Colonization and population dynamics of total, viable, and culturable cells of two biological control strains applied to apricot, peach, and grapevine crops.

The ecological fitness of the biological control strains Bacillus velezensis A17 and Lactiplantibacillus plantarum PM411 was evaluated in different crops, geographical zones, and growing seasons. Both strains (2 g L-1 of dried formulation) were spray-inoculated on apricot trees, peach trees, and grapevines. Depending on the crop, flowers, fruits, and leaves were picked at several sampling time points. The population dynamics of viable, viable but non-culturable, and dead cells were studied by comparing viability qPCR (v-qPCR), qPCR, and plate counting estimations. A17 showed high survival rates in apricot, peach, and grapevine organs. The A17 viability was confirmed since qPCR and v-qPCR estimations did not significantly differ and were rather constant after field applications. However, higher population levels were estimated by plate counting due to the non-selective characteristics of the medium used. The viability of PM411 was constrained by plant organ, crop, and climate conditions, being higher in apricot than in grapevine. PM411 survival declined after field application, indicating difficulties in its establishment. The PM411 population level was made up of dead, culturable, and viable but non-culturable cells since significant differences between the three methods were observed. In conclusion, A17 and PM411 differ strongly in their survival in grapevine, peach, and apricot.

Bacteria as Biological Control Agents of Plant Diseases.

Biological control is an effective and sustainable alternative or complement to conventional pesticides for fungal and bacterial plant disease management. Some of the most intensively studied biological control agents are bacteria that can use multiple mechanisms implicated in the limitation of plant disease development, and several bacterial-based products have been already registered and marketed as biopesticides. However, efforts are still required to increase the commercially available microbial biopesticides. The inconsistency in the performance of bacterial biocontrol agents in the biological control has limited their extensive use in commercial agriculture. Pathosystem factors and environmental conditions have been shown to be key factors involved in the final levels of disease control achieved by bacteria. Several biotic and abiotic factors can influence the performance of the biocontrol agents, affecting their mechanisms of action or the multitrophic interaction between the plant, the pathogen, and the bacteria. This review shows some relevant examples of known bacterial biocontrol agents, with especial emphasis on research carried out by Spanish groups. In addition, the importance of the screening process and of the key steps in the development of bacterial biocontrol agents is highlighted. Besides, some improvement approaches and future trends are considered.

Grapevine response to a Dittrichia viscosa extract and a Bacillus velezensis strain.

The present study aims to evaluate the response of the three Mediterranean local grapevines 'Garnacha Blanca', 'Garnacha Tinta', and 'Macabeo' to treatments with biocontrol products, namely a botanical extract (Akivi, Dittrichia viscosa extract) and a beneficial microorganism (Bacillus UdG, Bacillus velezensis). A combination of transcriptomics and metabolomics approaches were chosen in order to study grapevine gene expression and to identify gene marker candidates, as well as, to determine differentially concentrated grapevine metabolites in response to biocontrol product treatments. Grapevine plants were cultivated in greenhouse under controlled conditions and submitted to the treatments. Thereafter, leaves were sampled 24h after treatment to carry out the gene expression study by RT-qPCR for the three cultivars and by RNA-sequencing for 'Garnacha Blanca'. Differentially expressed genes (DEGs) were investigated for both treatments and highly influenced DEGs were selected to be tested in the three cultivars as treatment gene markers. In addition, the extraction of leaf components was performed to quantify metabolites, such as phytohormones, organic acids, and phenols. Considering the upregulated and downregulated genes and the enhanced metabolites concentrations, the treatments had an effect on jasmonic acid, ethylene, and phenylpropanoids defense pathways. In addition, several DEG markers were identified presenting a stable overexpression after the treatments in the three grapevine cultivars. These gene markers could be used to monitor the activity of the products in field treatments. Further research will be necessary to confirm these primary results under field conditions.

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

The use of lactic acid bacteria (LAB) to control multiple pathogens that affect different crops was studied, namely, Pseudomonas syringae pv. actinidiae in kiwifruit, Xanthomonas arboricola pv. pruni in Prunus and Xanthomonas fragariae in strawberry. A screening procedure based on in vitro and in planta assays of the three bacterial pathogens was successful in selecting potential LAB strains as biological control agents. The antagonistic activity of 55 strains was first tested in vitro and the strains Lactobacillus plantarum CC100, PM411 and TC92, and Leuconostoc mesenteroides CM160 and CM209 were selected because of their broad-spectrum activity. The biocontrol efficacy of the selected strains was assessed using a multiple-pathosystem approach in greenhouse conditions. L. plantarum PM411 and TC92 prevented all three pathogens from infecting their corresponding plant hosts. In addition, the biocontrol performance of PM411 and TC92 was comparable to the reference products (Bacillus amyloliquefaciens D747, Bacillus subtilis QST713, chitosan, acibenzolar-S-methyl, copper and kasugamycin) in semi-field and field experiments. The in vitro inhibitory mechanism of PM411 and TC92 is based, at least in part, on a pH lowering effect and the production of lactic acid. Moreover, both strains showed similar survival rates on leaf surfaces. PM411 and TC92 can easily be distinguished because of their different multilocus sequence typing and random amplified polymorphic DNA profiles.

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.

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.

Enhancing water stress tolerance improves fitness in biological control strains of Lactobacillus plantarum in plant environments.

Lactobacillus plantarum strains PM411 and TC92 can efficiently control bacterial plant diseases, but their fitness on the plant surface is limited under unfavourable low relative humidity (RH) conditions. To increase tolerance of these strains to water stress, an adaptive strategy was used consisting of hyperosmotic and acidic conditions during growth. Adapted cells had higher survival rates under desiccation than non-adapted cells. Transcript levels and patterns of general stress-related genes increased immediately after the combined-stress adaptation treatment, and remained unaltered or repressed during the desiccation challenge. However, there were differences between strains in the transcription patterns that were in agreement with a better performance of adapted cells of PM411 than TC92 in plant surfaces under low RH environmental conditions. The combined-stress adaptation treatment increased the survival of PM411 cells consistently in different plant hosts in the greenhouse and under field conditions. Stress-adapted cells of PM411 had similar biocontrol potential against bacterial plant pathogens than non-adapted cells, but with less variability within experiments.