown on iron, and green when grown on zinc-amended agar media. Boron (50 g ha-1) and iron (50 g ha-1) applied as iron sulfate were effective at reducing frogeye leaf spot (Cercospora sojina) in the one year the disease occurred. While many treatments were effective in suppressing disease severity in the field and reduced fungal growth in culture, there were no significant effects on soybean yield by treatment for either CLB or SBR. Future work identifying the mechanisms of disease suppression as well as more pronounced effects from commercial formulations and technologies surrounding minor element nutrition could yield sustainable strategies for managing soybean diseases. Research is planned on the siderophore-like properties of the toxin, cercosporin.

Bacterial panicle blight is among the three most limiting diseases of rice production in the United States. Yield loss up to 40% has been reported from rice fields severely infested with bacterial panicle blight. The disease is caused by Burkholderia glumae and B. gladioli. Although, both species produce similar symptoms and are closely related, but B. glumae strains are more aggressive and cause more severe symptoms on rice. Bacterial panicle bight is difficult to manage in the absence of effective chemical control measures. Rapid and early disease detection is needed to avoid severe yield losses. Several techniques have been developed for bacterial identification, but these methods are time consuming and some require high-precision instruments for amplification and analysis of target DNA. We developed a Loop-Mediated Isothermal Amplification (LAMP) protocol for rapid detection of B. glumae using a set of six primers from the gyrB housekeeping gene. Several commercially available dyes including, PicoGreen, Hydroxynaphthol Blue (HNB) and Calcein were compared to analyze the LAMP product. The LAMP detection method resulted in rapid and accurate detection of B. glumae. Among the different detection dyes, PicoGreen and HNB produced reliable results in the detection of B. glumae. Although, both produced accurate results; however, HNB is more cost-effective.