There is an increasing demand by consumers for nutritious foods that improve physical performance and reduce risks of diseases. Vegetables represent a widely consumed food worldwide. Solanum lycopersicum better known as tomato is one of the most important vegetable plants in the world (1). Tomato is a healthy food that supplies a wide range of vitamins needed for the organism, since it contains high levels of zinc, potassium, anthocyanins and lycopene, which provide a high antioxidant power. Along with other compounds they reduce risk of contracting cancer, among other benefits according to several epidemiological-food- and health studies (1, 2). On the other hand, Lactuca sativa better known as lettuce is another important crop with a growing interest from people due to its healthy and beneficial properties and richness in antioxidants (e.g., vitamins C, E and carotenoids) (3).
Agriculture in the last century has faced multiple challenges, including the need to produce more food to feed a growing population, adapting to climate change, controlling crop diseases that cause significant losses, and adopting more efficient and sustainable production methods. An increased number of fungi, bacteria and viruses are causing plant diseases, several of which are the reason of major economic losses (4). For this reason, food security has become one of the main points of attention in human-driven development, and therefore any plant pathogen causing substantial crop yield losses needs to be minimized (5). Currently, tomato and lettuce production losses due to biotic agents (insect pests, microbe -or- virus caused diseases and weeds) have been controlled mainly by spraying crops with a vast amount of synthetic chemical pesticides. However, the extensive use of them caused adverse side effects which represent a serious threat to living organisms and the environment. In addition, for many plant pathogens, fungicide-resistant populations have made many fungicides ineffective (6).
Some mammalian pathogens, such as Bacillus cereus, attach and form biofilm on lettuce leaf surfaces posing a risk of causing disease in humans upon consumption (7). Klebsiella pneumoniae and Escherichia coli are other known pathogens that have been reported as causative agents of foodborne diseases, being found in different sources including raw vegetables (8). Contamination by pathogenic bacteria of vegetables can occur during the harvesting period, post harvesting, handling, storage, transportation, and processing by customers. To ensure safety level of vegetables and others, research of biocontrol agents against mammalian pathogen is needed as well.
It is now well established that plant-associated microorganisms play essential roles in plant health and development and contribute to the environmental equilibrium (9). The use of beneficial microorganisms is a promising method to fight against crop diseases and increase yields to ensure sufficient crop production (10). The structural and functional analysis of microbial genomes and the proteins encoded by genes of important plant-associated microbes, which can be possible biocontrol agents will provide insights into several aspects of these molecular interactions and will be crucial for the development of more efficient control measures (11).
Historically, research investigating the factors associated with plant microbe interactions has focused on the rhizosphere, which comprises the area in the soil around plant roots, but much less is known about the phyllosphere. The phyllosphere represents the aboveground or aerial parts of the plants dominated by the leaves in contrast to the rhizosphere that represents the below-ground part of plants. Environmental factors, including UV radiation, changes in relative humidity, temperature, leaf wetness, pollution, nitrogen fertilization as well as biotic factors, such as leaf age and the presence of other microorganisms are factors that microbe endure in such environment (12).
Gram-positive, aerobic spore forming bacteria, like Bacillus and Paenibacillus spp., have been widely reported to be effective in stimulating plant growth and are well known as producers of a broad array of antimicrobials having between 5 to 8% of the total genome devoted to the biosynthesis of secondary metabolites (13).
Bacillus and closely related species antimicrobials production is highly diverse, depending on their biosynthesis pathways, and chemical nature. Antimicrobial compounds can be classified into bacteriocins (both linear and ribosomally synthesized and postranslationally modified peptides (RiPPs)), non- ribosomally synthesized peptides (NRPs), and Polyketides (14). NRPs and PKs natural products are synthesized via multi domain mega enzymes known as non-ribosomally synthesized peptides synthases (NRPSs) and polyketide synthases (PKSs) which are arranged into units called ‘modules’ that work in an assembly-line system to build polymeric peptide chains with a determined function (15). PKSs gather small acetic acid-type acyl construction blocks into polyketides through C–C bonds, and NRPSs gather amino acids into peptides through amide bonds. NRPSs and PKSs employ a similar strategy for the biosynthesis of different classes of natural products (16). On the other hand, bacteriocins are ribosomally synthesized. Bacteriocin BGCs are smaller than the previous mentioned compounds, and carry one or more precursor peptide gene(s), which allows a finer prediction of the ending product structure based on the properties of enzymes involved in their biosynthesis and on the chemical structure of the initial peptide substrate. Bacteriocins can be classified in peptides that undergo post-translational modifications (class I) also known as Ribosomally synthesized and post-translationally modified peptides (RiPPs), or largely unmodified peptides, that sometimes contain disulfide bonds (class II) (17).
Reported and well-known NRPs antimicrobials produced by B. subtilis and B. velezensis are cyclic lipopeptides exhibiting strong surfactant and antimicrobial activities, such as surfactins, bacillibactins and fengycins (18, 19). B. velezensis, the biocontrol model also employs polyketides such as macrolactin, bacillaene, and difficidin, which play significant roles in both pathogen suppression and plant growth promotion (20). NRPs fusaricidin and polymyxin produced by Paenibacillus strains, contributes to antagonism against phyopathogens like Erwinia spp. (21), while well studied Bacillus-originated bacteriocins include subtilocin and subtilomycin (22).
In the last decade volatile compounds (VOCs) produced by some plant-associated bacteria and their biological function, have attracted increased attention. Among other characteristics, they have been proved to have the potential as antimicrobials and have plant growth promoting properties. The VOCs 2,3-butanediol and acetoin, could trigger growth promotion in Arabidopsis thaliana rhizosphere. Bacillus megaterium XTBG34 produces 2-pentylfuran which promotes the growth of Arabidopsis thaliana plants after 15 days of treatment (23, 24). In our study we aimed to isolate and screen novel phyllospheric bacteria with antimicrobial properties, and further mined into their genomes to identify known or novel biosynthetic gene clusters (BGCs) that are potentially involved in phytopathogen, and plant-originated mammalian pathogen antagonism.