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nicotiana tabacum seeds

Smoking Tobacco Seed (Tabacum species)

Nicotiana tabacum is the most widely grown species of smoking tobacco. While native to the Americas, this plant is a traveler. There are dozens of unique cultivars available, many hailing from distinct parts of the world. Our strain originated from Strictly Medicinal Seeds in Williams, Oregon but can now properly be called a Mano Farm landrace as we’ve cultivated it on our land and saved seed from the plants for many years over.

Smoking Tobaccos are striking ornamental plants that can reach up to six feet in height. They produce abundant gorgeous, pink, trumpet-shaped flowers. However, with the proper nurturance and maintenance in the garden, plants will produce copious leaves that can be harvested and cured for smoking. Note that when tobacco is being grown for smoking, method is everything. Plants must be topped and suckered – essentially prevented from going to flower – throughout their entire life stage. Leaves must be harvested at the appropriate stage – a paling green/light yellow hint that is not easy to convey via words or pictures. And the curing process itself is an art – with a number of tried and trued methods, it’s best to do a lot of research ahead of time prior to harvest. Consult the Fair Trade Tobacco forum for more information.

When done right, home cured tobacco has an entirely different essence than anything that can be found commercially.

85 days from transplant to flower. Annual.
Seeds are certified organic by Oregon Tilth.

This is the most widely-grown species of Tobacco, and for good reason. Commonly known as Smoking tobacco, Nicotiana tabacum is our favorite variety to smoke, hands down. When these plants are nurtured in the garden, and the leaves cured well, the smooth s

Nicotiana tabacum seed endophytic communities share a common core structure and genotype-specific signatures in diverging cultivars

Xiaoyulong Chen

a Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, 550025 Guiyang, China

b College of Tobacco Science, Guizhou University, 550025 Guiyang, China

c Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Guizhou University, 550025 Guiyang, China

Lisa Krug

d Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria

Hong Yang

b College of Tobacco Science, Guizhou University, 550025 Guiyang, China

c Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Guizhou University, 550025 Guiyang, China

Haoxi Li

b College of Tobacco Science, Guizhou University, 550025 Guiyang, China

c Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Guizhou University, 550025 Guiyang, China

Maofa Yang

b College of Tobacco Science, Guizhou University, 550025 Guiyang, China

c Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Guizhou University, 550025 Guiyang, China

Gabriele Berg

d Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria

Tomislav Cernava

b College of Tobacco Science, Guizhou University, 550025 Guiyang, China

c Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Guizhou University, 550025 Guiyang, China

d Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria

Associated Data

The amplicon dataset that was used for this study was deposited at ENA (https://www.ebi.ac.uk/ena) under the accession number PRJEB31125.

Graphical abstract

Abstract

Seed endophytes of crop plants have recently received increased attention due to their implications in plant health and the potential to be included in agro-biotechnological applications. While previous studies indicated that plants from the Solanaceae family harbor a highly diverse seed microbiome, genotype-specific effects on the community composition and structure remained largely unexplored. The present study revealed Enterobacteriaceae-dominated seed-endophytic communities in four Nicotiana tabacum L. cultivars originating from Brazil, China, and the USA. When the dissimilarity of bacterial communities was assessed, none of the cultivars showed significant differences in microbial community composition. Various unusual endophyte signatures were represented by Spirochaetaceae family members and the genera Mycobacterium, Clostridium, and Staphylococcus. The bacterial fraction shared by all cultivars was dominated by members of the phyla Proteobacteria and Firmicutes. In total, 29 OTUs were present in all investigated cultivars and accounted for 65.5% of the combined core microbiome reads. Cultivars from the same breeding line were shown to share a higher number of common OTUs than more distant lines. Moreover, the Chinese cultivar Yunyan 87 contained the highest number (33 taxa) of unique signatures. Our results indicate that a distinct proportion of the seed microbiome of N. tabacum remained unaffected by breeding approaches of the last century, while a substantial proportion co-diverged with the plant genotype. Moreover, they provide the basis to identify plant-specific endophytes that could be addressed for upcoming biotechnological approaches in agriculture.

1. Introduction

In human history the cultivation of Nicotiana tabacum L. represents a long tradition across different cultures and continents. Its seeds were recovered from historic settlements (1500–1000 BC), providing evidence for tobacco cultivation during the early Chiripa phase in Southern America [1]. Centuries of genetic selection and breeding resulted in new cultivars which have larger leaves and higher nicotine content than earlier wild varieties [2]. Nowadays, its cultivation mainly relies on hybrid plants that originate from Southern as well as Northern America [3]. In addition to the important role as a model plant, N. tabacum is still an important agricultural crop in different countries that provide suitable environmental conditions for its cultivation [4]. The four most important cultivation areas are currently found in Asia and the Americas; China, Brazil, India, and the United Sates are currently the countries with the largest cultivation areas [5].

Similar to other plants from the Solanaceae family, various fungi, oomycetes, bacteria, viruses, as well as nematodes, can cause high yield losses during tobacco cultivation [6]. Due to the limitations of plant breeding towards more resistant cultivars and conventional disease management, the plant microbiota has emerged as a promising basis to increase plant resilience against prevailing pathogens. Recently, the tobacco microbiome was explored in fields with different incidences of bacteria wilt caused by Ralstonia solanacearum [6], [7], [8]. The authors concluded that the soil microbiota plays an important role in the bacteria wilt incidence as well as its management [9], [10]. Recent studies with different crop plants have shown that early colonizers of germinating seedlings can substantially contribute to plant development and that they originate from different sources [11]. Here, the indigenous seed microbiota constitutes a core element, because it is intrinsically connected to its host and it can be vertically transmitted between plant generations [12]. For a long time, it was assumed that a plant seedling’s microbiome is exclusively recruited from microbes located in the surrounding environment; especially from soils. This hypothesis was based on the concept of sterile seeds and omitted potential legacy effects of parental lines, because modern seed production includes surface sterilization as an integral process step [12]. However, distinct microorganisms colonize the integument, endosperm or embryo of seeds and thus are not affected by the robust sterilization procedures [13]. These seed endophytes are largely uncultivable and plant metabolites that are released when seeds are disrupted further aggravate the detection of microbes based on cultivation-dependent methods [14]. Therefore, seed endophyte studies have substantially profited from the recent development of cultivation-independent methods that allow community-level assessments [15].

So far, seed endophytes were explored in several plants, including maize, pumpkins, beans, and radish [e.g. [16], [17], [18]]. Each of the plant species harbored distinct bacterial communities that showed substantial differences in their composition at phylum level. However, relatively high proportions of Gammaproteobacteria and Enterobacteriaceae therein were identified as a common feature irrespective of species and cultivar. Studies that focused on plant breeding have shown that it not only affects root-associated microbial communities and the biocontrol potential of beneficial microbes against pathogens [19], [20], but also has an impact on the seed microbiome [16]. Tomato plants were used as model systems to verify vertical transmission of beneficial endophytes from one generation to the next [21]. However, little is known about the structure of seed-associated microbial assemblages in other plants of the Solanaceae family, which includes tobacco as a prominent member. The tobacco plant offers a highly specific microenvironment for endophytes due to the presence of high nicotine concentration in its tissues, which can substantially vary among different cultivars [22]. Diverging breeding approaches within the same plant species provide an ideal basis to explore the impact of the plant genotype on seed-endophytic communities. In the present study, we used four different tobacco cultivars, originating from Asia and the Americas, to characterize their seed-endophytic bacterial communities and to provide new insights into genotype-specific differences related to their diversity and structure. The cultivars originate from different breeding approaches, which is reflected by their phenotypes. We aimed to investigate the implication of different N. tabacum genotypes on the composition and structure of their seed-endophytic bacterial communities.

2. Materials and methods

2.1. Sample description

N. tabacum seeds were acquired during the growing season in 2018 from the seed producing companies Yuxi Zhong Yan Seed Co., Ltd (Yuxi, Yunnan; N24°19′54.32″, E102°31′44.95″) and Variety Production Base owned by Guizhou Tobacco Company (Guiyang, Guizhou; N26°52′30.48″, E107°05′50.79″). All obtained seeds were produced for conventional cultivation in January 2018 and treated with a specific seed coating as required by China’s State Tobacco Monopoly Administration. The coatings include colored talcum powder as structural element and following nutrients and trace elements: NO3, NH4, P2O5, K2O, Mg, S, Fe, Mn, B, Cu, Zn, and Mo. In addition, the coating includes carbendazim as fungicide to protect the seedlings during germination. Exact proportions are not provided for any of the components (Chinese patent: CN1561740A). The hybrid cultivars K326 and PVH1452 (obtained from Yuxi Zhong Yan Seed Co., Ltd) as well as Yunyan87 and Bina1 (obtained from Variety Production Base of Guizhou Tobacco Company) are currently the prevalent N. tabacum genotypes in China’s main cultivation areas and were therefore selected as models for the present study. They originate from different breeding lines and show different levels of resistance towards nematodes, oomycetes, and viruses ( Table 1 ). For the Brazilian cultivar PVH1452, the hybrid’s pedigree was not publicly available, while the other relevant information was directly obtained from the seed producer.

Table 1

Cultivar details for the utilized N. tabacum seeds. All seeds were produced in the same season at the indicated seed production facilities in China. Information related to disease resistance was obtained from the State Tobacco Monopoly Administration (http://www.tobacco.gov.cn). The nicotine content refers to the proportion in dry tobacco leaves that were cultivated in Guizhou/China (Xia et al., 2017).

Genotype name Breeding type Pedigree Geographic origin of seeds Location of seed production/harvest year Disease resistance of cultivar Nicotine content [%]
Bina1 Hybrid Yunyan2 × K326 (mutant line) Guizhou, China 26°52′56″ N, 107°05′6″ E/2017 Black shank: moderately resistant;
Nematodes: moderately resistant;
TMV: moderately susceptible
1.69
K326 Hybrid McNair30 × NC95 Kentucky, USA 24°20′2″ N, 102°31′50″ E/2017 Black shank: highly resistant;
Nematodes: moderately resistant;
TMV: highly resistant
2.24
PVH1452 Hybrid Information
not available
Rio Grande do Sul, Brazil 24°20′2″ N, 102°31′50″ E/2017 Black shank: moderately resistant;
Nematodes: moderately susceptible;
TMV: moderately susceptible
2.39
Yunyan87 Hybrid Yunyan2 × K326 Yunnan, China 26°52′56″ N, 107°05′6″ E/2017 Black shank: moderately resistant;
Nematodes: moderately resistant;
TMV: highly susceptible
2.02

2.2. Extraction of total community DNA from seeds and construction of the amplicon library

In the first processing step, the seed coating was removed from all seeds by washing seeds in sterile ddH2O. The washing step was conducted in 50-ml tubes with manual shaking until no residues were visible by visual inspection. Subsequently, a surface sterilization was conducted to remove all non-endophytic microorganisms from the seed surface. The seeds were collected with a sterile sieve and transferred into 20 ml of an aqueous sodium hypochlorite solution (4% NaOCl) in sterile 50-ml reaction tubes. After placing the tubes horizontally on a shaker (120 rpm) for 3 min, the seeds were again collected with a sterile sieve and washed three times in 20 ml sterile ddH2O for 3 min at 120 rpm. For each composite sample a total of 20 seeds were pooled and treated for 5 min with an automatic homogenizer (Sangon Biotech, China) and sterile, disposable pestles. The total community DNA was extracted with the FastDNA SPIN Kit for Soil (MP Biomedicals, USA) from five composite samples from each tobacco cultivar following the manufacturer’s protocol. Subsequently samples were analyzed with NanoDrop (Thermo Fisher Scientific, USA) to monitor the DNA recovery. The samples were amplified with the primers 515f (5′ GTGYCAGCMGCCGCGGTAA) and 806r (5′ GGACTACHVGGGTWTCTAAT) according to the Earth Microbiome Project protocol (www.earthmicrobiome.org) [23] with sample-specific barcodes and Illumina sequencing adaptors. In addition, specific peptide nucleic acid (PNA) oligomers were added to the PCR mix to prevent the amplification of mitochondrial (mPNA) or plastidial (pPNA) RNA from eukaryotes [24]. The sequencing was conducted on an Illumina PE250 platform (2 × 250 bp paired-end reads) by Novogene (Beijing, China) with a minimum output of 100,000 quality-filtered (Q30 ≥ 75%) reads per sample.

2.3. Demultiplexing of the 16S rRNA gene fragment amplicons and OTU table construction

The data was subjected to a standardized workflow for further dereplication and quality filtering. In order to demultiplex the 16S rRNA gene fragment library, paired-end reads were assigned to samples based on their unique barcode and truncated by removing their barcode and primer sequence from the raw reads. Corresponding paired-end reads were merged using FLASH v1.2.7 [25] before further bioinformatic processing. Subsequently, quality filtering of the raw tags was performed under strict filtering conditions to obtain high-quality tags [26]. The resulting output was used to identify and remove all chimeric sequences using the UCHIME algorithm [27], [28]. The taxonomic analysis was performed after the sequences were clustered at 97% similarity with the Uparse software [29]. The assignments were based on a naïve-Bayes RDP classifier [30] clustered at 97% similarities with the Greengenes Database 13.8 [31].

Nicotiana tabacum seed endophytic communities share a common core structure and genotype-specific signatures in diverging cultivars Xiaoyulong Chen a Key Laboratory of Green Pesticide and