Youjian Lin

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.

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 <detection limit (DL)-309, < DL-102, < DL-106, and < DL-15.7 microg/L, respectively. Even though median concentrations of lead (<DL), zinc (6.45 microg/L), copper (2.53 microg/L), and cadmium (<DL) were lower than their respective U.S. Environmental Protection Agency (Washington, D.C.) limits for freshwater (2.5 microg/L for lead, 120 microg/L for zinc, 9.0 microg/L for copper, and 0.25 microg/L for cadmium), pulse concentrations of the metals significantly exceeded these limits. Correlations of lead, zinc, copper, and cadmium concentrations in the TMC to other environmental factors, including the results of principal component analysis, suggest that there are different sources of metals. These sources include surface runoff from agricultural lands or urban wastewater, geological backgrounds, and tidal flow. Water-, M1-, and M3-extractable lead, zinc, copper, and cadmium concentrations in river sediments indicate the possibility that river sediment serves as an internal source of the metals for the TMC. This hypothesis was supported by positive correlations between concentrations of copper, lead, and zinc in the river water and their respective water-extractable concentrations in the sediments.

Nitrogen versus phosphorus limitation of phytoplankton growth in Ten Mile Creek, Florida, USA

Ten Mile Creek (TMC) is a major tributary of the Indian River Lagoon (IRL), one of the largest and most ecologically diverse estuaries of the east coast of Florida. Recent algal blooms within the IRL have focused attention on the role of different watersheds playing in the supply of growth-limiting nutrients. The goal of this study was to determine the nutrient-limiting status of the TMC outflow, which is influenced by both agricultural input and urban development. Four laboratory experiments were conducted with water samples from TMC, adding different concentrations of phosphorus (P) and nitrogen (N) under controlled conditions. The results showed that turbidity and phytoplankton biomass (in terms of chlorophyll a concentration) in TMC water samples were responsive to N additions. Turbidity and phytoplankton biomass increased with addition of available N, but were not affected by addition of reactive P. The results indicate that available N is the limiting nutrient for the growth of phytoplankton in the TMC.

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.

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.

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.