UC Riverside International Organization of Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Distribution of Xylella fastidiosa within Sweet Orange Trees : Influence of Age and Level of Symptom Expression of Citrus Variegated Chlorosis Permalink

The internal distribution of Xylella fastidiosa in 2-, 4and 6-yr-old trees of Pera sweet orange with citrus variegated chlorosis (CVC) symptoms levels (I, II and III) was evaluated . The aim of this study was to determine the X. fastidiosa distribution within the citrus to support pruning height recommendations as a control method. Plants were segmented into five parts and three samples per segment were analyzed. The diagnostic methodology was based on PCR, using specific primers for X. fastidiosa strains that cause CVC. In plants with symptom levels II and III, independent of the age, the bacteria were detected in the trunk and even near the primary branches. Plants with level I symptom expression, mainly the 4-yr-old, showed the lowest frequency of positive PCR results in the woody tissue. The high frequency of positive PCR results in asymptomatic leaves suggests a need for a constant inspection in the orchard, since our data showed that previous identification of CVC symptoms facilitates integrated management of CVC. Citrus variegated chlorosis (CVC) was detected in Brazil in 1987 and has spread quickly in all Brazilian citrus-growing regions. This disease is caused by the xylemlimited bacterium Xylella fastidiosa (4, 6), which is transmitted by sharpshooter leafhoppers (Hemiptera: Cicadellidae) (8). Because of the economic and social importance of Brazilian citrus culture, and the damage caused by CVC in Brazilian orchards, a great deal of effort has been done by researchers and growers to solve this problem. In São Paulo State, about 40% of the sweet orange plants show CVC symptoms, with the greatest percentage of plants having mild symptoms (FUNDECITRUS, unpublished data, 1997). The systemic occurrence of X. fastidiosa and limitation to xylem vessels hinder the development of chemical or biological control. On the other hand, its apparent slow growth in plant and reduced movement against the flow of water in the xylem could be the basis for a possible control by eliminating infected branches, thus reducing the systemic infection as well as reducing the inoculum source within the orchard. Such practice has been done by some growers (13), but it is costly. Therefore, better knowledge of how X. fastidiosa is distributed within affected citrus would help growers make correct decisions about pruning. The objective of this work was to evaluate, using specific PCR-based diagnostic assays, the distribution of the X. fastidiosa in Pera sweet orange at different ages and levels of symptom expression of CVC growing in the field. MATERIALS AND METHODS Plant material and sampling methodology. Plants of different ages (2, 4 and 6 yr), and different CVC symptoms expression levels (EL) were analyzed: I = one branch with mild symptoms on one leaf); II = two branches with mild symptoms on several leaves; and III = branches with symptoms on all leaves. Three plants of each age and level of symptoms were used. Five sections in the canopy were sampled as shown in Fig. 1: main trunk; primary branches; first, second and third secondary branches. 244 Fourteenth IOCV Conference, 2000—Insect-Transmitted Procaryotes Woody tissue of each segment and leaves above the third secondary branch were sampled. Three samples per section were analyzed. Samples of the woody tissues and petioles of leaves were collected for DNA extraction. Selected trees were taken from two orchards located in the North (Barretos) and Central (Araraquara) regions of São Paulo State, Brazil. Both orchards containing trees with the three EL symptoms and included trees without symptoms. DNA extraction and PCR amplification. The samples were ground to a fine powder in liquid nitrogen and the DNA was extracted from tissues according to modifications of methodology described by Murray and Thompson (11) and used by Machado et al. (9). The DNA samples were redissolved in 15 μ l of 1/10 TE (1 mM Tris-HCl pH8.0; 0.1 mM EDTA) containing 10 μ g/ μ l RNAse DNA free and used for PCR analysis. Amplification of the samples was conducted in volumes of 25 μ l with 1/10 volume of 10 × buffer (100 mM Tris-HCl pH 8.3; 500 mM KCl; 25 mM MgCl 2 ; 0.01% gelatin); 200 mM of each dATP, dCTP dGTP dTTP nucleotides; 50 ng of DNA; 0.3 units of Taq polymerase; and primers CVC-1 and 272-2 int (12). The amplifications were performed in a thermocycler (RoboCyclerTM, STRATAGENE) programmed with a first denaturation at 94 ° C for 2 min followed by 30 cycles of 1 min at 94 ° C; 1 min at 60 ° C; 2 min at 72 ° C, with a final extension at 72 ° C for 10 min. Following PCR, aliquots of 10 μ l of amplification products were analyzed by electrophoresis in 1.0% agarose gels containing 0.5 μ g/ml of ethidium bromide. Positive and negative controls obtained from leaves with CVC symptoms and leaves from shoottip-grafting plant growing under greenhouse conditions (without CVC symptoms), respectively, were used for all the DNA extraction procedures, PCR amplification and gel electrophoresis. DNA from X. fastidiosa (isolates from coffee, periwinkle, plum and grape) and DNA from endophytic bacteria ( Methylobacterium sp. and Curtobacterium sp. (W. Araújo, personal communication)) isolated from citrus were used to confirm primer specificity.