The use of beneficial bacterial endophytes for improving biomass yield and nutritive value of vegetables

Abstract

Production and consumption of vegetables can help to reduce food insecurity and malnutrition amongst the global growing population. However, the overuse of synthetic fertilizers and pesticides in agriculture has led to their widespread presence, posing a threat to both the environment and global food security. Beyond plant growth promotion (PGP), bacterial endophytes have been proven to improve the quality of crops and provide sustainable alternatives in traditional agricultural practices. As research on the application of endophytes in agriculture expands, it is important to conduct investigations that will help lessen the burden of food insecurity. Such approaches include the application of endophytes in vegetable farming, shown to improve crop productivity through nutrient uptake, stress tolerance, and disease protection. The present study investigated the potential of bacterial endophytes to enhance the biomass yield and nutritional value of vegetable crops, thereby promoting productivity and the nutritional quality of vegetables. A total of 60 vegetable crops including cabbage (Brassica oleracea) (n=20), lettuce (Lactuca sativa) (n=20), and spinach (Spinacia oleracea) (n=20) were collected from four commercial farming sites in the Gauteng and North West Provinces of South Africa. The diversity of endophytic bacteria associated with cabbage, lettuce, and spinach were identified by metabarcoding analysis of 16S rRNA gene. Furthermore, the macro- and micronutrients of the selected vegetable crops were assessed to evaluate their impact on bacterial endophyte communities in these vegetables. A total of 1,280 Amplicon Sequence Variants (ASVs) was obtained, mainly Proteobacteria, Actinobacteriota, Firmicutes, and Bacteroidota, whose distribution indicated vegetable organ and its nutrient content as the determinants of the endophytic bacterial community structures. In all three vegetables, the most represented genera included Pseudomonas, Pantoea, Bacillus, Cutibacterium, Nocardioides, Rickettsia, Streptomyces, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium reported already with proven plant development and protection traits. The analysis of alpha and beta diversity indicated that

the distribution of bacterial endophytes in lettuce and spinach is influenced by the type of plant part. Moreover, the vegetable organs of lettuce and spinach indicated a filtering effect when a host plant selects bacterial communities according to the plant organ type. Interestingly, the core microbiota analysis also revealed a selective and non-systemic ascent of certain endophytic genera from the roots to the leaves. For example, the content of zinc (Zn) in cabbage and spinach showed a significant influence on the distribution of endophytic genera such as Cutibacterium, Streptomyces, Lechevalieria, and LWQ8. Additionally, the results revealed a significant influence of calcium (Ca) on endophyte diversity in lettuce. Such findings on nutrients potentially influencing the assemblage of endophytic bacterial communities in plant organs, enlighten this study to demonstrate further the role of bacterial endophytes on promoting nutritive value and biomass yield of vegetable crops. Endophytic bacteria inhibiting vegetable crops are inadequately evaluated for their benefit on promoting growth and quality of these vegetable crops. Yet, their significant roles in sustainable agriculture have the opportunity to improve vegetable productivity and health. Bacterial endophytes were isolated and screened using a culture-dependent method for their potential to promote plant growth through various mechanisms, including nitrogen fixation, phosphate solubilization, siderophore production, and indole acetic acid (IAA) production. The three isolates that exhibited plant growth promotion traits in vitro were obtained from Beta vulgaris (SK1), and, other two from Aloe longibracteata (SK2 and SK3). The genomic DNA of the three isolates sequenced on illumina Miseq platforms assessed for quality reads using FastQC yielded 12,819,948 pb sequence reads Enterococcus lactis [SK1]; 17,005,458 bp sequence reads for Bacillus safensis [SK2]; and 9,306,034 pb for Priestia megaterium [SK3]. The draft assemblies for E. lactis [SK1], B. safensis [SK2], and P. megaterium [SK3] were annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Genome annotation revealed multiple PGP putative enzyme and protein-encoded genes including (i) Nitrogen regulatory protein P-ll (N-reg PII),

and Carbon-Nitrogen hydrolase for nitrogen assimilation and regulation, (ii) phoH, phoR, and phoU for phosphorus uptake, (iii) sirA, sirB, feoA, and feoB for siderophore and iron acquisition, and (iv) IAA acetyltransferase and tryptophanyl-tRNA synthetase for IAA regulation. Secondary metabolite gene clusters associated with PGP traits were predicted by antiSMASH software. All three strains revealed cluster genes coding for non-ribosomal peptide synthetases (NRPS), NI-siderophore, phosphonate, ribosomally synthesized and post-translationally modified peptides (RiPP-like), terpene, and type III polyketide synthases (T3PKS). The predicted putative genes revealed multifunctional PGP traits associated with E. lactis [SK1], B. safensis [SK2], and P. megaterium [SK3] as promising relevant features in agricultural practices and food security. To further demonstrate their potential as growth promoters of vegetable crops, E. lactis [SK1], B. safensis [SK2], and P. megaterium [SK3] were investigated for their effects on cabbage, lettuce, and spinach biomass yield and nutrients content in a greenhouse pot trial. The three endophytic bacteria were transformed with pUC19-mCherry plasmid before inoculation and monitoring their multiple PGP functions in the greenhouse. A confocal microscopic analysis was used to visualise their distribution in cabbage, lettuce, and spinach leaves. All three endophytic bacterial strains showed significant differences on the wet weight of cabbage and spinach leaves biomass compared to non-inoculated (control). Outstandingly, P. megaterium [SK3] showed a similar significant difference when cabbage was inoculated with this strain individually and in a mix with E. lactis [SK1] and B. safensis [SK2]. Priestia megaterium [SK3] also showed significant difference in wet weight of spinach root biomass compared to the control. In lettuce, E. lactis [SK1] showed a higher significant difference in comparison to the control. Moreover, endophytic bacterium inoculum in selected vegetable crops showed an increase in some of the investigated macro- and micronutrient content. Potassium (K) content, which was the highest in all three vegetable crops, was higher in lettuce leaves than the control (3.11 %) when inoculated with B. safensis [SK2] (4.46 %), P. megaterium [SK3] (3.99 %), E. lactis

[SK1] (3.86 %), and mix of the three isolates (3.83 %). The nitrogen (N) content was higher in lettuce leaves and roots inoculated with P. megaterium [SK3], B. safensis [SK2], E. lactis [SK1], and mix of the three isolates, respectively in comparison to the control. In the overall micronutrients, the three endophytic strains had an effect on higher iron (Fe) content in the roots of cabbage, lettuce, and spinach, compared to the control. Through further in vitro assessments on the safety and efficacy of these isolates in selected and similar vegetable crops for human consumption, their application in agriculture could be effective for the availability and nutrition security of vegetable crops.