Draft Genome Sequence Resource of the Citrus Stem-End Rot Fungal Pathogen Lasiodiplodia theobromae CITRA15.

Lasiodiplodia theobromae is a fungal pathogen associated with perennial tropical fruit plants worldwide. In citrus, L. theobromae causes stem-end rot (Diplodia stem-end rot), a damaging postharvest disease that is aggravated when trees are also infected with the citrus greening bacteria 'Candidatus Liberibacter asiaticus'. Due to the latent infection of L. theobromae during the preharvest stage, it becomes difficult to control the disease by chemical or physical treatment. In the current study, we sequenced and assembled strain CITRA15, the first genome of L. theobromae obtained from diseased Citrus paradise 'Flame' grapefruit in Florida, and thereby provided a genomic resource for future research on diagnostics, and postharvest and preharvest disease management of citrus and other fruit crops.

Effects of a larval mosquito biopesticide and Culex larvae on a freshwater nanophytoplankton (Selenastrum capricornatum) under axenic conditions.

The effects of microbial biopesticides used for mosquito control on autotrophic microorganisms such as nanophytoplankton are equivocal. We examined impacts of mosquito biopesticides and mosquito larvae on primary producers in two independent experiments. In the first experiment, we examined the effects of a commonly used microbial biopesticide formulation (VectoMax® CG) on a unicellular microalga, Selenastrum capricornatum Printz, under axenic laboratory conditions. The biopesticide treatments included two concentrations (0.008 and 0.016 g liter-1 ) of VectoMax® CG and two controls (one untreated and another with autoclaved 0.016 g VectoMax® CG liter-1 ) in replicated axenic experimental microcosms. Spectrophotometric analysis of chlorophyll a (proxy for algal biomass) and direct enumeration of algal cells following the treatments revealed no significant effects of the microbial biopesticide on algal population growth during the four-week study. In the second experiment, we tested the effects of different densities of Culex larvae on the population of S. capricornatum. Effects of mosquito larvae feeding on S. capricornatum were significant with a curvilinear relationship between larval density and algal abundance in the water column. Together, these studies demonstrated a lack of direct cytological/toxicological effects of Bacillus-based microbial pesticides on freshwater primary production and support the hypothesis that the reduction in algal primary production previously reported when Bti products were applied to aquatic environments was likely independent of the Bacillus-based larvicidal toxins. Instead, it was likely mediated by microbial interactions in the water column and the trophic cascade effects that resulted from the removal of larval mosquitoes. These studies suggest that mosquito larvae independent of pesticide application can influence primary production. Our method of evaluating biopesticides against small photoautotrophs can be very useful for studying the unintended effects on autotrophic microorganisms of other pesticides, including herbicides and pesticides applied to aquatic environments.

Influence of Three Citrus Herbicides on Potential Production of Sorghum bicolor 'Topper 76-6' as a Bioenergy Crop.

Planting bioenergy crops on land previously used for citrus production may offer an alternative source of revenue for growers looking for alternative-to-citrus crops. However, residual herbicides used in citrus production may adversely affect alternative crops. This study evaluated effects of three herbicides (bromacil, norflurazon, and simazine) commonly used in citrus production on the bioenergy crop Sorghum bicolor 'Topper 76-6'. Plants were exposed to herbicides in soil for 1-5 weeks and observations of effects on photosynthetic quantum yield, leaf greenness, height, and biomass were made. Results indicate that concentrations of bromacil and norflurazon greater than 0.09 and 0.07 mg/kg and simazine >0.46 mg/kg will impair growth and development in similar soils. Concentrations below these may also be toxic.

Occurrence and ecological risks from fipronil in aquatic environments located within residential landscapes.

This study investigated the occurrence of fipronil and its metabolites in aquatic environments in residentially-developed landscapes, including five canals and three retention ponds. Fipronil was detected at four of the sites, with concentrations of 0.5-207.3 ng L(-1). Fipronil sulfone and fipronil sulfide were detected at three sampling sites, with concentrations ranging from 0.46 to 57.75 and 0.40-26.92 ng L(-1), respectively. Multiple risk assessment methods were performed to characterize potential ecological risks, including deterministic screening and probabilistic risk assessment techniques. The deterministic method indicated no risk to certain biotic groups (i.e. aquatic plants, fish, molluscs, and algae-moss-fungi), but did indicate risks to larval insects and crustaceans. Results from the probabilistic risk assessment indicated significant ecological risks (acute and chronic) ranging from 0.75 to 58.9% and 3.9-35.0% when organisms were exposed to the maximum and median concentrations detected, respectively. The potentially affected fraction of species (PAF) likely to be acutely impacted ranged from 4.6 to 8.1% (fipronil), 0.2-1.6% (fipronil sulfone), and 1.9-3.1% (fipronil sulfide) in the ponds with frequent detectable concentrations. The PAF likely to be impacted at chronic toxicity levels ranged from 16.5 to 23.8% for fipronil. Joint probability curve analysis indicated that concentrations exceeded the LC50 of the most sensitive 5% of species 8.5-18.8% of the time at two of the sites with the most frequent detections. Using the more conservative NOEC/LOEC values, there was a 75-78% probability that concentrations were high enough to negatively affect the most sensitive 5% of species at the same two sites, indicating significant risks for chronic toxicity. JPCs indicated a ≤2.6% probability of fipronil sulfone exceeding the LC50 concentrations for the most sensitive 5% of species at the same two sites; and a 4.3-6.8% probability of fipronil sulfide exceeding the LC50 concentrations at the same sites. Results indicate that fipronil and its sulfone and sulfide degradation products may present significant risks to aquatic organisms in some residentially-developed areas.

Norflurazon and simazine removal from surface water using a constructed wetland.

Norflurazon and simazine are pre-emergent herbicides detected frequently in surface water associated with South Florida agricultural canals and drainage water. This study investigated the potential use of a 1.34 ha constructed wetland for removing these herbicides from surface water. The total length of the wetland was 400 m and width was 35 m. A surface water flow rate of 740 L/min was maintained in the system using a pump. The plant community within the system consisted primarily of Panicum repens, Alternanthera philoxeroides, and Bacopa caroliniana. Norflurazon and simazine, derived from commercial formulations, were injected (51.1 g active ingredient each) directly into the water pumped into the wetland over a 2 h period. Water samples were collected from the wetland upstream of the dosing system at 3 h intervals from the beginning through 360 h and at the exit point at 1, 2, and 3 h intervals for the periods of 0-24, 25-48 and 49-360 h after dosing, respectively. The herbicides were extracted using C-18 cartridges and were analyzed by GC-TSD. The total mass of each herbicide discharged from the system was estimated by multiplying the concentration by the total volume discharged during the sampled period. Neither herbicide was detected in the inflow water during the entire study. Norflurazon was first detected at the exit 19 h after dosing and simazine after 23 h. Discharge patterns of the two herbicides differed dramatically. Norflurazon tended to bleed off from the wetland with no distinct peak concentration. However, the mobile fraction of simazine was discharged over a 58 h period. Mean/maximum/median detectable concentrations of the herbicides were 3.9 ± 1.7/8.1/3.4 µg L(-1) for norflurazon, and 11.9 ± 6.8/23.6/12.0 µg L(-1) for simazine, respectively. The total masses of norflurazon and simazine discharged from the exit during the 15 day study were 51.7 and 26.9 g, indicating 0% and 47.4% removal from the surface water by the system.

Copper clean-up procedure for ultrasonic extraction and analysis of pyrethroid and phenylpyrazole pesticides in sediments by gas chromatography-electron capture detection.

A rapid ultrasonic extraction method coupled with a heated-copper clean-up procedure for removing interfering constituents was developed for analyzing pyrethroid and phenylpyrazole pesticides in sediments. Incubation of the 60 mL extract with 12 g copper granules at 60 °C for 2h was determined to be the optimal conditions for removing the interfering constituents. Eleven pyrethroid and phenylpyrazole pesticides were spiked into sediment samples to determine the effectiveness of the ultrasonic extraction method. The average recoveries of pyrethroids and phenylpyrazoles in sediment at 4 °C storage on day 0, 1, 7, 14, and 21 ranged from 98.6 to 120.0%, 79.2 to 116.0%, 85.0 to 119.7%, 93.6 to 118.7%, and 92.1 to 118.2%, respectively, with all percent relative standard deviations less than 10% (most <6%). This illustrated the stability of pyrethroids and phenylpyrazoles in sediment during sediment aging at 4 °C. Recoveries of the pesticides ranged from 98.6% to 120.0% for lowest fortification level (2-16 µg kg⁻¹), from 97.8% to 117.9% for middle fortification level (10-80 µg kg⁻¹), and from 94.3% to 118.1% for highest fortification level (20-160 µg kg⁻¹). Relative standard deviations of pesticide recoveries were usually less than 7%. Method detection limits of target pesticides ranged from 0.22 µg kg⁻¹ to 3.72 µg kg⁻¹. Furthermore, field sediment samples collected from four residential lakes during a three-month monitoring period were analyzed to evaluate the effectiveness of this method. Bifenthrin was detected in all of sediment samples (highest concentration 260.33±41.71 µg kg⁻¹, lowest concentration 5.68±0.38 µg kg⁻¹, and fipronil sulfone was detected at least once in sediment samples collected from three sites with concentrations ranging from 1.73±0.53 to 7.53±0.01 µg kg⁻¹.

Effective liquid-liquid extraction method for analysis of pyrethroid and phenylpyrazole pesticides in emulsion-prone surface water samples.

The distribution of pyrethroid and phenylpyrazole pesticides in the water environment has raised public concerns because of their potential risks to ecosystem and human health. However, co-extraction of emulsifier type compounds (by liquid-liquid extraction, LLE) present in environmental samples can present a challenge for quantifying typically low concentrations of pesticides. Several methods were evaluated for breaking emulsions in problematic environmental surface water samples extracted by LLE using methylene chloride. Target pesticides included 11 typical pyrethroid and phenylpyrazole pesticides commonly used in agricultural and landscape insect pest control. The most effective method was selected for validation in fortification studies with GC-ECD analysis. The average recoveries of spiked pyrethroid and phenylpyrazole pesticides were 88.2-123.4% for water samples with moderate emulsions and 93.0-117.4% for water samples with severe emulsions. Recoveries of the pesticides ranged 81.0-126.4% (water samples with moderate emulsions) and 95.9-110.6% (water samples with severe emulsions) for lowest fortification level (5-20 ngL(-1)), 88.2-123.4% (water samples with moderate emulsions) and 93.0-117.4% (water samples with severe emulsions) for middle fortification level (10-40 ngL(-1)), and 90.2-119.9% (water samples with moderate emulsions) and 91.2-105.9% (water samples with severe emulsions) for highest fortification level (50-200 ngL(-1)). Relative standard deviations of pesticide recoveries were usually < 10%. Results indicate that this method is a robust and reproducible option for LLE of pyrethroid and phenylpyrazole pesticides from emulsion-prone surface water samples.

A Nylon Membrane Bag Assay for Determination of the Effect of Chemicals on Soilborne Plant Pathogens in Soil.

A new nylon membrane bag (NMB) assay was developed for studies to determine the effect of chemicals added to soil on survival of soilborne plant pathogens. The rapid and effective assay can be used to study organisms for which there are no selective media or for which a selective medium is expensive or difficult to prepare. This assay consists of placing pathogens inside a bag made of small-pore (0.22-µm) nylon filtration membrane, which is placed in soil and later retrieved to determine survival of the pathogens on nonselective media. Chemicals but not other microorganisms can enter the bag from the soil. Using this assay, Streptomyces scabies, Fusarium oxysporum f. sp. lycopersici race 3, and Ralstonia solanacearum were successfully recovered from soil after 72 h as demonstrated by growth on a semiselective Streptomyces medium (S. scabies) or nonselective potato dextrose agar medium (F. oxysporum f. sp. lycopersici race 3 and R. solanacearum) with minimal microbial contamination. Addition of acetic acid (200 mM) to soil killed 100% of S. scabies. SPK (a mixture of organic chemicals) at a concentration of 1,500 mg kg-1 of soil killed 83.3% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 100% of F. oxysporum f. sp. lycopersici race 3 spores, and 97.2% of R. solanacearum cells. SPK at 1,000 mg kg-1 of soil killed 50% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 68.2% of F. oxysporum f. sp. lycopersici race 3 spores, and 12% of R. solanacearum. Benlate (500 to 1,500 mg kg-1 of soil) did not kill the culture plugs of F. oxysporum f. sp. lycopersici race 3 but reduced the growth rate of F. oxysporum f. sp. lycopersici race 3. Benlate (500, 1,000, and 1,500 mg kg-1 of soil) reduced F. oxysporum f. sp. lycopersici race 3 spore germination by 39.4, 49.3, and 50.4%, respectively. Streptomycin sulfate (1,500, 800, 400, and 200 mg kg-1 of soil) caused 75.3, 21, 11.9, and 0.9% mortality, respectively, of R. solanacearum.

Organic-solvent-free extraction method for determination of carbamate and carbamoyloxime pesticides in soil and sediment samples.

This study evaluated the usefulness of monochloroacetic acid buffer (MCAAB) for extracting several carbamate/carbamoyloxime pesticides from a silt-loam soil and sediment, and an organic clay soil. The MCAAB extraction method, relative to acetonitrile and methanol extractants, was more accurate and precise for extraction of aldicarb, aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, carbofuran, 3-hydroxy-carbofuran, and propoxur; with recoveries ranging from 78.8% to 121.1%. Recoveries of carbaryl and methiocarb ranged from 0% to 64.1%. The MCCAB extraction method did not perform well for extraction of most compounds from the organic clay soil, with recoveries ranging from 0% to 66.7%.

Temporal and spatial variations of copper, cadmium, lead, and zinc in Ten Mile Creek in South Florida, USA.

Lead (Pb), zinc (Zn), copper (Cu), and cadmium (Cd) often seriously deteriorate water quality. Spatial and temporal fluctuations of the metal concentrations in the Ten Mile Creek (Florida) (TMC) were monitored on a weekly basis at 7 sampling sites, from June 2005 to September 2007. River sediment samples were also collected from these sites in April, June, and October 2006 and January 2007, and analyzed for water, Mehlich 1 (M1), and Mehlich 3 (M3)-extractable metals (Mehlich, 1953, 1984), to examine the role of sediments as sources or sinks of the metals. The concentrations of lead, zinc, copper, and cadmium in the water samples were

Temporal and spatial variations of nutrients in the Ten Mile Creek of South Florida, USA and effects on phytoplankton biomass.

Water quality throughout south Florida has been a major concern for many years. Nutrient enrichment in the Indian River Lagoon (IRL) is a major surface water issue and is suggested as a possible cause of symptoms of ecological degradation. In 2005-06, water samples were collected weekly from seven sites along Ten Mile Creek (TMC), which drains into the Indian River Lagoon, to investigate and analyze spatial and temporal fluctuations of nutrients nitrogen (N) and phosphorus (P). The objective of this study was to understand the relationships among chlorophyll a concentration, nutrient enrichment and hydrological parameters in the surface water body. High median concentrations of total P (TP, 0.272 mg L(-1)), PO4-P (0.122 mg L(-1)), and dissolved total P (DTP, 0.179 mg L(-1)); and total N (TN, 0.988 mg L(-1)), NO3(-)-N (0.104 mg L(-1)), NH4+-N (0.103 mg L(-1)), and total Kjeldahl N (TKN, 0.829 mg L(-1)), were measured in TMC. The concentrations of TP, PO4-P, DTP, TN, NO3(-)-N, NH4+-N, and TKN were higher in summer and fall than in winter and spring. However, chlorophyll a and pheophytin concentrations during this period in TMC varied in the range of 0.000-60.7 and 0.000-17.4 microg L(-1), with their median values of 3.54 and 3.02 microg L(-1), respectively. The greatest mean chlorophyll a (10.3 microg L(-1)) and pheophytin (5.71 microg L(-1)) concentrations occurred in spring, while the lowest chlorophyll a (1.49 microg L(-1)) and pheophytin (1.97 mug L(-1)) in fall. High concentrations of PO4-P (>0.16 mg L(-1)), DTP (>0.24 mg L(-1)), NO3(-)-N (>0.15 mg L(-1)), NH4+-N (>0.12 mg L(-1)), and TKN (>0.96 mg L(-1)), occurred in the upstream of TMC, while high concentrations of chlorophyll a (>6.8 mug L(-l)) and pheophytin (>3.9 microg L(-l)) were detected in the downstream of TMC. The highest chlorophyll a (11.8 mug L(-l)) and pheophytin (6.06 microg L(-l)) concentrations, however, were associated with static and open water conditions. Hydrological parameters (total dissolved solid, electrical conductivity, salinity, pH, and water temperature) were positively correlated with chlorophyll a and pheophytin concentrations (P < 0.01) and these factors overshadowed the relationships between N and P concentrations and chlorophyll a under field conditions. Principal component analysis and the ratios of DIN/DP and TN/TP in the water suggest that N is the limiting nutrient factor for phytoplankton growth in the TMC and elevated N relative to P is beneficial to the growth of phytoplankton, which is supported by laboratory culture experiments under controlled conditions.

Prereaction of Citrus tristeza virus (CTV) Specific Antibodies and Labeled Secondary Antibodies Increases Speed of Direct Tissue Blot Immunoassay for CTV.

An improved direct tissue blot immunoassay (DTBIA) procedure for detection of Citrus tristeza virus (CTV) within 1 h is described. Prints of fresh young stems of citrus plants that were infected or not infected with CTV were made by gently and evenly pressing the fresh-cut surface of the stems onto a nitrocellulose membrane. The tissue blots were air-dried for 5 min, incubated with prereaction solutions of CTV-specific antibodies and labeled secondary antibodies, goat anti-mouse Ig (H+L)-alkaline phosphatase conjugate or goat anti-rabbit IgG alkaline phos-phatase conjugate, for up to 20 min, rinsed with PBST buffer for 5 min, and immersed into an NBT-BCIP substrate solution for 15 to 20 min. Then the blots were rinsed in water for a few seconds to stop the reactions, and the results were observed and recorded under a light microscope. All samples from greenhouse plants that were infected with CTV decline inducing isolate T-36 were positive to CTV-specific polyclonal antibody 1212 (PCA 1212) and monoclonal antibodies 17G11 (MAb 17G11) and MCA13 (MAb MCA13), whereas samples from greenhouse plants infected with non-decline-inducing isolate T-30 were positive to PCA 1212 and MAb 17G11, but not to MAb MCA13. The noninfected greenhouse plants were negative to all of the antibodies. The improved DTBIA was at least as reliable as other immunological procedures and almost as reliable as polymerase chain reaction for detecting CTV in field trees. The improved DTBIA enables the detection of CTV within 1 h by having a prereaction of CTV-specific antibodies and labeled secondary antibodies in solutions before they are applied to the tissue blots. This DTBIA procedure may be useful in detecting other plant viruses and other pathogens such as bacteria and fungi.

In Situ Immunoassay (ISIA) of Field Grapefruit Trees Inoculated with Mild Isolates of Citrus tristeza virus Indicates Mixed Infections with Severe Isolates.

Ten grapefruit trees that had been inoculated with a mild isolate of Citrus tristeza virus (CTV) and maintained in the field for 18 years were found in a previous study to be declining and infected with severe isolates of CTV, or symptomless and infected with mild isolates of CTV, using enzyme-linked immunosorbent assay (ELISA). They were assayed with an in situ immunoassay (ISIA) procedure using monoclonal antibodies 17G11 (reacts with most Florida isolates of CTV) and MCA13 (reacts with severe, but not Florida mild isolates of CTV). All the grapefruit trees were 17G11 positive by ELISA and ISIA. The five trees that showed moderate decline symptoms were MCA13 positive by ELISA and ISIA. The five symptomless trees were MCA13 negative by ELISA. However, four of the five symptomless trees were MCA13 positive by ISIA, which showed that ISIA with MCA13 had greater sensitivity in detecting severe CTV isolates than ELISA. These results suggested that the cross-protected grapefruit trees, regardless of symptoms, were infected with both mild and severe isolates of CTV.

In Situ Immunoassay for Detection of Citrus tristeza virus.

An in situ immunoassay (ISIA) is described for detection of Citrus tristeza virus (CTV). Sections from stems, petioles, or leaf veins of citrus plants that were healthy or infected with CTV were fixed with 70% ethanol and incubated with specific polyclonal antiserum (PCA) 1212 or with monoclonal antibodies (MAbs) MCA13 or 17G11. Bound antibodies were labeled with enzyme-conjugated species-specific secondary antibodies and exposed to a substrate mixture (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate). Presence of CTV antigens was indicated by the development of a purple color, which could be visualized by light microscopy, in the phloem tissues of infected citrus plants. No purple color was observed in the phloem tissues of healthy plants. All isolates used in this study, both severe and mild, were detected by ISIA with the PCA 1212 and the broad spectrum MAb 17G11, but only severe isolates were detected by the strain selective MAb MCA13. Location of CTV antigens could be determined directly and accurately by ISIA in both fresh tissues and samples stored in plastic bags at 4°C or frozen for 4 weeks. Sensitivity of ISIA for detecting CTV in infected plants compared favorably with that of direct tissue blot immunoassay (DTBIA). ISIA is a simple, rapid, specific, and practical procedure for CTV identification applicable to both research and diagnostic needs.