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Endophytic Bacteria Associated with Origanum heracleoticum L. (Lamiaceae) Seeds

ABSTRACT: Seed-associated microbiota are believed to play a crucial role in seed germination, seedling establishment, and plant growth and fitness stimulation, due to the vertical transmission of a core microbiota from seeds to the next generations. It might be hypothesized that medicinal and aromatic plants could use the seeds as vectors to vertically transfer beneficial endophytes, providing plants with metabolic pathways that could influence phytochemicals production. Here, we investigated the localization, the structure and the composition of the bacterial endophytic population that resides in Origanum heracleoticum L. seeds. Endocellular bacteria, surrounded by a wall, were localized close to the aleurone layer when using light and transmission electron microscopy. From surface-sterilized seeds, cultivable endophytes were isolated and characterized through RAPD analysis and 16S RNA gene sequencing, which revealed the existence of a high degree of biodiversity at the strain level and the predominance of the genus Pseudomonas. Most of the isolates grew in the presence of six selected antibiotics and were able to inhibit the growth of clinical and environmental strains that belong to the Burkholderia cepacia complex. The endophytes production of antimicrobial compounds could suggest their involvement in plant secondary metabolites production and might pave the way to endophytes exploitation in the pharmaceutical field.

SUBMITTER: Semenzato G

PROVIDER: S-EPMC9612275 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Endophytic Bacteria Associated with Origanum heracleoticum L. (Lamiaceae) Seeds

Semenzato Giulia G Faddetta Teresa T Falsini Sara S Del Duca Sara S Esposito Antonia A Padula Anna A Greco Claudia C Mucci Nadia N Zaccaroni Marco M Puglia Anna Maria AM Papini Alessio A Fani Renato R

Microorganisms 20221021 10

Seed-associated microbiota are believed to play a crucial role in seed germination, seedling establishment, and plant growth and fitness stimulation, due to the vertical transmission of a core microbiota from seeds to the next generations. It might be hypothesized that medicinal and aromatic plants could use the seeds as vectors to vertically transfer beneficial endophytes, providing plants with metabolic pathways that could influence phytochemicals production. Here, we investigated the localiza ...[more]

PMID: 36296360

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Project description:BackgroundIntragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner.MethodsTotal genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5-V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the 'number of AluI bands' and 'type of eletropherogram'.ResultsTwo types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.ConclusionThe approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

Project description:Seeds offer an internal microbial niche, termed the endosphere, colonized by communities of endophytic bacteria. To elucidate the functions of seed endophytes during germination and early plant growth, studies with culturable isolates are essential. Conventional growth media favor few fast-growing taxa, while micro organisms with restricted nutrient requirements are usually outcompeted prior to isolation. Consequently, current knowledge of the interaction between seeds and their endophytes remains limited to only few bacterial taxa, despite a "black box" of unculturable isolates colonizing the endosphere. Here, we designed various solid media to mimic the endosphere of germinating soybean (Glycine max L.) seeds and assessed their effect on the diversity of culturable endophytic bacteria. The embryonic axis (i.e., the future plant) possessed higher richness and harbored more unique genera (i.e., Brevundimonas, Methylobacterium, Microbacterium, Pseudoclavibacter, and Rathayibacter) than cotyledons (i.e., seed storage organs). Overall, media containing germinating and ground seeds enabled culturing and isolation of the broadest diversity of endophytic bacteria, viewed through the molecular identification of 246 isolates. The use of multiple tailored media helped uncover trophic adaptation of the core taxa. Furthermore, comparison of seeds from four lots of distinct cultivars and origin revealed few overlapping taxa, indicating that the parental environment, including soil and fertilization regime, influenced seed endophytic diversity. Extended diversity of native seed endophytic bacteria revealed the functional relevance of unique Arthrobacter, Bacillus, and Curtobacterium strains to seed germination under salt stress, exemplifying the importance of enhanced culturing approaches to elucidate the role of microbiota in seed germination. IMPORTANCE Plant growth-promoting endophytic isolates that appear to advance seed germination are often obtained from plant niches other than the seed endosphere. Isolating pure cultures of native endophytes from seeds during germination is crucial to investigate their function during early plant growth. Here, the diversity of endophytic bacteria isolated from seeds during soybean germination was enhanced by combining media tailored to the nutritional composition of the seed endosphere, including pregerminated seeds themselves. Our results show that isolation from distinct soybean seed compartments affected such diversity, with the embryonic axis harboring more unique taxa while displaying higher endophytic richness. Furthermore, using pools of seeds from separate lots, each corresponding to a certain cultivar and field site, supported isolation of further unique strains that often unveiled substantial effects on germination performance. Such findings are relevant to assist studies on the interactions between seeds and their native endophytic bacteria.

Dataset Information

Endophytic Bacteria Associated with Origanum heracleoticum L. (Lamiaceae) Seeds

Publications

Endophytic Bacteria Associated with Origanum heracleoticum L. (Lamiaceae) Seeds

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