Molecular Assay Development to Monitor the Kinetics of Viable Populations of Two Biocontrol Agents, Bacillus subtilis QST 713 and Gliocladium catenulatum J1446, in the Phyllosphere of Lettuce Leaves

Abstract

Optimising the use of biocontrol agents (BCAs) requires the temporal tracking of viable populations in the crop phyllosphere to ensure that effective control can be achieved. No sensitive systems for quantifying viable populations of commercially available BCAs, such as Bacillus subtilis and Gliocladium catenulatum, in the phyllosphere of crop plants are available. The objective of this study was to develop a method to quantify viable populations of these two BCAs in the crop phyllosphere. A molecular tool based on propidium monoazide (PMA) (PMAxx™-qPCR) capable of quantifying viable populations of these two BCAs was developed. Samples were treated with PMAxx™ (12.5-100 μM), followed by 15 min incubation, exposure to a 800 W halogen light for 30 min, DNA extraction, and quantification using qPCR. This provided a platform for using the PMAxx™-qPCR technique for both BCAs to differentiate viable from dead cells. The maximum number of dead cells blocked, based on the DNA, was 3.44 log₁₀ for B. subtilis and 5.75 log₁₀ for G. catenulatum. Validation studies showed that this allowed accurate quantification of viable cells. This method provided effective quantification of the temporal changes in viable populations of the BCAs in commercial formulations on lettuce leaves in polytunnel and glasshouse production systems.

Keywords: PMA; biocontrol; commercial formulations; phyllosphere; qPCR; quantitative method; viable cells.

Figure 1

(a) The propidium monoazide (PMA)-treated standard curve for B. subtilis QST 713 of Ct to copy number generated from genomic DNA; (b) The PMA-treated standard curve for B. subtilis QST 713 of Ct to log₁₀ total number of viable populations.

Figure 2

Effect of PMAxx™ on blocking DNA amplification from dead BCA cells for (a) B. subtilis and (b) G. cantenulatum. The black circular points show the mean of six replicates for the non-PMA treatment, and the mean of 24 replicates for the PMA treatment. Each replicate was carried out in triplicate on the qPCR plate. The bars represent the standard error of the means.

Figure 3

(a) Impact of PMAxx™ concentrations on blocking DNA amplification of increasing log₁₀ total dead cells from B. subtilis; (b) Impact of PMAxx™ concentrations on blocking DNA amplification of increasing log₁₀ total dead cells from G. catenulatum. Each treatment combination contained two independent biological replicates, and each biological replicate was carried out in triplicate on the qPCR plate. The mean is represented by the black circles, and the bars represent the standard error of the means.

Figure 4

(a). Dose response of cycle threshold (Ct) to increasing PMAxx™ concentrations on the same total numbers of B. subtilis dead cells; (b) Dose response of cycle threshold (Ct) to increasing PMAxx™ concentrations on the same total numbers of G. catenulatum dead cells. The points show the mean of two independent biological experiments carried out with three replicates for the qPCR plate. The bars represent the standard error. The detection limit was Ct < 35.

Figure 5

(a) Effect of increasing PMAxx™ dose on viable plate counts; (b) Linear standard curves of the relationship between CFUs mL⁻¹ before and after PMAxx™ treatment. The PMAxx™ concentration response with R2 values for B. subtilis are between 0–36%, while for G. catenulatum ranged between 0–16%. Each biological replicate was plated four times to confirm CFUs mL⁻¹, and therefore each treatment combination contains four technical replicates. The standard error for each treatment was <1.25.

Figure 6

(a) Interval plot of overall mean ΔCt (signal reduction) to overall mean log₁₀ total dead cell number of B. subtilis; (b). The interval plot of overall mean ΔCt (signal reduction) to overall mean log₁₀ total dead cell number of G. catenulatum. Log₁₀ mean numbers of total dead cells for all treatments were determined from the difference between total cell counts using a haemocytometer and the viable plate counts. The bars represent the standard error of the means.

Figure 7

(a) Temporal viable population of B. subtilis in glasshouse and polytunnel grown lettuce; (b) the temporal viable population of G. catenulatum in glasshouse and polytunnel grown lettuce. The standard errors were <1.25.