Phyllis A. Rundell

Detection and Isolate Differentiation of Citrus tristeza virus in Infected Field Trees Based on Reverse Transcription-Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was compared with enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) for detection of non-decline-inducing and decline-inducing isolates of Citrus tristeza virus (CTV) in 21 field sweet orange and grapefruit plants on sour orange rootstock in Fort Pierce, FL. Among these samples, seven, six, and eight were infected with decline-inducing, non-decline-inducing, and both decline-inducing and non-decline-inducing isolates of CTV, respectively. However, there was not a good correlation between field symptoms and detection of the decline-inducing isolate. The results confirmed that RT-PCR is not only able to detect and differentiate decline-inducing and non-decline-inducing isolates of CTV in Florida, but also can detect both isolate types in a single field sweet orange or grapefruit tree. For most samples, results from RT-PCR, ELISA, and DTBIA were the same. However, the 320-bp fragments produced only from decline-inducing isolates were amplified from two sweet orange and two grapefruit samples that did not react with decline-inducing CTV-specific monoclonal antibody MCA13 in ELISA or DTBIA, indicating that RT-PCR has a higher sensitivity than these immunological tests for field sweet orange or grapefruit samples. Thus, RT-PCR is a simple, rapid, and specific procedure for CTV identification applicable to both research and diagnostic needs.

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.

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.

Cross-Protection of Grapefruit from Decline-Inducing Isolates of Citrus Tristeza Virus

The ability of three mild isolates of citrus tristeza virus (CTV) to prevent natural infection of 84 Ruby Red grapefruit on sour orange rootstock by aphid-transmitted, decline-inducing isolates of CTV was assessed by symptoms and verified by enzyme-linked immunosorbent assay (ELISA) after 16 years. Of 21 trees in each of four treatments protected by the DD 102 bb, Guettler HS, and DPI 1-12-5-X-E mild CTV isolates, 14, 10, and 14% were infected by severe isolates (MCA13 monoclonal antibody reactive) compared with 67% for unprotected control trees. The health of trees protected by the DD 102 bb CTV isolate was significantly better than that of unprotected control trees as measured by decline, tree ratings, and tree height. These data suggest that infection by certain mild isolates of CTV can cross-protect grapefruit trees on sour orange rootstock from decline-inducing isolates of CTV that are prevalent in the Indian River region of Florida.

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.