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.

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.