The biocontrol agent Lactiplantibacillus plantarum AMBP214 is dispersible to plants via bumblebees.

IMPORTANCE: Plant protection products are essential for ensuring food production, but their use poses a threat to human and environmental health, and their efficacy is decreasing due to the acquisition of resistance by pathogens. Stricter regulations and consumer demand for cleaner produce are driving the search for safer and more sustainable alternatives. Microbial biocontrol agents, such as microorganisms with antifungal activity, have emerged as a promising alternative management strategy, but their commercial use has been limited by poor establishment and spread on crops. This study presents a novel system to overcome these challenges. The biocontrol agent Lactiplantibacillus plantarum AMBP214 was spray-dried and successfully dispersed to strawberry flowers via bumblebees. This is the first report of combining spray-dried, non-spore-forming bacteria with pollinator-dispersal, which scored better than the state-of-the-art in terms of dispersal to the plant (CFU/flower), and resuscitation of the biocontrol agent. Therefore, this new entomovectoring system holds great promise for the use of biocontrol agents for disease management in agriculture.

Latilactobacillus fragifolii sp. nov., isolated from leaves of a strawberry plant (Fragaria x ananassa).

Thirteen Gram-positive, catalase-positive, rod-shaped single colonies were obtained after culturing a strawberry leaf on de Man-Rogosa-Sharpe agar. Based on 16S rRNA gene and rpoA gene sequence similarities, ranging between 99.0-100% and 96.5-100%, respectively, the 13 isolates were found to be closely related to each other. Two of the independent isolates, AMBP162T and AMBP252, were whole-genome sequenced, and showed to be undistinguishable with an average nucleotide identity (ANI) value of 100 %. Compared to the reference genomes for all species in the family Lactobacillaceae, the AMBP162T genome was most similar to the reference strain of Latilactobacillus curvatus with ANI of only 89.5 %, indicating they were a different species. Based on genotypic and phenotypic data, a novel Latilactobacillus species, Latilactobacillus fragifolii sp. nov., with the type strain AMBP162T (=LMG 32285T=CECT 30357T) is proposed.

The Greenhouse Phyllosphere Microbiome and Associations with Introduced Bumblebees and Predatory Mites.

Greenhouses are highly productive environments in which conditions are regulated to optimize plant growth. The enclosed character of greenhouses usually results in reduced microbial diversity, while it is known that a diverse microbiome is important for plant health. Therefore, we explored the phyllosphere microbiome of tomatoes and strawberries grown in greenhouses. We observed that the microbiome of both crops was low in diversity and abundance and varied considerably over time and space. Interestingly, the core taxa of tomatoes were Snodgrasella and Gilliamella, genera typically associated with bumblebees. The same amplicon sequence variants (ASVs) were found on reared bumblebees, indicating that the bumblebees, present in the sampled greenhouses to pollinate flowers, had introduced and dispersed these bacteria in the greenhouses. Overall, we found that 80% of plants contained bumblebee-associated taxa, and on these plants, bumblebee-associated reads accounted for up to a quarter of the reads on tomatoes and a tenth of the reads on strawberries. Furthermore, predatory mites had been introduced for the control of spider mites. Their microbiome was composed of a diverse set of bacteria, which varied between batches ordered at different times. Still, identical ASVs were found on mites and crops, and these belonged to the genera Sphingomonas, Staphylococcus, Methylobacterium, and Pseudomonas. These new insights should now be further explored and utilized to diversify ecosystems that are characterized by low diversity and abundancy of microbes. IMPORTANCE Greenhouses, though highly effective agricultural environments, are characterized by reduced sources of bacterial diversity and means of dispersal compared to more natural settings. As it is known that plant health and productivity are affected by associated bacteria, improving our knowledge on the bacterial communities on greenhouse crops is key to further innovate in horticulture. Our findings show that tomato and strawberry crops cultivated in greenhouses harbor poor and variable bacterial communities. Furthermore, commonly implemented biological solutions (i.e., those based on living organisms such as bumblebees and predatory mites) are important sources and means of dispersal of bacteria in greenhouses. This study shows that there is great potential in using these biological solutions to enrich the greenhouse microbiome by introducing and dispersing microbes which have beneficial effects on crop production and protection, provided that the dispersed microbes have a beneficial function.

Bacterial Succession and Community Dynamics of the Emerging Leaf Phyllosphere in Spring.

Every year, deciduous trees shed their leaves, and when new leaves emerge next spring, they establish a characteristic bacterial leaf community. In this exploratory study, we assessed the bacterial phyllosphere (aboveground plant surfaces) of eight London plane trees (Platanus × acerifolia) in Antwerp and Milan by sampling weekly during leaf emergence and expansion. We sampled the surfaces of different tree compartments: leaves, leaf buds, branches, and trunk, for up to 6 weeks. Phyllosphere community composition was most strongly determined by tree compartment. Only the communities on the emerging leaves showed changing dynamics over time. The rate of change in the leaf phyllosphere composition, expressed as the beta dissimilarity between consecutive time points, was very high following leaf emergence, with decreasing speed over time, indicating that these communities stabilize over time. We also identified cooccurring groups of bacteria associated with potential stages of ecological succession on the leaves and accordingly named them general cluster, early cluster, middle cluster, and late cluster. Taxa of the general cluster were not only more abundant than the others on leaves, but they were also widespread on other tree compartments. The late cluster was most pronounced in trees surrounded by trafficked urban land use. This study mainly generates hypotheses on the ecological succession on the emerging leaves of deciduous trees in urban environments and contributes to understanding the development of the tree leaf phyllosphere in spring. IMPORTANCE Improving our understanding of phyllosphere ecology is key in successfully applying bacterial biological agents or modulating the leaf microbiome in order to achieve valuable ecosystem services, such as plant protection, plant growth, air purification, and developing a healthy human immune system. Modulation of the phyllosphere microbiome in the field works only with variable success. To improve the impact of our applications in the field, a better understanding of the ecological principles governing phyllosphere dynamics is required. This exploratory study demonstrates how the combination of different analyses of a chronosequence of bacterial communities can provide new ecological insights. With a limited number of sampled trees, we demonstrated different indications of ecological succession of bacterial communities in the leaves and observed a potential impact of intensely trafficked land use becoming apparent in the leaf bacterial communities approximately 3 weeks after leaf emergence, consisting of a separate stage in community development.

Modes of Action of Microbial Biocontrol in the Phyllosphere.

A fast-growing field of research focuses on microbial biocontrol in the phyllosphere. Phyllosphere microorganisms possess a wide range of adaptation and biocontrol factors, which allow them to adapt to the phyllosphere environment and inhibit the growth of microbial pathogens, thus sustaining plant health. These biocontrol factors can be categorized in direct, microbe-microbe, and indirect, host-microbe, interactions. This review gives an overview of the modes of action of microbial adaptation and biocontrol in the phyllosphere, the genetic basis of the mechanisms, and examples of experiments that can detect these mechanisms in laboratory and field experiments. Detailed insights in such mechanisms are key for the rational design of novel microbial biocontrol strategies and increase crop protection and production. Such novel biocontrol strategies are much needed, as ensuring sufficient and consistent food production for a growing world population, while protecting our environment, is one of the biggest challenges of our time.