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.

Leaf scald, caused by Xanthomonas albilineans, is a major sugarcane disease worldwide that is controlled primarily with host plant resistance. Since visual evaluation of disease resistance can be uncertain due to erratic symptom expression, a more reliable screening method is needed for resistance research. A quantitative polymerase chain reaction (qPCR) assay was developed previously with demonstrated potential for resistance screening. However, only four cultivars with extreme reaction against the disease (two highly susceptible and two highly resistant) were compared. Therefore, additional research was conducted to demonstrate assay specificity, compare bacterial populations in 31 clones with varying levels of susceptibility at different times after inoculation, and evaluate the correlation with the visual rating method currently used for resistance evaluation. SYBR Green and TaqMan qPCRs were tested against DNA from different bacteria and a fungus, and the assays showed high specificity amplifying only X. albilineans isolates. Inhibitors of amplification during PCR were not detected in DNA extracts from four cultivars. Comparison between the bacterial population quantified by qPCR and visual symptom severity ratings showed variable results with the highest correlation between the data at 8 weeks after inoculation (ñ = 0.62, P < 0.0001). In order to measure consistency, the correlation was determined among three different inoculations for data obtained with the same method at different times after inoculation. The qPCR assay was more consistent among the different inoculations (r = 0.77, P < 0.0001) compared with the visual rating system (r = 0.53, P = 0.0026) at 8 weeks after inoculation. Bacterial quantification was more consistent in field inoculations compared to greenhouse inoculation. The high specificity and consistency suggest that qPCR can provide an improved method to evaluate resistance to leaf scald in sugarcane.

Plant viruses have shown to be the cause of the yellow vein symptoms on the foliage of several ornamental plants. In this study five ornamental plants: oxalis (Oxalis debilis var. corymbosa) cv. Golden Veined Oxalis, geranium (Pelargonium peltatum) cv. Crocodile, pseuderanthemum (Pseuderanthemum carruthersii) cv. Golden Net Bush (narrow leaf and broad leaf variants), honeysuckle (Lonicera japonica) cv. Yellow Net Honeysuckle and coleus (Solenostemon scutellarioides) cv. Electric Lime, showing foliar yellow vein were used. Graft transmissions indicated that, except for coleus, the foliar yellow vein of all other plants was caused by infectious agents, likely plant viruses. With the exception of Golden Veined Oxalis, attempts to identify a virus as the causal agent of the foliar yellow vein in geranium, pseuderanthemum, and honeysuckle failed. Successful whitefly (Bemisia tabaci biotype B) transmission of the foliar yellow vein of Golden Veined Oxalis suggested that a begomovirus was the causal agent. Therefore, PCR amplifications were conducted using degenerate primers for the genus Begomovirus. A 1.2 kb PCR product was obtained using the primer set PAL1v1978/ PAR1c496 and a 0.7 kb product with the primer set AV494/AC1048. These sets of primers have been used widely to detect and identify begomoviruses. Sequencing of the fragments confirmed the begomovirus nature of the amplified DNA from Golden Veined Oxalis. Sequence information and phylogenetic analyses revealed that the Golden Veined Oxalis begomovirus was closely related to Tomato yellow spot virus and Sida mottle virus, two begomoviruses reported to infect solanaceous and legume crops. The host range of the virus isolated from Golden Veined Oxalis begomovirus, designated Oxalis yellow vein virus, is not known. This finding illustrates the potential spread of plant viruses to different geographical areas through the commercialization of virus- infected ornamental plants.

In 2011 and 2012, 100 residential lawns in the Baton Rouge, LA area were sampled to document the incidence of plant-parasitic nematodes, in addition four full-season microplot experiments and four 71-day duration greenhouse experiments were conducted to evaluate their pathogenicity on St. Augustine and centipede turfgrasses. Nematode genera associated with both turfgrasses included Criconemella, Helicotylenchus, Meloidogyne, Pratylenchus, Tylenchorynchus and Tylenchus spp. In microplot trials in 2012, nematodes did not cause significant damage to either turfgrass, but soil exhibited an effect on plant growth parameters. In 2013, when there was significant nematode related injury to both turfgrasses, there were no significant effects of soil on plant growth parameters. Greenhouse based pathogenicity trials were conducted separately with Meloidogyne incognita and Pratylenchus zeae. Across two levels of nematode infestation, reductions below controls for St. Augustine and centipede averaged 24% and 28% for M. incognita and 37.0% and 39.3% for P. zeae; indicating that overall, P. zeae was more damaging than M. incognita to both turfgrasses.

Cercospora leaf blight of soybean (Glycine max) is a serious problem in the mid-south of the United States and is present in most soybean-growing regions of the world. The causal organisms, Cercospora kikuchii, C. cf. flagellaris and C. cf. sigesbeckiae, utilize the photo-activated toxin cercosporin as the primary pathogenicity factor. The disease has historically been understood to progress from a purpling or bronzing of the leaves to a blight in which tissue dies. Previous literature showed possible binding of cercosporin to metals and previous work in our group showed possible use of minor element nutrition in the plant to reduce disease severity. Through the use of UV-vis spectrometry, nuclear magnetic resonance spectroscopy, and mass spectrometry evidence is given in support of cercosporin-metal binding. From this work, it is hypothesized that the metals bind at the phenol group of the cercosporin molecule, yielding a dark purple compound. Cercosporin appears to have higher affinity for iron, especially the Fe(III) form, over the other plant minor elements but appears to have some minor binding affinity for aluminum, copper, manganese, and zinc. Cercosporin and iron combinations show greatly reduced toxicity compared to cercosporin alone, in support of previous reports in the literature. Manganese added to cercosporin resulted in more necrosis of the soybean tissue. Soybean plants fertilized with iron sufficient to increase tissue concentrations by 20 mg/kg dry weight resulted in a significantly higher resistance to the toxin. The iron fertilization also resulted in a roughly 60 mg/kg dry weight reduction in manganese, which may also help account for the reduction in toxicity. The symptoms also were less similar to the typical blight symptoms and more similar to the purple leaf symptoms. Isolates of C. cf. flagellaris grown on split chrome azurol sulfate (CAS) plates elicited the signature color shift indicative of siderophore production. Cultures of C. cf. flagellaris also were grown in liquid minimal medium amended with cercosporin, iron, or both. In broths containing both cercosporin and iron, fungal growth was higher, total cercosporin production was lower, and iron content of the mycelium was significantly higher than that of controls. This demonstrates that the fungus is capable of up-taking and incorporating iron in this form. When only cercosporin was provided, mycelial concentrations of aluminum, copper, and boron were higher than other treatments. This indicates that cercosporin may be involved in other metal uptake and these metals may compete with iron. While previous work indicated cercosporins ability to bind iron, none gave conclusive evidence, analyzed the mechanisms, or investigated the purpose. Findings presented herein suggest that cercosporin is capable of acting as a siderophore; serving both as the primary pathogenicity factor and as an iron acquisition mechanism. Furthermore, the role of iron in disease development and symptom progression should be re-evaluated along with its effects on yield. The symptoms of purpling and blight appear to be correlated with the amount of iron in soybean tissue. Further work should investigate ways to practically manipulate these results to lower damage caused by these pathogens.

To study the effect of Silicon (Si) with and without a fungicide on anthracnose development of sorghum, several experiments were conducted in the greenhouse and field. In the initial study, different Si rates [0 (control), 0 (lime control), 200, 400, 600, 800 kg Si/ha] were used in a low-Si Alfisol to determine if inoculum densities were affected by Si levels. No differences were observed between inoculum densities of 1*105 and 1*106 conidia/ml in affecting anthracnose development in the greenhouse. Anthracnose severity was found to be lowest in plants treated with 800 kg Si/ha, regardless of inoculum density. In another study, the effect of Si on moderately susceptible (Pioneer 84G62) and moderately resistant (Pioneer 84P80) hybrids was also examined with lower inoculum concentration under greenhouse conditions. Fungicide (Pyraclostrobin) was also included to suppress the anthracnose development. Silicon had a significant effect on plant Si concentration and anthracnose development. Anthracnose severity was reduced as plant and soil Si levels increased. The highest Si application rate (800 kg Si/ha) reduced Final Disease Severity (FDS) and Area Under Anthracnose Progress Curve (AUAPC) by 18 and 36% as compared to the control for the first greenhouse experiment (p<0.05). Likewise, it reduced FDS and AUAPC of the 2nd greenhouse experiment by 76 and 67%, respectively (p<0.001). Pyraclostrobin effectively reduced AUAPC by 50 and 36%, respectively, for the two greenhouse experiments. Similar Si + pyraclostrobin experiments were conducted under field conditions at Dean Lee (Inceptisols) and Winnsboro (Alfisols), Louisiana with higher soil Si levels (120 µg/g and 40 µg/g respectively). Even though soil Si increased with higher Si rates for both fields, no significant increase in Si accumulation in sorghum leaves or grains was observed. At Dean Lee, pyraclostrobin reduced AUAPC by 44 and 39% and FDS by 50 and 48%; respectively, for Pioneer 84G62 and Pioneer 84P80 (p<0.001). However, pyraclostrobin had no effect in reducing anthracnose at Winnsboro. Yield was higher for Pioneer 84G62 than Pioneer 84P80 at Dean Lee. Silicon had a greater impact in suppressing anthracnose development on low-Si soils under greenhouse conditions than high-Si soils of the field.