Greenhouse and microplot studies were conducted to evaluate the influence of soil texture on reproduction and pathogenicity of Rotylenchulus reniformis (reniform nematode) on cotton. A 45 day duration greenhouse experiment confirmed the pathogenicity of an isolate of R. reniformis from Avoyelles Parish on Stoneville LA887 cotton. A series of greenhouse experiments were conducted with three geographic isolates of R. reniformis (identified as Avoyelles, Evangeline, and Rapides to indicate the Parish of origin) on Stoneville LA887, Stoneville 5288B2F, and Phytogen 375WF cotton growing in soils with varying textures for 60 days. Soil types with sand, silt, and clay contents ranging from 74.4 to 7.8, 20.7 to 66.3, and 4.9 to 25.9, respectively, were employed in this research. Two experiments were conducted with Stoneville LA887 cotton, three soil types and Avoyelles isolate of reniform nematode for 150-152 days in a microplot environment. In the greenhouse, variations in soil texture significantly affected plant height and dry weights in both Stoneville 5288B2F and Phytogen 375WF, but did not have any significant effect on plant growth of Stoneville LA887, except in the 45-day duration experiment. Stoneville 5288B2F plants were significantly taller throughout the experiment in soil with 31.4% sand and 13.3% clay. Phytogen 375WF cultivar showed the same pattern, but the difference in plant height was not observed at harvest. Stoneville 5288B2F and Phytogen 375WF had significantly reduced dry root and shoot weights in sandier soils. Soil type had a significant effect on nematode reproduction on all three cotton cultivars. The interaction between soil type and reniform isolate significantly affected population densities of all reniform isolates tested among the multiple soil types in the cultivars Stoneville 5288B2F and Phytogen 375WF, but no effect was observed in Stoneville LA887 cultivar. In the microplot, plants growing in soil with more clay content had significantly greater root and shoot weights than the others. The number of bolls open and seed cotton weight were significantly reduced by R. reniformis in the microplots. Population densities of R. reniformis in the microplot followed the same pattern observed in greenhouse experiments.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice, which is becoming a major threat to global rice production. The genome of a highly virulent B. glumae strain, 336gr-1 that was isolated from rice in Louisiana, was sequenced to better understand the genome-scale characteristics, particularly that of its pathogenicity. Comparative genomic analyses with another strain, BGR1 that was isolated from Korea, revealed several unique regions present in the genomes of these two geographically separated phytopathogenic bacteria. Genome plasticity, primarily caused by a horizontal gene transfer, was observed in these closely related strains of Burkholderia that are capable of infecting the same host plant. The highly conserved nature of chromosome 2, along with the presence of important virulence determinant gene coding regions in it, such as those involved in type III secretion and toxoflavin production, indicates its importance in pathogenesis. The presence of multiple genomic islands, detectable pseudo genes, insertions, deletions, and paralogous genes indicates recent adaptation to diverse ecological niches and reduced selection pressures in specific regions of the B. glumae genome. These findings would help explain the genotype and host range diversity of B. glumae and augment characterization of its ecology and pathogenesis. Characterization of ECF σ70 gene and σ54 dependent response regulator gene in B. glumae has revealed that they are not directly involved in pathogenicity in this phytopathogenic bacterium. Global transcriptome analysis of B. glumae strain 336gr-1 has revealed that the expression of 87 genes is influenced by the quorum sensing genes tofI/tofR and their intergenic region, orf1. Especially, the genes for the type II secretion system and diguanylatecyclase activity, which play important roles in the pathogenesis of B. glumae, were found to be regulated by quorum sensing mechanism.

Sweetpotato potyviruses [Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2)] commonly infect sweetpotato and weedy morning glories in the USA. These viruses are transmitted in a non-persistent manner by various aphid species and cause up to 15% yield loss. Sweetpotato is vegetatively propagated, and in the USA growers are supplied with virus tested propagation material to minimize impact of viruses. However the rapid re-infection of these materials with viruses warranted further studies to determine factors that influence the epidemiology of these viruses. The objectives of this study were: (i) to determine if differences in acquisition hosts, aphid species and infection status influenced transmission of SPFMV; (ii) to determine how aphid abundance, aphid species diversity and virus titers relate to the spread of potyviruses in Louisiana sweetpotato fields; (iii) to determine the effects of virus infection on the population dynamics of aphids on sweetpotato and morning glories; and (iv) to determine the effects of virus infection on stylet penetration behaviors of aphids. SPFMV was transmitted at a greater rate from morning glories which also had greater virus titers compared with sweetpotato and from mixed infection sources than from singly infected sources, and Aphis gossypii was the most efficient vector. Aphids were captured in fields during the entire crop cycle, and A. gossypii and Rhopalosiphum padi, were the most abundant species occurring throughout the growing season. Virus infection of sentinel plants occurred mainly during the months of June to August when virus titers were high in sweetpotato plants. SPFMV was more commonly detected than SPVG or SPV2 in sentinel plants. Myzus persicae had a significantly greater reproduction on sweetpotato cvs. Beauregard and Evangeline with mixed virus infection compared with non-infected plants. Stylet penetration behaviors were variable depending on host and virus infection status. Differences in virus transmission rates depending on host plant, aphid species, virus species and virus titers, and pattern of spread in sweetpotato fields suggest the dissemination of sweetpotato potyviruses is influenced by the source of inoculum, the quantity of inoculum, virus species and aphid species vectors.

A study documenting basidiomycete yeast biodiversity was conducted in Baton Rouge, Louisiana during 2010 and 2011. Using the spore-fall method, the leaf surfaces of seven ferns were sampled at biweekly intervals. Maximum likelihood phylogenetic analyses of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) of nuclear ribosomal DNA were used to identify 463 isolates representing 81 species spanning 12 orders within six classes in Basidiomycota. Nearly 30 of these isolates appear to be species new to science. Data indicate fern leaf developmental stage has an effect on the number of yeasts present. A significant difference exists between the number of isolates recovered from young versus senescent leaves. On average, more yeast isolates were recovered from young leaves than from senescing ones for all classes of yeasts except Ustilaginomycetes, which were more abundant on senescing leaves. The number of yeasts recovered from lower (abaxial) versus upper (adaxial) leaf surfaces did not differ significantly, though isolates were recovered more frequently from abaxial surfaces for all classes expect Microbotryomycetes and Tremellomycetes. For all six classes there was a trend for non-fertile frond portions to yield more isolates than those with sori, but this difference is not statistically significant. Monthly records of temperature and precipitation were compiled and while they show no statistically significant correlation with yeast abundance, several patterns may be meaningful. A general downward trend was observed in the number of isolates recovered with respect to decreasing temperature and increasing precipitation levels. More isolates were recovered during the months with the highest average temperatures and lowest levels of precipitation. Additionally, two new yeast species collected during this survey are provisionally described. SA209 and SA575 represent previously undescribed yeast species in the genera Farysia and Sporisorium (Ustilaginales). The LSU and ITS regions of these isolates were compared to available sequences of Farysizyma and Pseudozyma species and related taxa in Farysia, Sporisorium and Ustilago. SA209 is part of a Farysizyma/Farysia clade in Anthracoideaceae and SA575 is sister to Sporisorium chrysopogonis and S. heteropogonicola within a clade of Sporisorium species that includes the type (S. sorghi).

ABSTRACT Isolates of Cercospora kikuchii, the causal agent of Cercospora leaf blight (CLB) and purple seed stain (PSS), were used to determine baseline sensitivities to selected quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides by conducting radial growth assays on fungicide-amended media. The effective concentration to inhibit 50% radial growth (EC50) for each isolate was calculated by linear interpolation of the dose-response relationship. All baseline distributions were non-normal with outliers towards the less sensitive ends of the spectra, and median EC50 values for azoxystrobin, pyraclostrobin, trifloxystrobin, flutriafol, propiconazole, and tetraconazole were 0.081, 0.013, 0.012, 0.273, 0.143, 1.47 µg/ml, respectively. When compared to baseline sensitivities, median EC50 values for isolates exposed to azoxystrobin, pyraclostrobin, and trifloxystrobin in 2011/2012 were significantly higher at 37.2/57.6, 10.1/12.2, and 20.1/29.1 µg/ml, respectively. Cross-resistance to all three QoI fungicides was observed in the 2011 and 2012 populations. Discriminatory doses of 10 µg/ml were developed for all three QoI fungicides to distinguish between sensitive and resistant isolates. Approximately 83% of all isolates screened in 2011 and 2012 were resistant to QoI fungicides, and isolates from 21 of 27 parishes tested positive for resistance. Median EC50 values for isolates exposed to flutriafol, propiconazole, and tetraconazole in 2011/2012 were 0.41/0.54, 0.33/0.24, and 0.75/0.73 µg/ml. Significant shifts from the baseline towards less sensitivity were detected in isolates exposed to flutriafol and propiconazole. Additionally, outliers towards less DMI sensitivity were detected for all three DMI fungicides 2012. Strong, positive, and significant cross-sensitivity was observed among all three DMI fungicides. At a discriminatory dose of 5 µg/ml thiophanate methyl, methyl benzimidazole carbamate (MBC) resistance was detected in the 2000, 2011, and 2012 populations at 23.3, 44.8, and 35.7%, respectively, with resistant isolates in 19 of 27 parishes. Isolates exhibiting multiple resistance to QoI and MBC fungicides also were detected in 15 of 27 parishes. Ninety-eight percent of MBC-resistant isolates also were resistant to QoI fungicides. Based on results from this research, CLB/PSS management strategies with QoI and MBC fungicides should be reconsidered in areas where resistance has been confirmed, and C. kikuchii populations should be further monitored for shifts in DMI sensitivity.

Loblolly pine (Pinus taeda L.) decline (LPD) has been present in upland sites of central Alabama since the 1960s. Symptoms of LPD (fine root deterioration, short chlorotic needles, sparse crowns, reduced radial growth) begin in the 30-40 yr age class, resulting in premature death at ages 35-50. Previously, declining loblolly was diagnosed as littleleaf disease (LLD); however, site conditions associated with LPD are different from LLD sites. Littleleaf disease only occurs on eroded, heavy clay soils and is secondarily associated with the fungus, Phytophthora cinnamomi. In contrast, LPD occurs on sandy, well-drained soils and is associated with Leptographium spp., as well as with root-feeding bark beetles and weevils. In the present study, 17 species (eleven newly reported) of subcortical root- and lower-stem feeding beetles were identified as vectors of Leptographium species, of which Hylastes salebrosus, H. tenuis, Hylobius pales and Pachylobius picivorus were statistically more abundant (F_3,14=13.90, p=0.003) in LPD sites. Leptographium terebrantis, L. procerum, L. lundbergii, and L. serpens were isolated from the roots and insects. Pathogenicity studies suggested that L. lundbergii and L. serpens, fungi not previously reported in the U.S., were more virulent on loblolly pine. Spatial analysis correlated LPD to site and stand physical factors. Slope and aspect were the predominant predictive variables of LPD in central Alabama. Convexity and elevation were predictive only in combination with other topographical factors. These analyses have allowed the creation of LPD risk maps to accurately predict areas of loblolly decline, providing a vital new tool for managing southern forests for predetermined purposes.

Nitrogen nonutilizing mutants were used to assess vegetative compatibility of 58 isolates of Cercospora kikuchii, 55 of which were isolated from soybean plants in Louisiana. Only 16 of 56 self-compatible isolates were assigned to six multi-member vegetative compatibility groups (VCGs), 01-06, with two or three isolates in each VCG. The other 40 isolates were not vegetatively compatible with any isolates other than themselves. All six multi-member VCGs contained isolates from different soybean cultivars, and three included isolates from different locations. Only one VCG included isolates both from soybean leaves and from seeds, while two and three multi-member VCGs included isolates only from leaves or from seeds, respectively. Population structure of C. kikuchii was further examined in 164 isolates, 161 of which were from Louisiana, with random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR). All isolates in the VCG study were included. Based on analysis of molecular variances, isolates from different host cultivars or different locations in Louisiana were not significantly different, but the Louisiana population was significantly different from isolates collected outside the state. Leaf and seed populations were significantly different. In the clustering analysis, isolates from Louisiana were grouped into four lineages, clades A-D. Clades A-C were further grouped into a large clade (ABC) with moderately strong bootstrap support. Clade B was the most dominant lineage in Louisiana. Only seven isolates from Louisiana were in the lineage, clade D, that included all three isolates from outside sources. Multilocus gametic disequilibrium tests did not reject the null hypothesis of random mating in clade B, but it was rejected in Clades A and D and the total collection. Some isolates within a VCG were closely related, but isolates within a VCG were not clustered together according to VCG in general. Representive isolates in clades A, B, and D were used to screen six commercial soybean cultivars, HBK R5588, AG5701, DP 5806 RR, TV59R85, SS RT 6299N and DP 6880 RR. Cultivars AG5701 and TV59R85 were significantly more resistant than other cultivars, and cultivar DP 6880 RR was most susceptible. Clade D was significantly more virulent than the other two lineages.

Four hundred and two bacterial isolates isolated on the semi-specific S-PG medium from diseased rice tissues showing symptoms of panicle blighting. These isolates were purified using serial dilution in sterile water and replating on S-PG medium. A total of 420 single isolates were obtained. These isolates were subjected to pathogenicity tests on rice (Oryza sative L. cv. Cypress). Based on these tests, 339 isolates were used in BiologTM tests and identified to the species level. Bacterial strains from 39 species in 16 genera were identified, including 52 isolates representing15 Pseudomonas species and 261 isolates representing six Burkholderia species. The remaining 26 isolates included 14 other genera. Of 261 Burkholderia strains, 103 isolates were B. gladioli, 68 isolates were B. glumae, 60 isolates were B. multivorans, 25 isolates were B. plantarii, three isolates were B.cocovenenans and three isolates were B. vietnamiensis. The pathogenicity tests revealed that 69% or 234/339 isolates were on Cypress rice, causing seedling infection, sheath rot and/or panicle blighting. Most of the pathogenic strains were in the genera Burkholderia and Pseudomonas. The four most common species, B. glumae, B. gladioli, B. multivorans, and B. plantarii, comprised 90% of all of the pathogenic bacteria, suggesting that a complex of Burkholderia species were causing the panicle blight/sheath rot syndrome recently found in Louisiana. Five Pseudomonas species, with total of 16 isolates, were found to associate with this disease, including two isolates of P. syringae pv zizanize, three isolates of P. fluorescens, three isolates of P. pyrrocinia, one isolate of P. spinosa and seven isolates of P. tolaasii. The symptoms of the disease on rice were only produced by the indicated bacterial strains. Symptoms included brown, margined flag leaf sheath lesions, grain rot, sterile florets, grain discoloration, and leaf and sheath rot on inoculated seedlings. It was impossible to distinguish among Burkholderia species based on symptoms and colony morphology. Pathogenic Burkholderia strains produced a yellow pigment in Kings B medium. Avirulent strains did not produce this pigment. The isolates from rice were grouped by species and pathogenicity. It appeared that B. glumae and B. gladioli were the most main pathogenic species.

Sheath blight (SB), caused by Rhizoctonia solani K¨¹hn, is a major rice disease internationally and in the southern rice area of the Unites States, including Louisiana. Breeders have incorporated partial resistance into commercial rice varieties to control the disease, but a higher level of resistance is needed. It has been demonstrated that the pathogenesis-related (PR) proteins ¦Â-1, 3-glucanase and chitinase are components of effective defense mechanisms for protecting plants against fungal pathogens. This research was conducted to co-transform the ¦Â-1, 3-glucanase, chitinase and bar genes into the rice variety Taipei 309 using the hpt gene for resistance to hygromycin B as a selective marker. Transformed calli and regenerated plants were screened with hygromycin B, and the plants were then further tested for resistance to Liberty herbicide and Rhizoctonia solani. Methods were developed to screen transgenic plants for resistance to hygromycin B and Liberty herbicide using dip and cut in toxicant solutions. Five of 99 plants in the field test and 51 of 55 plants in greenhouse test were highly resistant to Liberty herbicide. The tooth-pick inoculation method was used to test transformed plants for SB resistance. Seventeen transgenic plants in the field test and 10 transgenic plants from greenhouse tests were highly resistant to SB. Fourteen of the17 SB resistant plants were also resistant to hygromycin B, one of the plants was highly resistant to Liberty herbicide, and 9 of the 17 SB resistant plants had moderate resistance to Liberty. Panicle blight, caused by Burkholderia glumae, has been an important bacterial disease in rice worldwide and in Louisiana. No effective pesticides are available to control this disease. The PR protein thionin is reported to control certain bacterial diseases in plants. In this study, the thionin production, bar, and hpt genes were co-transformed to the rice variety Lafitte. Resistance to hygromycin B, Liberty herbicide, Xanthomonas oryza and B. glumae were expressed in selected transformed Lafitte plants. This research has created, through transformation, new sources of resistance to two major rice pathogens that cause major losses to rice. These resistances can be transferred to commercial varieties by conventional breeding methods.

Viral diseases, especially those caused by mixed infections, are among the economically most important diseases of sweetpotato. Real-time PCR assays were developed for the detection and quantification of the potyviruses Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), Ipomoea vein mosaic virus (IVMV); the crinivirus Sweet potato chlorotic stunt virus (SPCSV), and the begomovirus Sweet potato leaf curl virus (SPLCV) directly from infected sweetpotato plants. Titers of SPFMV, IVMV, and SPVG were lower in singly-infected sweetpotato plants compared to singly-infected plants of the standard indicator host Brazilian morning-glory (Ipomoea setosa) and the standard propagation host I. nil cv. Scarlet O Hara plants. The effect of SPSCV on titers of potyviruses infecting sweetpotato in the U.S. was investigated in a separate study. Titers of all potyviruses evaluated were enhanced in the presence of SPCSV suggesting that a conserved mechanism may underlie the enhancement of different potyviruses. Although titers of the common strain of SPFMV (SPFMV-C) were enhanced similarly to the russet crack strain (SPFMV-RC), SPFMV-C did not cause typical sweet potato virus disease (SPVD) symptoms when co-infecting with SPCSV, whereas SPFMV-RC with SPCSV caused severe SPVD symptoms. Titers of SPCSV were lower when coinfecting with potyviruses compared to plants infected with SPCSV alone. Expression analysis using cDNA microarrays revealed that the number of differentially expressed genes in plants infected with either SPFMV or SPCSV alone compared to virus-tested plants was 3 and 14, respectively. These findings were in stark contrast with SPVD-affected plants where over 200 genes were differentially expressed. SPVD-responsive genes are involved in various cellular processes including several that were identified as pathogenesis- or stress-induced. Even though titers of the U.S. isolate of SPLCV (SPLCV-US) were greater in the presence of potyviruses compared to titers of SPLCV in single infections, they were statistically different only when co-infecting SPFMV-RC and IVMV. Quantification of SPLCV in sweetpotato cultivars revealed that titers were significantly lower in cultivars known to be tolerant of the effects of SPLCV on yield. Real-time PCR was a more sensitive and specific detection method for the viruses evaluated compared to conventional PCR or ELISA assays.