Multiplex Nested PCR for Detection ofXanthomonas axonopodispv. allii from Onion Seeds

Abstract

Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes (pilW and pilX) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444-to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 ؋ 10 2 CFU g ؊1 or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen.

Study on the stability and antioxidant effect of the Allium ursinumwatery extract

“…This paper, besides presenting an impressive number of chemically characterised compounds, confirms previous data according to which the main component with a therapeutic action is allicin [9,10]. A. ursinum (ramson) and other representatives of the Allium species, contain 1–5% nonprotein secondary metabolites of aminosulfuric acids [11]. In the cell, the stable precursor of the antibacterial principle of Cavallito, the S–oxyde (+/−) of S–2–propenyl cysteine and the S–oxyde of S–alkenyl cysteine (odour and flavour precursors) are located in the cytoplasm while the enzyme alliinase in the vacuole [12].…”

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

“…) or using nested PCR (Robène‐Soustrade et al . ) has been shown to improve the efficiency and sensitivity of multiplex PCR. These processes have been effective in reducing PCR inhibition factors as the formation of primer dimers that may occur before the start of thermocycling (4–25°C).…”

“…; Robène‐Soustrade et al . ). The methods or protocols described so far for tomato bacterial spot have not been able to accomplish this simultaneous detection and identification of the different species causing the disease.…”

Xanthomonas axonopodis pv. allii

“…Xanthomonas axonopodis pv. allii can survive and retain some culturability for at least 10 years in association with onion seed stored in a cool place (4°C) (Robene-Soustrade et al, 2005). Xanthomonas axonopodis pv.…”