Microplot studies were conducted to evaluate the effects of cotton (LA. 887), soybean (Pioneer 96B21), and three endemic weed species, pitted morningglory (Ipomoea lacunosa), hemp sesbania (Sesbania exaltata), and johnsongrass (Sorghum halepense), on reproduction of the reniform nematode, (Rotylenchulus reniformis). Over two microplot trials the co-culture of cotton with any of the three weeds suppressed numbers of reniform nematode juveniles in soil. When grown singly, reniform nematode reproductive values after 60 days on cotton averaged 69.0, while those for morningglory, hemp sesbania, and johnsongrass averaged 42.0, 23.5, and 18.0, respectively. Reproductive values for cotton co-cultured with morningglory averaged 38.7. Those for the cotton-hemp sesbania and cotton-johnsongrass combinations averaged 23.5 and 26.2, respectively. Reniform reproduction data for soybean cultured alone or with the three weeds in two trials showed reduced reproduction of reniform nematode only in the presence of johnsongrass. Suppression of reniform nematode reproduction likely resulted from the secretion of allelopathic compounds by weed roots and from crowding due to the increased amount of biomass present in microplots containing two plant species. Data from subsequent greenhouse experiments conducted with cotton and soybean and leachates from each of the three weed species supported the allelopathy hypothesis. Reniform reproduction on cotton and soybean plants irrigated with leachates from the roots of morningglory, hemp sesebania and johnsongrass was significantly reduced compared to soybean irrigated with water. Laboratory experiments conducted in which reniform nematode eggs were exposed to leachates from roots of morningglory, hemp sesbania and johnsongrass, nonfiltered and filtered through a .45 μm and a .80um filter unit resulted in suppression of hatch and delayed development of reniform eggs in the nonfiltered portions of both filter units and the filtered portion of the .80 μm filter.

Recalcitrant seeds, which die when desiccated, can be difficult to study because of their generally large size, high metabolism, and poor storage properties. However, recalcitrant seeds from the salt-marsh grass Spartina alterniflora are unique when compared to most other recalcitrant species because the seeds are dormant and small; Spartina pectinata and S. spartinae, which produce orthodox seeds, can be used as controls. Because of these somewhat rare characteristics, S. alterniflora is a good model system to study recalcitrance. In the present study, the following physiological parameters were examined: Cardinal temperatures for germination, a viability test to determine if seeds are dormant or dead, stratification needed to alleviate dormancy, and the effects of dry down rates on viability. For non-dormant seeds, the fastest germination rates occurred between 27-34C. For dormant seeds, viability was established by cutting the coleoptile, which caused live seeds to germinate. Dormancy was alleviated with stratification, with average times to 50% germination of 2.1 and 2.6 months when seeds were stored at 2 and 10C, respectively. Finally, S. alterniflora seeds lost viability when desiccated below 45% moisture content on a dry weight basis (DWB); however, drying rate did not influence death. To investigate the causes of recalcitrant seed death, the putative role of oxidative stress was examined by assays for lipid peroxidation, leakage of cell components, total water-soluble antioxidant capacity (TAR), protein carbonylation and DNA fragmentation as Spartina seeds were dried. While lipid peroxidation was not associated with recalcitrant seed death, artifactual damage was observed when seeds were not freeze-clamped prior to extraction. TAR decreased during initial desiccation of orthodox and recalcitrant Spartina seeds. Protein carbonyl amounts (an indicator of protein oxidation) increased as S. alterniflora and orthodox S. spartinae seeds were desiccated. However, rehydration of dry, orthodox S. pectinata, and subsequent drying, did not alter the TAR or protein carbonyls. DNA fragmentation was not evident during desiccation. These results suggest that lipid peroxidation, membrane damage and DNA fragmentation do not play a role in death due to drying. While protein oxidation and loss of antioxidant capacity changed, these are general responses to drying, rather than to recalcitrance.

Leaf curl virus diseases have been reported in sweetpotato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) which belongs to the genus Begomovirus (family Geminiviridae). In the United States, SPLCV has been found infecting an ornamental sweetpotato and several breeding lines but not in sweetpotatoes grown for commercial production. SPLCV does not cause symptoms on Beauregard, the predominant sweetpotato cultivar in the US, but it can reduce its yield. Since SPLCV could become an important constraint for sweetpotato production; diagnosis, identification, and characterization are essential steps to develop an effective management program. The variability among begomoviruses obtained from 11 sweetpotato genotypes was evaluated through the analysis of the nucleotide sequence of a fragment of the replication-associated protein gene (AC1). Ten of these begomoviruses were closely related to SPLCV and one was closely related to Sweet potato leaf curl Georgia virus (SPLCGV). These results suggest that in the US, SPLCV may be more common in sweetpotato genotypes than SPLCGV. Phylogenetic analysis using the obtained nucleotide sequences of the AC1 and the full length nucleotide sequences of the coat protein gene (AV1) clustered all sweetpotato begomoviruses together. However, SPLCV and SPLCGV were placed in different groups supporting their status as different species. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E, and transformed into E. coli XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 ìg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltose-binding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the CP of SPLCV.

Aspergillus flavus is an opportunistic fungal pathogen that infects peanuts, cotton, corn and tree nuts. Aspergillus flavus is a major problem globally due to the production of acutely toxic and carcinogenic aflatoxins. Louisiana climatic conditions lead to annual threats of corn aflatoxin contamination. The purpose of this study was to determine the specific ability of different strains of A. flavus to infect corn. Five soil samples and 10 corn ears were collected from each of seven corn fields throughout Louisiana. In addition, Francis Deville of Monsanto Company collected 7, 6, 2, and 4 soil samples and corn ears from four additional fields in Louisiana. Six hundred twelve and 255 A. flavus colonies were isolated from the corn and soil samples, respectively. Isolates were characterized by vegetative compatibility groups (VCGs), sclerotia size, aflatoxin B1 (AFB1) production, mating type and 8 simple sequence repeat loci polymorphisms. Eighty-eight percent of corn isolates belonged to two VCGs, whereas only 5% of soil isolates belonged to the same two VCGs. Ninety-five percent of corn isolates did not produce any sclerotia, whereas 56% and 41% of soil isolates produced small and large sclerotia, respectively. The mean AFB1 production on rice for corn kernel isolates was 2314 ± 7455 ppb and 10248 ± 11430 ppb for the soil isolates. Ninety-six percent of corn isolates were in the Mat1-2 mating type whereas only 52% of soil isolates were Mat1-2. SSR fingerprints revealed 26 haplotypes in the corn sample isolates and 78 in the soil sample isolates. All characteristics differed significantly between the soil and the corn kernel populations. Differences between the corn and soil populations indicate that not all soil isolates are as capable of infecting corn and that some isolates have become specialized to infect corn. Further understanding of virulence of A. flavus is important for the development of a better biocontrol against toxigenic A. flavus and possibly more resistant hybrids of corn.

Natural epidemics of brown rust, caused by Puccinia melanocephala, affecting sugarcane were studied to determine the crop and/or environmental factors that affect epidemic onset, severity and eventual decline. Environmental and crop growth variables were monitored along with disease severity in two susceptible cultivars, LCP 85-384 and Ho 95-988, each grown at a different location in Louisiana. During two seasons, correlation and multiple regression analyses identified leaf wetness and temperature as important determinants of disease severity for both cultivars. The results suggested that crop growth variables were not determinants for epidemics. Controlled conditions experiments assessing the interaction of leaf wetness and temperature demonstrated that changes in one variable will influence the effect of the other and identified minimum and maximum values required for infection. Increasing leaf wetness duration from 7 to 10 or 13 hours resulted in greater infection at an optimal temperature range of 17 to 27 C. Minimum requirements for leaf wetness and temperature were 7 hours and 17 C. Minimal infection occurred at 29 and 31 C. Severe epidemics in both cultivars began to decline once maximum ambient daily temperature was 32 C or higher. Lower disease severity during the 2010 epidemic in Ho 95-988 allowed an analysis of the effects of conducive and limiting conditions on brown rust severity. Lower severity resulted from a combination of unfavorable temperature and leaf wetness conditions that delayed onset then reduced the rate of disease increase. An accumulation of 23-25 leaf wetness conducive days after the daily minimum temperature exceeded 17 C preceded the onset of disease on young leaves in all three epidemics suggesting cumulative leaf wetness days might provide an epidemic predictor. The study results suggest that the occurrence of limiting temperatures determines the initiation and decline of a brown rust epidemic under Louisiana climatic conditions. The availability of leaf wetness is then the most important determinant of disease severity during the epidemic period. The study results suggest that temperature and leaf wetness can provide the basis for a disease advisory or forecasting system that predicts the threat of a severe epidemic and improves recommendations for fungicide use.

Burkholderia glumae is the bacterial pathogen causing bacterial panicle blight disease of rice (BPB). The pathogen, whose growth and pathogenicity is favored by high temperatures, has become a serious threat to rice production around the world possibly due to the current global climate changes. In this study, previously reported avirulent B. glumae strains were characterized in respect to the production of known virulence factors, toxoflavin, lipase and motility, and potential virulence factors, including polygalacturnase and type III secretion system (hypersensitive response). Considerable phenotypic variation was observed among the strains tested. Virulence of the B. glumae strains was closely related to their ability to produce various virulence factors. Interestingly, all the confirmed avirulent strains were defective in multiple virulence factors and most of them lost their ability to produce acyl-homoserine lactone (AHL) quorum-sensing signals implying that mutation in global regulatory system(s) for the virulence factors may be the major cause of the occurrence of avirulent B. glumae strains in nature. We developed a high throughput method for virulence tests of B. glumae by using onion bulb scales instead of rice panicles. The results indicated that the onion assay system can be a convenient alternative way for initial and preliminary virulence testing of B. glumae. In addition, some B. glumae strains produce melanin-like brown pigment. In order to identify the biosynthetic pathway and regulatory genes of this mechanism and role in virulence, the B. glumae 411gr-6, brown pigment producing strain genome was randomly mutagenized with a mini-Tn5 derivative, mini-Tn5gus. From this mutagenesis, several novel regulatory elements for B. glumae virulence factors were identified, including putative sensor histidine kinase, Clp protease, histone H1 like protein, prephenate dehydrogenase, and a putative sigma54 dependent response regulator. The melanin-like brown pigment of B. glumae may be involved in the hypervirulence of the pathogen providing a survival advantage under adverse climatic condition over the melanin non-producing strains.

In 2007, a filamentous fungus was recovered from sori of soybean rust (SBR), caused by Phakopsora pachyrhizi, collected from Louisiana and Florida. This fungus was identified as Simplicillium lanosoniveum on the basis of ITS sequence data and morphological traits. Simplicillium lanosoniveum was found coiling within sori and around urediniospores and showed a trophic attraction to rust sori, extending from sorus to sorus. In co-inoculated soybean leaves, the fungus did not grow or establish on leaf surfaces until sori erupted. Similarly, S. lanosoniveum colonized within 3 days and sporulated within 4 days on leaves showing disease symptoms. In field studies, when soybean leaves were inoculated with S. lanosoniveum during the latent stages of infection of SBR, disease progression was significantly limited. Additionally, sori became increasingly red-brown, which appeared to represent accelerated aging of sori. In the presence of S. lanosoniveum, urediniospores turned brown and failed to germinate. To examine the mode of action by which S. lanosoniveum antagonized urediniospores, we used scanning and transmission electron microscopy as well as confocal microscopy to characterize the interaction. Putative penetration points were observed over germ pores, and hyphae penetrated urediniospores through germ pores within the first 24 hours. By the third day, hyphae exited urediniospores and sporulated on the surface of colonized urediniospores. These studies provide evidence of a mycoparasitic interaction between S. lanosoniveum and P. pachyrhizi. Implications of this mycoparasitic relationship include potential use of S. lanosoniveum as a component of an integrated pest management program or as a biological control agent in organic soybean production.

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.