Development and application of a SCAR marker to monitor and quantify populations of the postharvest biocontrol agent Pantoea agglomerans CPA-2

Abstract

Pantoea agglomerans CPA-2 is an effective biocontrol agent of postharvest diseases of citrus and pome fruit. A monitoring technique was developed for its identification and to quantify its populations. The methodology used consisted of (i) searching for a semi-selective medium, (ii) identification of molecular markers and (iii) monitoring population dynamics in a commercial trial. As a semi-selective medium, Malonate Broth Agar supplemented with tetracycline hydroxychloride and incubation at high temperature (max. of 40 • C) facilitated the selective recovery of P. agglomerans CPA-2 colonies. The RAPD technique was applied to a collection of 13 strains of P. agglomerans, including CPA-2. Among the 12 primers tested, OPL-11 amplified a fragment (about 720 bp) specific to strain CPA-2. On the basis of this fragment, two SCAR markers were amplified using a primer pair derived from OPL-11 elongation. A first SCAR marker of 720 bp was specifically amplified for the strain CPA-2 and a second one of 270 bp was obtained for all P. agglomerans strains tested, including CPA-2. Commercial trials demonstrated a significant reduction of decay with the treatment of formulated cells of P. agglomerans CPA-2. Population dynamics of CPA-2 in commercial trials were determined on fruit surfaces and in the environment using both the classical plating technique and PCR with SCAR primers. In general, no significant differences were observed between results obtained from the two methods. On fruit surfaces, 1 day after CPA-2 applied its population by classical methods was 4.37 × 10 6 cfu wound −1 and at the end of the experiment the population increased to 5.8 × 10 5 cfu wound −1 . The percentages of colonies identified as P. agglomerans CPA-2 at these sampling times using SCAR primers were 90 and 95%, respectively. Population dynamics in the environment to evaluate the environmental fate of P. agglomerans CPA-2 showed that it has a limited persistence and limited capacity for dispersion.

Biological control of postharvest diseases of fruit

“…In most reports on biocontrol of postharvest diseases a quantitative relationship has been demonstrated between the antagonist concentration in the wound and the efficacy of the biocontrol agent Nunes et al 2002a). Nunes et al (2008) reported that in oranges treated with the bacteria P. agglomerans, maintained during 24 h at 20°C before cold storage, better decay control was achieved when compared to fruit immediately stored at cold temperature, detecting an increase of P. agglomerans of more than 10-fold. The authors suggest that this lap of time is necessary to this biocontrol agent colonize the fruit surface.…”

Development of SCAR markers and a semi-selective medium for the quantification of strains Ach 1-1 and 1113-5, two Aureobasidium pullulans potential biocontrol agents

“…SCAR markers have thus been successfully developed for several biological agents, including those belonging to A. pullulans species (Schena et al, 2002;De Clercq et al, 2003;Pujol et al, 2005;Nunes et al, 2008;Loncaric et al, 2008). However, the fact that such kinds of markers may not differentiate dead cells from living cells, and therefore might overestimate the viable cells, constitutes their most important drawback.…”

“…Although this medium does not distinguish between our strains and other closely related ones, the application of a simple PCR on the plated colonies using the specific SCAR primers will specifically identify strains Ach 1-1 and 1113-5. Other studies have also exploited the benefits of the dilution plating method on a semi-selective medium with strain-specific identification by SCAR markers (Abbasi et al, 1999;De Clercq et al, 2003;Pujol et al, 2005;Nunes et al, 2008). The combined use of at least two different methods has been considered as a necessary and powerful tool for the monitoring, detection and quantification of biocontrol agents (Atkins et al, 2003;.…”

“…In the case of biocontrol agents, a strain-specific detection level is needed as they are released into the environment where other strains of the same species of the biocontrol agent may be present. Among microbiological monitoring methods, counting colony forming units (cfu) after dilution plating on selective or semi-selective media has been frequently and successfully used to monitor population size of biocontrol agents (Donegan et al, 1991;Teixido et al, 1999;Nunes et al, 2008). This method, although simple and cheap, is based on morphological and cultural characteristics of strains and may thus not be able to differentiate between strains having the same characteristics.…”

“…Rather than using one of the above-mentioned monitoring methods, more and more work reports on the combination of the advantages of dilution plating methods on (semi-)selective media with strain-specific identification by SCAR markers (Abbasi et al, 1999;De Clercq et al, 2003;Pujol et al, 2005;Nunes et al, 2008). In this context, the main objective of the present study was to develop (i) specific SCAR markers using the RAPD technique, and (ii) a semiselective medium as a mixed monitoring system to identify and quantify populations of A. pullulans strains Ach 1-1 and 1113-5.…”