Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR.

AIMS: To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot. METHODS AND RESULTS: We verified the specificity and sensitivity of the previously reported sets of primers designed for strains of the four species of Brazilian tomato bacterial spot xanthomonads, consisting of 30 of Xanthomonas euvesicatoria, 30 of X. vesicatoria, 50 of X. perforans and 50 of X. gardneri. Furthermore, we tested a multiplex PCR protocol for the purpose of concurrent species identification. The possibility of direct detection of the pathogens in diseased leaf samples was also verified. The primers were highly specific, amplifying only target DNA. The sensitivity of the primers in conventional PCR was 50 pg µl(-1) for purified DNA and ranged from 5 × 10(2) to 5 × 10(4) CFU ml(-1) when bacterial suspensions were analysed. The multiplex PCR was suitable for the detection of all four species and showed similar sensitivity to conventional PCR when tested on purified DNA. When using bacterial suspensions, its sensitivity was similar to conventional PCR only when a biological amplification step (Bio-PCR) was included. Both methods were able to detect the pathogens in symptomatic tomato leaves. CONCLUSIONS: Brazilian Xanthomonas strains causing tomato bacterial spot can be differentiated and identified at species level by a PCR-based method and by a multiplex PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This protocol may be a feasible alternative tool for the identification and detection of these pathogens in plant material and may be used for routine diagnostic purposes in plant pathology laboratories.

First Report of a Bacterial Leaf and Fruit Spot of Cashew Nut (Anacardium occidentale) Caused by Xanthomonas campestris pv. mangiferaeindicae in Brazil.

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.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.