Biological plant protection presents a promising and exciting alternative to chemical methods for safeguarding plants against the increasing threats posed by plant diseases. This approach revolves around the utilization of biological control agents (BCAs) to suppress the activity of significant plant pathogens. Microbial BCAs have the potential to effectively manage crop disease development by interacting with pathogens or plant hosts, thereby increasing their resistance. However, the current efficacy of biological methods remains unsatisfactory, creating new research opportunities for sustainable plant cultivation management. In this context, microbial consortia, comprising multiple microorganisms with diverse mechanisms of action, hold promise in terms of augmenting the magnitude and stability of the overall antipathogen effect. Despite scientific efforts to identify or construct microbial consortia that can aid in safeguarding vital crops, only a limited number of microbial consortia-based biocontrol formulations are currently available. Therefore, this article aims to present a complex analysis of the microbial consortia-based biocontrol status and explore potential future directions for biological plant protection research with new technological advancements.

Dickeya solani and Pectobacterium carotovorum subsp. brasili¬ense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologues of four nfs genes, that confer production of a nonfluorescent siderophore by P. donghuensis HYST. Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homologue of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predicting the presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.

“The Great Five” (GF) is an artificial bacterial consortium developed to protect potato tubers from soft rot caused by Pectobacterium spp. and Dickeya spp. To investigate the commercialization potential of the GF, we developed liquid and powder formulations of the consortium and of each of the comprising strains (Serratia plymuthica strain A294, Enterobacter amnigenus strain A167, Rahnella aquatilis strain H145, Serratia rubidaea strain H440, and S. rubidaea strain H469). To form powders, the cells were lyophilized using a newly developed lyoprotectant: Reagent PS. The shelf life of the formulations stored at 8 and 22 °C was monitored for a period of 12 months. The longest shelf life was obtained for formulations stored at 8 °C; however, the viability of all formulations was negatively affected at 22 °C. For the consortium, a 2.5 log₁₀ cfu (colony forming units) drop in cell number was recorded for the liquid formulation after 6 months, while in case of powders, the drop remained below 1 log₁₀ cfu following 12 months. The ability of the powder formulations to preserve biocontrol activity of the consortium was tested on potato tubers treated with the formulations and a mixture of the soft rot pathogens. The inoculated tubers were stored for 6 months at 8 °C to mimic commercial storage conditions. Soft rot severity and incidence on potato tubers treated with formulations were significantly reduced (62–75% and 48–61%, respectively) in comparison to positive control with pathogens alone. The potential use of the newly developed formulations of “The Great Five” for the biocontrol of soft rot is discussed. Key Points: • An innovative reagent to protect bacterial cells during lyophilization was developed. • Powder formulations of “The Great Five” prolonged its shelf life. • The powder-formulated “The Great Five” was active against soft rot bacteria on potato tubers.

Ochrobactrum spp. are ubiquitous bacteria attracting growing attention as important members of microbiomes of plants and nematodes and as a source of enzymes for biotechnology. Strain Ochrobactrum sp. A44^T was isolated from the rhizosphere of a field-grown potato in Gelderland, the Netherlands. The strain can interfere with quorum sensing (QS) of Gram-negative bacteria through inactivation of N-acyl homoserine lactones (AHLs) and protect plant tissue against soft rot pathogens, the virulence of which is governed by QS. Phylogenetic analysis based on 16S rRNA gene alone and concatenation of 16S rRNA gene and MLSA genes (groEL and gyrB) revealed that the closest relatives of A44^T are O. grignonense OgA9a^T, O. thiophenivorans DSM 7216^T, O. pseudogrignonense CCUG 30717^T, O. pituitosum CCUG 50899^T, and O. rhizosphaerae PR17^T. Genomes of all six type strains were sequenced, significantly expanding the possibility of genome-based analyses in Ochrobactrum spp. Average nucleotide identity (ANIb) and genome-to-genome distance (GGDC) values for A44^T and the related strains were below the single species thresholds (95% and 70%, respectively), with the highest scores obtained for O. pituitosum CCUG 50899^T (87.31%; 35.6%), O. rhizosphaerae PR17^T (86.80%; 34.3%), and O. grignonense OgA9a^T (86.30%; 33.6%). Distinction of A44^T from the related type strains was supported by chemotaxonomic and biochemical analyses. Comparative genomics revealed that the core genome for the newly sequenced strains comprises 2731 genes, constituting 50–66% of each individual genome. Through phenotype-to-genotype study, we found that the non-motile strain O. thiophenivorans DSM 7216^T lacks a cluster of genes related to flagella formation. Moreover, we explored the genetic background of distinct urease activity among the strains. Here, we propose to establish a novel species Ochrobactrum quorumnocens, with A44^T as the type strain (= LMG 30544^T = PCM 2957^T).

Pseudomonas donghuensis P482 is a tomato rhizosphere isolate with the ability to inhibit growth of bacterial and fungal plant pathogens. Herein, we analysed the impact of the carbon source on the antibacterial activity of P482 and expression of the selected genes of three genomic regions in the P482 genome. These regions are involved in the synthesis of pyoverdine, 7-hydroxytropolone (7-HT) and an unknown compound (“cluster 17”) and are responsible for the antimicrobial activity of P482. We showed that the P482 mutants, defective in these regions, show variations and contrasting patterns of growth inhibition of the target pathogen under given nutritional conditions (with glucose or glycerol as a carbon source). We also selected and validated the reference genes for gene expression studies in P. donghuensis P482. Amongst ten candidate genes, we found gyrB, rpoD and mrdA the most stably expressed. Using selected reference genes in RT-qPCR, we assessed the expression of the genes of interest under minimal medium conditions with glucose or glycerol as carbon sources. Glycerol was shown to negatively affect the expression of genes necessary for 7-HT synthesis. The significance of this finding in the light of the role of nutrient (carbon) availability in biological plant protection is discussed.