Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola Are a Main Cause of Halo Blight of Beans in Spain and Escape Current Detection Methods

Abstract

From a collection of 152 pseudomonads isolated from diseased beans in Spain, 138 (91%) of the strains were identified as Pseudomonas syringae pv. phaseolicola and the rest as P. syringae pv. syringae. The P. syringae pv. phaseolicola strains produced typical water-soaked lesions on bean pods, although 95 of them did not produce phaseolotoxin in vitro. Ninety-four of these isolates did not produce the expected 0.5-kb product after polymerase chain reaction (PCR) amplification using primers specific for open reading frame (ORF) 6 of the phaseolotoxin (tox) gene cluster and did not contain DNA homologous to ORF 6 in Southern hybridization experiments. To our knowledge, this is the first report of the widespread occurrence in the field of strains of P. syringae pv. phaseolicola lacking the tox cluster, which contrasts sharply with the general belief that Tox(+) isolates are the only ones with epidemiological importance. Additionally, the tox(-) isolates were not specifically detected by a commercial polyclonal antisera in an enzyme-linked immunosorbent assay. Accordingly, it is possible that the certification of seed lots as free of the pathogen cannot be reliably done in Spain, or in any other country where tox(-) strains might occur frequently, using current PCR or serological protocols. The amplification of three avirulence genes by PCR allowed us to make predictions of the P. syringae pv. phaseolicola race structure, as confirmed by plant assays. Six races (races 1, 2, 5, 6, 7, and 9) were identified, with race 7 being the most prevalent (46.1%) followed by races 6 (21.3%) and 1 (9.0%). All the tox(-) isolates contained gene avrPphF, typical of races 1, 5, 7, and 9.

Short communication. Detection by multiplex PCR and characterization of nontoxigenic strains of Pseudomonas syringae pv. phaseolicola from different places in Spain

“…However, it was recently showed that more than 66% of the P. syringae pv. phaseolicola isolates causing field epidemics in Castilla y León, Spain, were nontoxigenic and did not contain DNA specific for the biosynthesis of phaseolotoxin (Rico et al, 2003;Oguiza et al, 2004). The strains of P. syringae pv.…”

“…Incidentally, all the P. syringae pv. phaseolicola tox-strains analyzed so far also lacked other genes of the phaseolotoxin biosynthesis cluster and failed to produce phaseolotoxin (Rico et al, 2003;Oguiza et al, 2004). Additionally, the tox-isolates also failed to react with one of the few commercial antibodies for the serological detection of P. syringae pv.…”

“…The strains of P. syringae pv. phaseolicola that lacked the phaseolotoxin gene ORF6 were designated as tox-, whereas those containing this gene were designated as tox+ (Rico et al, 2003). Consequently, this designation will be used from now on because it is helpful to distinguish those strains that can (tox+) or cannot (tox-) be detected by current PCR methods using primers directed to the phaseolotoxin cluster.…”

“…Additionally, the tox-isolates also failed to react with one of the few commercial antibodies for the serological detection of P. syringae pv. phaseolicola (Rico et al, 2003), all of which are polyclonal. In consequence, the efficient certification of bean seed in Spain using current highly sensitive methods, such as PCR and serological methods, will depend on the possible distribution of tox-populations.…”

“…phaseolicola tox-isolates (Table 3). The identification of the fluorescent colonies obtained from the diluted soakates from these five seed lots was performed essentially as described (Lelliott and Stead, 1987;Braun-Kiewnick and Sands, 2001) following the LOPAT scheme (Lelliott and Stead, 1987), with slight modifications (Cazorla, 1998;Rico et al, 2003). Degradation of casein was examined on MT plates (Goszczynska and Serfontein, 1998).…”