Abstract
The efficient control of halo blight, caused by Pseudomonas syringae pv. phaseolicola, is primarily based on the use of pathogen-free seed. Detection of the pathogen in seeds is currently carried out with high-sensitive methods based on the detection by PCR of genes involved in the biosynthesis of phaseolotoxin, which was believed to be produced by all strains of the pathogen with epidemiological importance. However, field epidemics of halo blight in the county of Castilla y León, Spain, are often associated to nontoxigenic isolates of P. syringae pv. phaseolicola, which cannot be detected using current molecular and serological methods. The results presented in this work show the existence of nontoxigenic isolates of P. syringae pv. phaseolicola in areas other than Castilla y León, indicating the need to establish a reliable methodology for seed certification. A simple two-step methodology is presented with the aim to identify both types of isolates that is based on a multiplex enrichment PCR of seed soakates and on pathogenicity assays.
Highlights
The most relevant bacterioses of bean are those caused by the Gram negative bacteria Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae and Xanthomonas campestris pv. phaseoli, which result in important economical losses worldwide (Smith et al, 1988; Saettler, 1991)
Several highly sensitive PCR-based methods have been designed for the detection of P. syringae pv. phaseolicola, and all of them rely on the detection of DNA sequences involved in the biosynthesis of phaseolotoxin (Tourte and Manceau, 1991; De la FuenteMartínez et al, 1992; Prosen et al, 1993; Schaad et al, 1995; Audy et al, 1996)
It is generally accepted that the genes for the biosynthesis of phaseolotoxin are present in all the strains of P. syringae pv. phaseolicola found causing field epidemics (Rudolph, 1995; Schaad et al, 1995)
Summary
P3004L and P3004R, which amplify a 0.24 kb fragment (GenBank acc no. AJ568001) that has been observed only in nontoxigenic strains (Oguiza et al, 2004). 100 μl aliquots of a 1/10 dilution of the concentrated seed soakate were cultured on MT plates for 24-48 h at 28oC, and putative colonies belonging to P. syringae pvs. Phaseolicola and syringae were identified by their fluorescence and the presence or absence of haloes due to the hydrolysis of casein (Goszczynska and Serfontein, 1998). Fluorescent colonies producing water-soaking or sunken-brown lesions on bean pods, characteristic of P. syringae pvs. No colonies were observed in 17 out of the 22 seed lots examined, or the few encountered were not fluorescent. In all these cases no amplification bands were produced by multiplexed PCR, and these seed lots were not further assayed.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
