Assessing the Movement of Cucurbit yellow stunting disorder virus in Susceptible and Tolerant Cucumber Germplasms Using Serological and Nucleic Acid-based Methods

Cucurbit yellow stunting disorder virus (CYSDV) is an emerging virus causing significant yield losses in cucurbits. Simple but reliable detection and quantification methods are important tools for disease management. In susceptible germplasm, CYSDV was detected 5 days postinoculation (dpi) by reverse transcriptase polymerase chain reaction (RT-PCR) or by tissue blot immunoassay (TBIA), and 8–9 dpi by dot blot immunoassay (DBIA) or enzyme-linked immunosorbent assay (ELISA). For CYSDV quantification, real-time RT-PCR was the most sensitive method and gave the best linear range of detection, over four orders of magnitude as compared to approximately two orders of magnitude for DBIA and nucleic acid hybridization. DBIA was more sensitive than ELISA and equally sensitive to nucleic acid hybridization with a non-radioactively labelled cDNA probe. Time course studies at 3, 5, 8 and 14 dpi using TBIA revealed that tolerance to CYSDV in three tolerant cucumber germplasms was not correlated with restricted or delayed virus movement. Introduction Cucurbit yellow stunting disorder virus (CYSDV) is a member of the genus Crinivirus (family Closteroviridae) efficiently transmitted by Bemisia tabaci, biotypes B (B. argentifolii) and Q (Berdiales et al., 1999). The natural host range of CYSDV is limited to the family Cucurbitaceae including important crops, such as cucumber, melon, watermelon and squash. The severe yellowing induced by CYSDV drastically affects fruit number and weight leading to a 30–50% yield reduction (Abou-Jawdah et al., 2000; López-Sése and Gomez-Guillamon, 2000). CYSDV was first reported in the United Arab Emirates in 1991, but may have caused cucurbit diseases in the Mediterranean region since the early 1980s (Hassan and Duffus, 1991). CYSDV has spread rapidly in the Arabic Peninsula, Eastern and Western Mediterranean basin Egypt, Israel, Jordan, Lebanon, Syria and Saudi Arabia (Duffus, 1995; Abou-Jawdah et al., 2000), Spain (Célix et al., 1996), Morocco (Desbiez et al., 2000), Portugal (Louro et al., 2000), France (Pacheco, 2005) and North America (Kao et al., 2000). CYSDV is considered a plant quarantine pest in several regions (Pacheco, 2005). Fast, simple, reliable and sensitive detection and quantification methods are required for monitoring of virus spread and host range, for determination of virus titre in resistant, tolerant or susceptible cucurbit genotypes, and for determination of virus load in insect vectors. Due to the labile nature of CYSDV particles, and the subsequent difficulty in development of serological tests, detection of CYSDV has relied for over a decade on nucleic acid hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). For CYSDV quantification, nucleic acid hybridization using chemiluminescent detection (CDP) system was predominantly used as it provides sensitive detection and is less hazardous than radioactive methods (Ruiz et al., 2002; Marco et al., 2003). Recently CYSDV coat protein (CP) was expressed in a bacterial expression system and used to develop CYSDV-specific antibodies that were used in serological assays to detect CYSDV (Hourani and Abou-Jawdah, 2003; Cotillon et al., 2005). Recently 124 cucumber accessions were evaluated for resistance against CYSDV. No immune or highly resistant accessions were detected, but some accessions (PI 605923, PI 293432 and PI 211589) were tolerant and showed delayed expression of symptoms, milder final symptoms, and lower percentages of infected plants compared with susceptible cucumber (Eid et al., 2006). Several mechanisms for virus tolerance have been reported, including resistance to virus movement between cells, resistance to long distance movement of www.blackwell-synergy.com J. Phytopathology 156, 438–445 (2008) doi: 10.1111/j.1439-0434.2007.01388.x 2008 The Authors Journal compilation 2008 Blackwell Verlag, Berlin virus within the plant and resistance to virus multiplication or to virus accumulation (Lecoq et al., 2004). We have investigated whether CYSDV tolerance in cucumber germplasms is correlated with delayed virus movement or accumulation, and evaluated the relative sensitivity of CYSDV serological detection methods as compared with nucleic acid-based methods. Materials and Methods Source of CYSDV infected tissue A CYSDV isolate collected from a CYSDV-infected cucumber plant grown in a greenhouse in North Lebanon was maintained in CYSDV-infected cucumber plants grown in 30-cm diameter plastic pots in insectproof glasshouse equipped with ventilation when temperature exceeds 30 C. New seedlings were periodically inoculated using whitefly transmission every 45–60 days. Leaf tissue samples were collected from plants showing prominent inter-veinal yellowing but before development of widespread yellowing. CYSDV detection by serological methods Tissue blot immunoassay Leaf midribs and ⁄ or petioles of CYSDV-infected and healthy cucumber plants were cut with a razor blade and immediately the pieces were blotted for 20 s onto positively charged Nylon membrane (Amersham Biosciences, Little Chalfont, Bucks, UK). Membrane blocking, application of primary and secondary antibodies and colorimetric detection methods were performed as described previously (Hourani and Abou-Jawdah, 2003). The dried membranes were examined under a Stemi DV4 Stereoscope (Carl Zeiss, Berlin, Germany) for colour precipitation in phloem cells where the virus is located. Dot blot immunoassays Leaf samples consisting of 1 g of leaf midrib were homogenized in 20 ml tris buffer saline pH 8.0+0.1% tween 20 (TBST) +0.1% sodium sulphite. Nylon membranes were soaked in distilled water for 10 min and then in extraction buffer for 1 min. After washing each well with 100 ll of TBST buffer; the sample extracts (6 ll of each leaf extract dilution+80 ll TBST) were applied under vacuum in the wells of the Bio-dot apparatus (Bio-Rad , Hercules, CA, USA). Serial dilutions (1 : 9 and 1 : 1) of infected leaf extracts in healthy leaf extracts were blotted on Nylon membranes. The membranes were blocked for 2 h with TBST buffer containing 5% skimmed milk (Regilait , Saint Martin Belle Roche, France)+1%bovine serum albumin (Fraction V, reagent grade, BIOREBA AG , Reinach, Switzerland). CYSDV was immunologically localized using purified CYSDV polyclonal antibodies (1 : 1000) as primary antibodies and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulins (Sigma, St Louis, MO, USA) as secondary antibodies (1 : 10 000) (Hourani and Abou-Jawdah, 2003). For detection, the CDP-star substrate (Amersham Biosciences) was diluted (1 ⁄ 20) and applied to the membranes for 5 min. Thirty minutes after draining off excess detection reagent, the best exposure time of X-ray films (AGFA CP-BU, Medical X-ray film blue; Agfa-Gevaert N.V, Morstel, Belgium) was usually approximately 20 min. To quantify the signal intensity of each dot, autoradiograms were scanned and computer analyzed using the Labworks imaging and analysis software (Ultra-Violet products Ltd., Cambridge, UK), the signal of the healthy control was considered as background reference.