Transplants of Spartina alterniflora Loisel. (smooth cordgrass, Poaceae) are effective in stabilizing and protecting vulnerable coastal wetlands from erosion. However, the expense and labor associated with propagation and transplanting restrict the widespread use of S. alterniflora in coastal restoration and protection projects. As an alternative, seeding of S. alterniflora by aircraft has been proposed as a more useful and economical approach for revegetating denuded marsh sites. However, a period of stalled growth, which lasts for 12 or more weeks, has been observed in smooth cordgrass seedlings, and this increases their susceptibility to being washed away by inundating tides. This stalled growth period must be overcome before aerial seeding can be implemented. As with any plant species, it is reasonable to assume that S. alterniflora has optimal nutritional requirements for seedling and mature plant growth. Previous nutritional research has involved only mature smooth cordgrass plants. These studies showed that applications of nitrogen and phosphorus fertilizers increased plant growth. The objectives of this study were to document the lag phase of seedling growth observed in field and greenhouse conditions; determine the possible role of soil microbes, including seed and seedling pathogens and mycorrhizal fungi, as a cause of delayed seedling growth; and investigate the role of nutrition in seedling growth. This study confirmed the existence of stalled seedling growth in S. alterniflora and concluded that pathogens are not the cause of this lag period. Furthermore, supplemental N and P (240 kg N ha-1, 49.5 kg P ha-1) reduced the lag phase from over 100 days to less than 50 days under ideal greenhouse conditions. However, nutrient additions did not completely overcome stalled seedling growth. Future research possibilities include investigating the effects of plant growth regulators (hormones) and seed preconditioning treatments on seedling growth.

A decline of loblolly pine (Pinus taeda L.), characterized by expanding areas of declining and dead trees, has become prevalent at Fort Benning, Georgia. A 3 year study was conducted to determine the kinds of fungi, insects, and site disturbances associated with this problem. The insects Dendroctonus terebrans, Hylastes salebrosus, Hylastes tenuis, Pachylobius picivorus and Hylobius pales were significantly more abundant in symptomatic than in asymptomatic loblolly pine plots. These root and lower stem-infesting insects consistently carried the fungi Leptographium terebrantis, L. procerum, and L. serpens. Root sampling revealed high levels of root damage and mortality, staining and infection with Leptographium species. This below-ground damage and mortality preceded the expression of above-ground symptoms, such as short chlorotic needles, sparse crowns, and reduced radial growth. A sequence of interactions among this complex of organisms and abiotic factors is proposed as the cause of loblolly pine decline. This study confirms the findings for loblolly pine decline at other geographic locations and validates the Loblolly Pine Decline Risk Map as described by Eckhardt (2003).

Atoxigenic Aspergillus flavus was demonstrated by others as a promising biocontrol agent to minimize preharvest aflatoxins in susceptible crops. But the mechanism was unclear. A filter insert-well plate system was used to study the mechanism in lab. There was no inhibition when toxigenic A. flavus isolate 53 and inhibitory atoxigenic isolates were separated by 0.4 µm membrane, approximately 50% inhibition occurred when separated by 12 µm membrane, and complete inhibition occurred when a 74 µm membrane was used. This result suggested that touching or close physical interaction is needed for toxin inhibition and the nutrient competition hypothesis was not supported. Isolate 53 and inhibitory atoxigenic isolate 51 were used to study the timing of intraspecific toxin inhibition. The result showed that inhibition occurred when 0 - 4 day old isolate 51 was added within the first 16-hour growth of isolate 53. However, two-day old isolate 51 inhibited toxin production by two-day old isolate 53 and twenty-four hour old isolate 51 inhibited toxin production by 48-hour old isolate 53. These results suggested that there is a 16-hour window for the conidial inhibition ability of atoxigenic isolate but for mycelia, the window is expanded to 48 hours. Isolate Af70-GFP was acquired to microscopically examine the touch inhibition interaction. Surprisingly, none of the completely inhibitory atoxigenic isolates from our collection or NRRL 21882 inhibited toxin production by Af70-GFP. Isolate K49 and two Australian isolates were shown to be able to inhibit toxin production by Af70-GFP. The inhibitory abilities of additional atoxigenic isolates were tested with toxigenic isolates 53, Af70s-GFP and NRRL 3357. Different patterns were obtained among those three isolates. These results showed that there was specificity in the touch inhibition interaction. Af70-GFP and isolate K49 were used to continue microscopy work. The growth of Af70s-GFP appeared to be inhibited and vacuoles present in Af70s-GFP were absent when it was paired with K49. Biocontrol once thought to be due to competitive exclusion probably requires close physical growth or touching and displays specificity. Multiple atoxigenic isolates each specific to a subset of the toxigenic isolate population may be needed for an effective biocontrol application.

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.