Determination and Confirmation of Nicotinic Acid and Its Analogues and Derivates in Pear and Apple Blossoms Using High-Performance Liquid Chromatography−Diode Array−Electrospray Ionization Mass Spectrometry

Abstract

Erwinia amylovora causes fire blight, a serious disease of apple and pear. The bacterial pathogen colonizes the flower stigma and hypanthium, where it multiplies and then invades through natural openings (nectarthodes). E. amylovora requires nicotinic acid as growth factor, and competition for nicotinic acid is being explored as a novel biocontrol strategy. The ability of E. amylovora to substitute nicotinic acid with analogues or derivates as growth factors has not been investigated yet. Furthermore, the presence and/or variable concentration of nicotinic acid and its analogues/derivates in the hypanthium could be associated with the different susceptibilities to fire blight of hosts and nonhosts and with the differential sensitivity to the disease among apple and pear varieties. Currently, no methods to specifically quantify nicotinic acid and nicotinic acid analogues/derivates in the hypanthium of apple and pear blossoms are available. This study demonstrates that E. amylovora can grow using nicotinamide and 6-hydroxynicotinic acid as alternative growth factors to nicotinic acid, but not using 2-hydroxynicotinic acid. A novel HPLC/ES-MS method was developed for the detection and quantification of nicotinic acid and its analogues/derivates directly in the hypanthium of apple and pear blossoms. Analyses established the presence of nicotinic acid and nicotinamide, whereas no detectable amounts of 6-hydroxynicotinic acid and 2-hydroxynicotinic acid were observed. Mean nicotinic acid content in the pear hypanthium was found to be approximately 2 orders of magnitude higher than in the apple hypanthium, which may contribute to the differential susceptibility of these two host species to fire blight. Contents of nicotinamide were in contrast similar. Nicotinic acid can therefore be considered a relevant factor in the pathogen establishment in pear blossoms, whereas nicotinamide could cover a primary role in apple blossoms.

Cytosine chemoreceptor McpC in Pseudomonas putida F1 also detects nicotinic acid

“…The use of nicotinic acid-degrading strains of Pseudomonas rhizosphaerae and Pseudomonas fluorescens as biocontrol agents to protect plants from the plant pathogen Erwinia amylovora, a nicotinic acid auxotroph, was reported recently (Paternoster et al, 2010). It is unknown whether the tested Pseudomonas strains are chemotactic to nicotinic acid, but it is possible, as the Fig.…”

“…Many human, animal and plant pathogens lack pathways for de novo biosynthesis of NAD, and are nicotinic acid or nicotinamide auxotrophs; a few examples include Shigella species, enteroinvasive E. coli strains, Salmonella enterica (serovar Dublin), group A streptococci and Erwinia amylovora (Bergthorsson & Roth, 2005;Paternoster et al, 2010;Prunier et al, 2007;Sorci et al, 2013). The use of nicotinic acid-degrading strains of Pseudomonas rhizosphaerae and Pseudomonas fluorescens as biocontrol agents to protect plants from the plant pathogen Erwinia amylovora, a nicotinic acid auxotroph, was reported recently (Paternoster et al, 2010). It is unknown whether the tested Pseudomonas strains are chemotactic to nicotinic acid, but it is possible, as the Fig.…”

Floral traits affecting fire blight infection and management

“…The characteristic differences between apple and pear in the levels of NicAc were supposed to contribute to the differential susceptibility of these two host species to fire blight (Paternoster et al 2009a). The amount of NicAc on pear hypanthia was positively correlated with the altitude of the growing site and was inversely correlated with the sum of the maximum temperatures in the 30 days before flowering (Paternoster et al 2011).…”

“…Their ability to control fire blight was tested by pear flower assays using P. agglomerans as a carrier for the bacteriophages. Paternoster et al (2009aPaternoster et al ( , 2011 demonstrated that nicotinic acid (NicAc) and/or nicotinamide (NicNH 2 ) are essential for E. amylovora growth, but the amounts of these chemicals on pear and apple flowers do not limit the establishment of the pathogen when competing bacteria are lacking. Degradation of NicAc and consequent inhibition of the pathogen by microbial biocontrol agents have been recently proposed as a new approach for disease control (Paternoster et al 2009a, b).…”

“…Paternoster et al (2009aPaternoster et al ( , 2011 demonstrated that nicotinic acid (NicAc) and/or nicotinamide (NicNH 2 ) are essential for E. amylovora growth, but the amounts of these chemicals on pear and apple flowers do not limit the establishment of the pathogen when competing bacteria are lacking. Degradation of NicAc and consequent inhibition of the pathogen by microbial biocontrol agents have been recently proposed as a new approach for disease control (Paternoster et al 2009a, b). E. amylovora can grow using NicNH 2 and 6-hydroxynicotinic acid as alternative growth factors to NicAc, but not using 2-hydroxynicotinic acid (Paternoster et al 2009b).…”