Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria

Abstract

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11%–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

“…Alternatively, a loss of mutualistic interaction with beneficial endophytic bacteria could contribute to reduced adaptation to in vitro conditions and inhibited shoot growth. Indeed, growth-promoting [ 5 , 9 ] stress-reducing activities [ 6 , 8 , 10 ] of bacteria on in vitro propagated plants have been reported previously. Our study revealed that antibiotic treatment significantly reduced the abundance of several families of order Rhizobiales as well as Caulobacteraceae and Sphingomonadaceae that involve many common environmental bacteria including beneficial plant-associated bacteria [ 47 , 48 , 49 ].…”

“…Bacterial DNA was isolated using the GeneJET Genomic DNA Purification kit (Thermo Fisher Scientific, Waltham, MA, USA). The 16S rRNA gene fragment was amplified using universal primers as described previously [ 10 ] and the sequence was queried at the NCBI BLAST server [ 88 ]. Isolate resistance to antibiotics was tested by cultivation on AIA medium supplemented with timentin (250 mg L −1 ), chloramphenicol (30 mg L −1 ), or rifampicin (25 mg L −1 ).…”

“…Some bacterial endophytes maintain habitually concealed lifestyles or, due to limited bacterial growth-supporting media conditions, remain latent over extended periods of in vitro tissue cultivation. In fact, several studies showed a beneficial effect of endophytic bacteria on the growth of in vitro cultures of tomato [ 5 ], grapevine [ 6 ], sweet cherry [ 7 ], apple [ 8 ], purple coneflower [ 9 ] and tobacco [ 10 ]. However, the formation of bacterial colonies on culture media and bacterial overgrowth of plant tissues are common and manifest as infection by a variety of pathogenic species or the non-fastidious proliferation of commensal endophytic bacteria that can be triggered by changes in environmental conditions or plant host physiology [ 11 , 12 , 13 ].…”

“…Cultivated tobacco ( Nicotiana tabacum L.) has well established in vitro culture conditions [ 33 ] and has been commonly used as a model species for genetic transformation studies as well as for practical application in molecular farming using in vitro techniques or contained greenhouse practices [ 34 , 35 , 36 ]. Recently, the composition of the endophytic microbiome of field-grown tobacco plants or seeds has been explored using cultivation and next-generation sequencing-based methods [ 37 , 38 ] and several endophytic species of the Bacillus genus were isolated from tobacco leaves [ 10 , 39 ]. However, the microbial diversity of in vitro cultivated tobacco tissues has not been addressed to date.…”

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

“…Some bacterial endophytes maintain habitually concealed lifestyles or, due to limited bacterial growth-supporting media conditions, remain latent over extended periods of in vitro tissue cultivation. In fact, several studies showed a bene cial effect of endophytic bacteria on the growth of in vitro cultures of tomato [5], grapevine [6], sweet cherry [7], apple [8], purple cone ower [9] and tobacco [10]. However, the formation of bacterial colonies on culture media and bacterial overgrowth of plant tissues are common and manifest as infection by a variety of pathogenic species or the non-fastidious proliferation of commensal endophytic bacteria that can be triggered by changes in environmental conditions or plant host physiology [11][12][13].…”