Seed-borne Bacterium Research Articles

New subspecies‐specific polymerase chain reaction‐based assay for the detection of Acidovorax avenae subsp. citrulli

New subspecies‐specific primers for the detection of Acidovorax avenae subsp. citrulli (Aac), the seedborne bacterium which causes bacterial fruit blotch of cucurbits, were designed based on PCR fragments obtained in ERIC‐ and BOX‐PCR profiles of Aac strains. PCR with both primer sets identified 30 strains from different locations and hosts, but did not amplify DNA from other closely related species and subspecies assessed, with the exception of DNA of one strain of A. avenae subsp. avenae, which was amplified by one of the primer sets, named BX‐S. This primer set, based on a BOX‐PCR fragment, performed under high‐stringency conditions without losing its detection sensitivity. The primers were also evaluated for their ability to detect the pathogen in contaminated watermelon and melon seed samples. The BX‐S primers facilitated the detection of the pathogen from washings of 5000‐seed samples with 0·02% infestation. This primer set was also assessed for detection using immunomagnetic separation polymerase chain reaction (IMS‐PCR) and was shown to be as sensitive as a previously described primer set (AACF2/R3), detecting 0·02% infestation in seed samples. This highly specific and sensitive primer set could be used to improve PCR‐based detection of this important pathogen.

Meio de cultura semi-seletivo para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em solo e sementes de feijoeiro

A murcha-de-curtobacterium, causada pela bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), é um importante problema para o cultivo de feijão (Phaseolus vulgaris L.) na região Centro-Sul do Brasil. A principal forma de disseminação da bactéria é por sementes contaminadas. Embora a ocorrência desta doença seja relativamente recente no Brasil, Cff tem sido disseminado rapidamente nas regiões produtoras de feijão do país. Este trabalho teve como objetivo ajustar o meio de cultura CNS (Corynebacterium Nebraskense Selective Medium) para recuperação e detecção de Cff em solo e sementes de feijoeiro. Suspensões provenientes da lavagem de amostras de solo e sementes contaminadas pela bactéria foram plaqueadas no meio de cultura CNS - modificado. Os ajustes realizados no meio de cultura possibilitaram a recuperação eficiente de Cff presente em solo e sementes contaminadas. As modificações estabelecidas para o meio CNS foram a redução da concentração de sulfato de polimixina para 16 mg/l, exclusão do componente ciclohexamida e substituição do fungicida Daconil 2787-F (530 mg/ml de chlorothalonil) pelo Dacostar 500 (500 mg/ml de chlorothalonil).

PCR‐based detection of Xanthomonas campestris pathovars in Brassica seed

Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR‐based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris. Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false‐negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed‐washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.

Inheritance of Resistance to Halo Blight Flower and Stem Color and Association in Common Beans

Halo blight is one of the most important bacterial diseases of common beans (Phaseolus vulgaris L.). It is serious under moderate temperature and high humidity conditions. The disease is caused by a seed-borne bacterium, Pseudomonas syringae pv. phaseolicola (Burkh.) Dowson (Psp). The inheritance of leaf reactions to Psp, flower, and stem color was studied using greenhouse-grown 109 F9 recombinant inbred lines (RI) from the P. vulgaris cross BelNeb 1 [resistant (R)] (USDA/NE) × A 55 [susceptible (S)] (CIAT). Two Psp strains, HB16 (NE) and 83-Sc2A (NE), were inoculated using the water-soaking method. A segregation ratio of 1 R:1 S RI lines were observed for disease reactions in leaves for both strains indicating major gene control. The presence of recombinants for SR, RS to the strains indicated that different genes were involved. Stem (SC) and flower (FC) color traits were each determined by two major genes. Linkages were found for reactions to the two Psp strains and also between FC and SC. No linkages were observed from FC and also SC with reactions to Psp strains.