Rust (Puccinia emaculata) Management and Impact on Biomass Yield in Switchgrass.

Rust, putatively caused by Puccinia emaculata, is a widespread and potentially damaging disease of switchgrass, a crop produced as feedstock for livestock and bioenergy. Azoxystrobin, chlorothalonil, and myclobutanil were applied at 1-, 2-, 3-, or 4-week intervals for 12 to 14 weeks to the vegetatively propagated switchgrass cultivar Cloud Nine to assess fungicide selection and application interval for the control of rust as well as the impact of this disease on switchgrass biomass yield. Although rust severity significantly differed among study years, azoxystrobin and myclobutanil were often equally and more effective than chlorothalonil at controlling rust, with superior disease control coming at shorter application intervals compared with extended application intervals. Year, product, application interval, and product × interval significantly impacted dry biomass yield, which was greatest in 2016 and lowest in 2014. Dry biomass yield protection was significantly better with azoxystrobin and myclobutanil applications than with chlorothalonil or no fungicide. Linear regression models with the final disease rating, as well as with the area under disease progress curve in each year, were significant, but coefficients of determination were low to moderate (0.21 < R2 < 0.60), indicating that rust response and subsequent disease impact on dry biomass yield were impacted by other factors. From our models, an estimated 3 to 5% biomass decline was calculated for each 10% increment in rust-related leaf necrosis observed at the final September rating date. With rust-related leaf necrosis ≥80% by 1 September in each of 4 study years, biomass yield may be reduced by 24 to 40% if rust problems are not managed in switchgrass crops.

Multiyear Regional Evaluation of Foliar Fungicide Applications for Cotton Target Spot Management in the Southeastern United States.

Fungicide programs for managing target spot of cotton caused by Corynespora cassiicola were evaluated over 15 site-years in the southeastern United States between 2014 and 2016. Two cultivars, hypothesized to vary in target spot susceptibility, PhytoGen 499WRF (PHY499) and Deltapine 1137B2RF (DPL1137), and four fungicides (azoxystrobin, flutriafol, pyraclostrobin, pyraclostrobin + fluxapyroxad) plus nontreated control, were compared. Fungicide programs consisted of 1) a single application at first flower or disease onset and 2) the first application followed by a second 14 days later. Treatments were applied in a factorial, randomized complete block design. Target spot onset and severity varied among site-years. Except when severity was low, target spot-associated defoliation was greater on PHY499 than on DP1137. Fungicides delayed disease development and defoliation, but application number had little impact. Based on a meta-analysis of 15 site-years, pyraclostrobin-based applications resulted in a 4 to 6% yield preservation, and yield preservation was greater at site-years with early disease onset and >40% target spot associated defoliation. Results suggest a single well-timed application of a pyraclostrobin-based fungicide reduces defoliation and protects cotton yield at locations with high target spot severity. Additional research is needed to identify risk factors for target spot-associated yield losses in cotton production systems.

Petrobactin Is Exported from Bacillus anthracis by the RND-Type Exporter ApeX.

Bacillus anthracis-a Gram-positive, spore-forming bacterium-causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen.IMPORTANCEBacillus anthracis requires iron for growth and employs the siderophore petrobactin to scavenge this trace metal during infections. While we understand much about petrobactin biosynthesis and ferric petrobactin import, how apo-petrobactin (iron free) is exported remains unknown. This study used a combination of bioinformatics, genetics, and mass spectrometry to identify the petrobactin exporter. After screening 17 mutants with mutations of candidate exporter genes, we identified the apo-petrobactin exporter (termed ApeX) as a member of the resistance-nodulation-cell division (RND) family of transporters. In the absence of ApeX, petrobactin accumulates inside the cell while continuing to export petrobactin components that are capable of transporting iron. Thus, the loss of ApeX does not affect the ability of B. anthracis to cause disease in mice. This has implications for treatment strategies designed to target and control pathogenicity of B. anthracis in humans.

Flying under the radar: The non-canonical biochemistry and molecular biology of petrobactin from Bacillus anthracis.

The dramatic, rapid growth of Bacillus anthracis that occurs during systemic anthrax implies a crucial requirement for the efficient acquisition of iron. While recent advances in our understanding of B. anthracis iron acquisition systems indicate the use of strategies similar to other pathogens, this review focuses on unique features of the major siderophore system, petrobactin. Ways that petrobactin differs from other siderophores include: A. unique ferric iron binding moieties that allow petrobactin to evade host immune proteins; B. a biosynthetic operon that encodes enzymes from both major siderophore biosynthesis classes; C. redundancy in membrane transport systems for acquisition of Fe-petrobactin holo-complexes; and, D. regulation that appears to be controlled predominately by sensing the host-like environmental signals of temperature, CO2 levels and oxidative stress, as opposed to canonical sensing of intracellular iron levels. We argue that these differences contribute in meaningful ways to B. anthracis pathogenesis. This review will also outline current major gaps in our understanding of the petrobactin iron acquisition system, some projected means for exploiting current knowledge, and potential future research directions.

Insect Damage, Aflatoxin Content, and Yield of Bt Corn in Alabama.

Isoline pairs of hybrid corn, similar except for presence or absence of a Bt trait, were planted at eight sites across Alabama over three years. This study evaluated insect damage, yield, and aflatoxin levels as affected by the Bt traits, YieldGard Corn Borer (expressing Cry1Ab), Herculex I (expressing Cry1F), Genuity VT Triple PRO (expressing Cry1A.105 and Cry2Ab2), Agrisure Viptera 3111 (expressing Vip3Aa20 and Cry1Ab), and Genuity SmartStax (expressing Cry1A.105, Cry2Ab2, and Cry1F). When examined over all sites and years, hybrids with any of the included Bt traits had lower insect damage and higher yields. However, insect damage was not consistently correlated to yield. Bt traits expressing multiple proteins provided greater protection from corn earworm feeding than did traits for single proteins. Yields and aflatoxin levels were highly variable among sites although irrigated sites had higher yields than nonirrigated sites. Aflatoxins commonly accumulate in corn in the southeastern United States because of prevailing high temperatures and frequent dry conditions. Aflatoxin levels were not consistently associated with any factors that were evaluated, including Bt traits.

First Report of Impatiens Downy Mildew Caused by Plasmopara obducens in Alabama.

During spring 2012, potted impatiens (Impatiens walleriana Hook.f.) plants with symptoms of a foliar disease were found in several commercial greenhouses in Mobile County, Alabama. Symptomatic leaves were chlorotic with no distinct lesions, and quickly wilted and abscised from erect green stems. In summer 2012 and 2013, numerous landscape impatiens plants with similar symptoms were observed in a large area from Mobile County north to Lee County, Alabama. A downy mildew was observed on the lower surfaces of symptomatic and abscised leaves from all locations. It consisted of hyaline, monopodial sporangiophores and ovoid, hyaline sporangia. Sporangiophores, which emerged from stomata, consisted of apical branches arranged at right angles to the supporting branches; they measured 69 to 90 µm long with individual branches measuring 7 to 14 µm long. Sporangia were borne on the tips of sporangiophore branches and measured 10 to 16.5 × 17 to 22.5 µm. No oospores were observed. In 2013, symptomatic plants were obtained from two separate locations in Alabama (Mobile and Tallapoosa counties). Total genomic DNA was extracted directly from symptomatic plant tissue and the large ribosomal subunit DNA was amplified by PCR using primers NL-1 and NL-4 (1). From both isolates, amplicons of 600 and 775 bp were obtained. DNA from each amplicon of both isolates was purified, sequenced, and the sequences were deposited in GenBank (Accession Nos. KF956518 to 21). The sequences of the 600-bp amplicons were 99% similar to that of I. walleriana (JX142135); the sequences of the 775-bp amplicons were 99% similar to Plasmopara obducens isolates from Florida (JX217746), Ohio (JX142134), Serbia (HQ246451), and the United Kingdom (AY587558). In pathogenicity tests, 10 potted impatiens plants, I. walleriana'Super Elfin,' were inoculated with a sporangial suspension (1 × 105 sporangia/ml washed from infected leaves) from the Mobile County isolate, by spraying until runoff. Controls were inoculated with sterile water. Plants were incubated in a moist chamber at 21°C for 48 h and then maintained in a greenhouse at 22 to 25°C until symptom development. All inoculated plants developed symptoms of downy mildew within 10 days. Microscopic examination of the symptomatic tissue revealed sporangiophores and sporangia similar to those observed in naturally infected plants. Control plants showed no symptoms. To our knowledge, this is the first report of downy mildew caused by P. obducens on impatiens in Alabama. This disease has been reported to have a significant economic impact for growers throughout the United States (2,3). Impatiens downy mildew is likely to be a recurring problem in Alabama. References: (1) K. O'Donnell. Curr. Genet. 22:213, 1992. (2) A. Palmateer et al. Plant Dis. 97:687, 2013. (3) S. Wegulo et al. Plant Dis. 88:909, 2004.

First Report of Corynespora cassiicola-Incited Target Spot on Cotton in Alabama.

Target spot symptoms were first observed on dryland and irrigated cotton (Gossypium hirsutum L.) statewide in Alabama in 2011. Leaf spots first appeared in the lower canopy and spread upward through the canopy toward the shoot tips. Individual leaf spots were roughly circular, formed concentric rings of alternating light and dark brown bands, and were up to 10 mm in diameter. Leaves with multiple lesions senesced prematurely. In 2012, target spot symptoms were observed as early as 68 days after planting in Tallapoosa County, Alabama. The possible combination of early disease onset and frequent showers/irrigation triggered rapid premature defoliation in some fields in excess of 75% in susceptible cultivars (Phytogen 499). Estimated yield losses in select cultivars (Deltapine 1050 and Phytogen 499) exceeded 336 kg/ha seed cotton. In 2012, symptomatic leaves were obtained from two separate locations in Alabama (Baldwin and Tallapoosa counties). The fungus was isolated from lesions by single spores plated on antibiotic V8 agar (1) and incubated at 21°C for 2 weeks under 12-h light cycles. Conidiophores arising from the gray, flocculose colonies were simple, erect, cylindrical, brown or olivaceous, unbranched, with two to seven septa. Conidia were borne singly, ranging from subhyaline to olivaceous, obclavate to cylindrical, straight to slightly curved, contained 4 to 15 pseudosepta, and were 50 to 209 µm long and 7 to 15 µm wide. These characteristics were consistent with the original description of Corynespora cassiicola on cotton (2). The internal transcribed spacer region (ITS) of two isolates, one representing each location, was amplified using primers 2234c and 3126t targeting a 550-bp region of the ITS1, 5.8S rRNA gene, and ITS2 (3). Sequences revealed 99% similarity to C. cassiicola in NCBI (Accession Nos. AY238606 and JQ717069). In greenhouse pathogenicity tests, 10 cotton seedlings (Phytogen 499) were inoculated by spraying a fungal suspension (2 × 104 spores/ml) of each of the two isolates prepared from 2-week-old cultures until runoff. Controls were inoculated with sterile water. Cotton seedlings were incubated in a moist chamber at 21°C for 72 h. All plants inoculated with the fungus developed leaf spot symptoms in 6 days. The fungus was reisolated from five inoculated plants. DNA was extracted from each isolate, amplified using primer pair 2234c/3126t, and sequenced. Sequences (550-bp) from all isolates shared 99% similarity to other C. cassiicola sequences in GenBank (Accession Nos. AY238606 and JQ717069). Nucleotide sequence data reported are available in GenBank under Accession Nos. KC544017 to 23. This pathogen has been reported previously to be economically important on a number of other hosts. To our knowledge, this is the first report of C. cassiicola on cotton in Alabama. Given the increasing prevalence of this disease in Alabama, its confirmation is a significant step toward developing management recommendations for growers. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) J. P. Jones. Phytopathology 51:305, 1961. (3) J. Sequerra et al. Mycol. Res. 101:465, 1997.

Lanthanide-based time-resolved luminescence immunoassays.

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized.

First Report of Phytophthora Blossom Blight of Chrysanthemum Caused by Phytophthora nicotianae.

In October 2000, chrysanthemums (Dendranthema × grandiflorum) cv. Debonair exhibiting blossom blight were submitted to the Plant Diagnostic Lab at Auburn University by a commercial greenhouse where most of the potted plants of this cultivar were symptomatic. At a local retail outlet, approximately 95% of the plants of the same cultivar of chrysanthemum had a similar blossom blight. Blighted petals were examined microscopically, and nonpapillate, internally proliferating sporangia (40 to 45 µm in length), characteristic of some species of Phytophthora, were observed. A species of Phytophthora was isolated repeatedly on PARP selective medium (corn meal agar containing pimaricin, ampicillin, rifamycin, and pentachloronitrobenzene). Isolates recovered were grown on V8 juice agar, under fluorescent lights and in darkness, at room temperature. These isolates were identified as Phytophthora nicotianae (= Phytophthora parasitica), on the basis of morphological and cultural characteristics. Sporangia were papillate (including some with dual apices), noncaducous, 45 to 60 µm in length, and spherical, ovoid, or obpyriform. Mycelium growth occurred at 36°C. Isolates were considered heterothallic because they did not produce oospores when grown on V8 juice agar in the dark for 2 weeks. Sporangia that were nonpapillate and proliferating internally were not observed on any of these isolates. Because we apparently did not isolate the Phytophthora spp. seen microscopically on petals, we cannot comment on its exact identity or significance in causing this disease. We did conduct pathogenicity tests to determine whether isolates of P. nicotianae were capable of causing the observed symptoms. These tests were conducted twice on chrysanthemum cultivars Debonair, Yellow Triumph, Spotlight, Raquel, Jennifer, Grace, and Hot Salsa. In the first test, two plants of each cultivar were sprayed to runoff with a zoospore suspension (105 spores per ml) in sterile, filtered water. Two plants of each cultivar were sprayed with sterile, filtered water as noninoculated controls. Individual plants were placed in loosely closed plastic bags, misted daily, and held at 23 to 24°C with indirect lighting (approximately 12 h per day) for 1 week. In the second test, four plants of each cultivar except Debonair were inoculated as described previously, four plants of each cultivar were left untreated as noninoculated controls, and one Debonair plant was inoculated and one remained noninoculated. Plants were held for 3 days in an environmentally controlled growth room, with 23°C days (11 h)-20°C nights (13 h), under a plastic tent where high levels of humidity were maintained with a humidifier and daily misting. A grow light provided a low level of lighting (4 to 6 µE · m-2 · s-1). All inoculated plants developed severe blossom blight similar to that observed initially. In the first test, symptoms were evident at 2 days. In the growth room, blossom blight first was observed at 24 h postinoculation. In both tests, blossom blight severity increased quickly in the 1 to 2 days after the initial occurrence of symptoms. Only blossoms became diseased; symptoms did not extend to other plant organs. P. nicotianae was reisolated consistently from symptomatic blossoms on selective medium. This is, we believe, the first report of blossom blight on chrysanthemum caused by a species of Phytophthora. Previously, P. nicotianae has been reported to cause leaf blight on artificially inoculated Chrysanthemum × morifolium (Dendranthema × grandiflorum) cultivars Capri and Vermilion in Florida (1) and twig and leaf blight on Chrysanthemum coronarium in India (2). References: (1) C. R. Semer and B. C. Raju. Plant Dis. 69:1005-1006, 1985. (2) N. Sushma and N. D. Sharma. J. Mycol. Plant Pathol. 27:345, 1997.

Number of Tebuconazole Applications for Maximizing Disease Control and Yield of Peanut in Growers' Fields in Alabama.

Disease and yields of peanut were monitored in growers' fields treated with zero, one, two, three, and four applications of tebuconazole. Yields ranged from 1,918 to 6,891 kg/ha and averaged 4,319 kg/ha over eight locations in 3 study years. Defoliation caused by peanut leaf spots (Cercospora arachidicola and Cercosporidium acutatum) and incidence of southern stem rot (Sclerotium rolfsii) were inversely related to number of tebuconazole applications, while yield was directly related to number of tebuconazole applications. Average yields with zero, one, two, and four tebuconazole applications (each application at 0.25 kg a.i./ha) were 3,609, 4,088, 4,526, and 4,868 kg/ha, respectively. Data from each location were best modeled by quadratic equations allowing determination of numbers of tebuconazole applications for minimal disease and maximal yields. Calculated spray numbers indicate that between three and four applications of tebuconazole is best for disease control and yield maximization.