In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Bacterial canker of grapevine (Vitis vinifera), caused by Xanthomonas campestris pv. viticola was first detected in Brazil in 1998, affecting grapevines in the São Francisco river basin, state of Pernambuco. The disease was also reported in Juazeiro, Bahia and later in Piauí and Ceará. Due to its limited geographical distribution and relatively recent detection in Brazil, very little is known about the pathogen's biology and diversity. Repetitive DNA based-PCR (rep-PCR) profiles were generated from purified bacterial DNA of 40 field strains of X. campestris pv. viticola, collected between 1998 and 2001 in the states of Pernambuco, Bahia and Piauí. Combined analysis of the PCR patterns obtained with primers REP, ERIC and BOX, showed a high degree of similarity among Brazilian strains and the Indian type strain NCPPB 2475. Similar genomic patterns with several diagnostic bands, present in all strains, could be detected. Fingerprints were distinct from those of strains representing other pathovars and from a yellow non-pathogenic isolate from grape leaves. The polymorphism observed among the Brazilian strains allowed their separation into five subgroups, although with no correlation with cultivar of origin, geographic location or year collected.

In 2003 and 2004, leaves and young fruits of cashew nut plants showing an undescribed disease symptom were observed on plants of an early-dwarf clone in a commercial orchard in Ceará and Piauí states in northeastern Brazil. Initial symptoms consisted of angular, water-soaked, dark-to-black spots on the leaf and at the mid-rib vein surrounding the leaf veins. Eventually, lesions also extended from the mid-rib to the secondary veins, delineating the vein system of the leaf. In young, green fruits, symptoms were large, dark, oily spots surrounded by conspicuous water-soaked areas. A yellow-pigmented colony was consistently recovered from the lesions on nutrient yeast-extract dextrose agar medium (3 g of meat extract, 5 g of peptone, 10 g of dextrose, 5 g of yeast extract, and 18 g of agar per liter). Physiological tests revealed colonies that were gram negative, strictly aerobic, oxidase negative, catalase positive, lacking fluorescent pigmentation on King's B medium, urea hydrolase negative, and able to grow on yeast dextrose calcium carbonate medium yielding yellow colonies. These tests indicated that the bacterium belonged to the genus Xanthomonas. PCR amplification of bacterial DNA using RST2 (1) and Xcv3R (3) primers resulted in identical band patterns to mango isolates Xanthomonas campestris pv. mangiferaeindicae. Restriction fragment length polymorphism analysis of PCR-amplified products of six isolates of X. campestris pv. mangiferaeindicae was conducted with HaeIII and showed different profile patterns on agarose gel, indicating genetic variability among these isolates. Pathogenicity was demonstrated by gently piercing and misting cashew leaves with a bacterial suspension adjusted to 106 CFU/ml. Inoculated plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (29 ± 1°C). Control plants were misted with sterile water and treated the same way. After 8 days, foliar symptoms similar to those observed in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be X. campestris pv. mangiferaeindicae. Control plants remained symptomless. To our knowledge, this is the first description of commercially grown cashew plants as host to X. campestris pv. mangiferaeindicae in Brazil. This disease may pose a serious problem to the cashew-growing industry in Brazil. This bacterial pathogen has been reported on mangoes (Mangifera indica) and cashew in India (2) under the former name of Pseudomonas mangiferae-indicae. References: (1) R. P. Leite, Jr. et al. Appl. Environ. Microbiol. 60:1068, 1994. (2) M. K. Patel et al. Curr. Sci. 17:189, 1948. (3) L. C. Trindade et al. Summa Phytopathol. 33:16, 2007.