Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method

Abstract

A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-specific molecular marker. Three primer sets with different amplicon lengths (92, 188, and 317 bp) and one TaqMan probe were designed. All the qPCR assays showed good linearity over a 4-log range and good efficiencies but differed in sensitivity. The nucleic acid-binding dye PEMAX was used to selectively detect and enumerate viable bacteria by v-qPCR. The primer set amplifying a 188-bp DNA fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed on apple blossoms, pear, strawberry, and kiwifruit leaves in potted plants under controlled environmental conditions, as well as pear and apple blossoms under field conditions, by comparing v-qPCR population estimations to those obtained by qPCR and specific plate counting on de Man-Rogosa-Sharpe (MRS)-rifampin. The population estimation did not differ significantly between methods when conditions were conducive to bacterial survival. However, under stressful conditions, differences between methods were observed due to cell death or viable-but-nonculturable state induction. While qPCR overestimated the population level, plate counting underestimated this value in comparison to v-qPCR. PM411 attained stable population levels of viable cells on the flower environment under high relative humidity. However, the unfavorable conditions on the leaf surface and the relatively dryness in the field caused an important decrease in the viable population.IMPORTANCE The v-qPCR method in combination with plate counting and qPCR is a powerful tool for studies of colonization and survival under field conditions, to improve formulations and delivery strategies of PM411, and to optimize the dose and timing of spray schedules. It is expected that PEMAX v-qPCR could also be developed for monitoring other strains on plant surfaces not only as biological control agents but also beneficial bacteria useful in the sustainable management of crop production.

Biological control of bacterial plant diseases with Lactobacillus plantarum strains selected for their broad‐spectrum activity

“…To achieve the BCA population required for biocontrol, repeated spray applications may be necessary. Therefore, monitoring viable cells could aid the design of a suitable delivery schedule of applications (Daranas et al, ). Likewise, the improvement of BCA ecological fitness could be implemented (Daranas, Badosa, et al, ).…”

Abundance, diversity and plant‐specific adaptations of plant‐associated lactic acid bacteria

“…betaine, carnitine and trehalose) and elevatedsynthesis of proteases, chaperones and peptidoglycan (Papadimitriou et al ., ). Recently, L. plantarum PM411, a strain isolated from a pear, was shown to induce the expression of genes coding for chaperones ( dnaK , groEL ), proteases ( clpB , clpC ), small heat shock proteins ( hsp1 , hsp3 ), cold shock proteins ( cspP , cspL ), stress factors ( ftsH , ctsR ), small redox proteins ( trxB1 ), adhesion protein ( efTU ) and bacteriocin synthesis ( plnE , plnJ ) after exposure to desiccation stress (Daranas et al ., ). The capacity to withstand low amounts of available water is also associated with EPS synthesis (Nwodo et al ., ).…”

“…To this regard, the characteristics of plant‐associated LAB appear to be similar to certain species of fungi, which are found in low numbers on plants, but are responsible for post‐harvest plant diseases and aid in the decomposition of plant litter (Dijksterhuis, ). For example, even though L. plantarum PM411 survived on apple blossoms and leaves of pear, strawberry and kiwifruit plants maintained under high relative humidities in a greenhouse, this strain did not multiply (Daranas et al ., ). Reductions in L. plantarum PM411 culturable cell numbers were also observed after application onto apple and pear blossoms in the field, with a fraction of PM411 cells entering into a viable but non‐culturable state (Daranas et al ., ).…”

“…For example, even though L. plantarum PM411 survived on apple blossoms and leaves of pear, strawberry and kiwifruit plants maintained under high relative humidities in a greenhouse, this strain did not multiply (Daranas et al ., ). Reductions in L. plantarum PM411 culturable cell numbers were also observed after application onto apple and pear blossoms in the field, with a fraction of PM411 cells entering into a viable but non‐culturable state (Daranas et al ., ). Similarly, plant‐associated P. pentosaceus B6N, L. mesenteroides BN10, L. plantarum MKR2, or L. lactis D119, did not grow on gnotobiotic‐ or field‐grown Napa cabbage (Miller et al ., ).…”

Development of a viability digital PCR protocol for the selective detection and quantification of live Erwinia amylovora cells in cankers

“…On the other hand, classical PCR detection neither discriminates between the live and dead E. amylovora cells nor allows their quantification. Improvement of molecular methods for pathogen quantification and/or selective detection of viable cells have been two important research topics in the last two decades [13][14][15][16][17][18] .…”