The role of exogenous glycinebetaine on some antioxidant activity of non-T and T tobacco (Nicotiana tabacum L.) under in vitro salt stress

<p class="042abstractstekst">Glycine betaine is an osmoprotectant compound which enhances cell tolerance in plant species in response to environmental stresses. This study aimed to investigate the effect of exogenous application of glycine betaine on some antioxidant activities of tobacco plants overexpressing <em>P5CS</em> gene. Sterile tobacco seedlings with four to six leaves were transferred to MS medium containing 0, 100, and 200 mM NaCl, after which glycine betaine (20 and 40 mg l^-1) were foliar sprayed on the surface of the plants. After four weeks, glycine betaine treatment enhanced the antioxidant capacity of the plant through activation of catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). In contrast, H₂O₂ content and MDA level were reduced by glycine betaine under similar conditions. Therefore, application of exogenous glycine betaine under salt stress improved stress tolerance in T and non-T plants. Meanwhile, our results indicated the positive effect of glycine betaine in T plants was greater than in non-T plants. On the other hand, this result suggested that the synergistic effects of glycine betaine and proline in plants enhanced the antioxidant defense system in T plants overexpressing <em>P5CS</em> gene.</p>

Molecular and Biochemical Characterization of Two 4-Coumarate: Coa Ligase Genes in Tea Plant (Camellia Sinensis)

Tea is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, p-coumaroyl-CoA, which is formed from p-coumaric acid catalyzed by p-coumaric acid: CoA ligase (4CL). Herein, we reported two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which were a member of class I and II, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, light and stress-inducible (LSI), and meristem-specific elements, while that of Cs4CL2 had AC and LSI elements only. Moreover, the promoter of Cs4CL1 had two more stress-inducible elements than Cs4CL2 had and the two promoters had six different light-inducible elements. These features suggested their differences in their responses to environmental conditions. Three stress treatments indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assay showed that both recombinant Cs4CL1 and Cs4CL2 transformed p-coumaric acid, ferulic acid and caffeic acid to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze caffeic acid, while the recombinant Cs4CL2 favored to catalyze p-coumaric acid. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

Management of Pythium myriotylum in Tobacco Transplant Production Greenhouses

Pythium root rot is a common disease that can threaten tobacco seedling production in greenhouses. However, management tools are limited in tobacco transplant production greenhouses. To identify additional Pythium control options, oomyceticide treatments (ethaboxam, mefenoxam, and copper ethanolamine complex) and non-oomyceticide (ultraviolet light and copper ion) water treatments were compared with etridiazole and an untreated control on TN 90LC tobacco seedlings inoculated with P. myriotylum in greenhouses. All the treatments in oomyceticide trials were applied to the bay water once before inoculation, when seedling roots had extended into the water. The inoculum was applied immediately before seeding in non-oomyceticide trials, where etridiazole was applied to bay water once, two weeks after seeding, as a positive control. Non-oomyceticide treatments were applied three times: 24 hours before, two weeks after, and four weeks after seeding. At the end of the tobacco transplant production season, ethaboxam and mefenoxam significantly (P<0.05) reduced root rot incidence and severity by as high as 100%, compared with the untreated control. Ethaboxam and mefenoxam also significantly (P<0.05) reduced oospores produced in infected root tissues, while significantly (P<0.05) increasing root length and weight. Ultraviolet radiation and copper ion treatments had no significant effects on tobacco seedling root length or weight compared with the untreated control, although the copper ion treatments significantly (P<0.05) reduced root rot severity and oospores produced in root tissues. Similar to etridiazole, ethaboxam and mefenoxam consistently reduced the AUDPC of Pythium root rot, but copper ion treatments only reduced AUDPC significantly (P<0.05) in one trial.

THE EFFECT OF BIOREGULATOR REGOPLANT ON TOBACCO PRODUCTIVITY DURING THE SEEDING AND FIELD PERIODS

We studied the effect of the growth regulator Regoplant on the mass of seedlings, the quality of seedlings and the productivity of tobacco. Soaking tobacco seeds before sowing in a 0.0001 % solution of the preparation at an exposure of 6 hours, and then spraying the plants at the stage of “auricles” and “seedling ready for planting” with a working solution of an agrochemical with a 0.00001 % concentration significantly improved the quality of seedlings. This technique increases the length of tobacco seedlings to the apical point by 66 %, to the end of elongated leaves by 29 %, the mass of stems by 53 %, the mass of roots by 32 %, the diameter of the stem by 38 %. After planting the plants in the field, developed under the influence of the Regoplant stimulator, we noted an increase in productivity by 16 % and an improvement in the quality of raw materials due to an increase in carbohydrates and a decrease in proteins. The economic effect against the background of the application of Regoplant preparation amounted to 106 thousand rubles.

Moderate Boron Concentration Beneficial for Flue-cured Tobacco Seedlings Growth and Development

Boron (B) is a micronutrient tobacco needs in minute amounts, and Boron insufficient supply can causes significant tobacco yield loss, however, the appropriate concentration for flue-cured tobacco seedlings to growth remains unknown. In this sense, a hydroponic experiment was conduct to measure the agronomic traits, dry matter mass, chlorophyll content, photosynthetic performance, antioxidant enzymes, boron ion and nicotine content of flue-cured tobacco seedlings K326 under different boron concentrations of 0.000mmol/L (B1, CK), 0.125mmol/L (B2), 0.250mmol/L (B3), 0.750mmol/L (B4), 5.000mmol/L (B5), 10.000mmol/L (B6), 20.000mmol/L (B7), 40.000mmol/L (B8) after 30 days. B significantly influenced flue-tobacco seedlings growth on agronomic traits, photosynthetic performance, the activities of antioxidant enzymes, boron ion and nicotine content aspects. B linearly enhanced the accumulation of boron ion by 24.00%~96.44%, and decreased nicotine content by 21.60%~82.03% in tobacco seedlings. Solution B concentration at 0.750 and 5.000mmol/L markedly improved tobacco seedlings maximum leaf length by 4.83%~82.03% and leaf width by 0.77%~24.36%, root weight by 13.64%~56.82%, stem weight by 12.26%~52.36%, leaf weight by 9.68%~36.56%, dry matter mass by 10.65%~38.92%, the Pn parameter by 1.22%~80.28%, the Cond paramete by 33.40%~75.86%, while decreased the activities of SOD by 10.44%~91.67%, POD by 21.32%~65.62% and CAT by 50.05%~96.44%, and MDA by 16.23%~75.16%. The B concentration concluded in this study enhanced the agronomy traits, photosynthetic and biochemical characteristics of flue-cured tobacco seedlings, which lays a scientific theoretical foundation for rational application of B in tobacco production and improve the internal quality of flue-cured tobacco.

First Report of Root Rot of Tobacco Caused by Fusarium brachygibbosum in China

Tobacco (Nicotiana tabacum L.) is an important cash crop in China, with an estimated production of 2.2 million tons every year (Berbeć and Matyka, 2020). In June 2020, a root rot disease was observed on tobacco (cv. Zhongyan 100) in four surveyed counties (Mianchi, Lushi, Duguan and Lingbao) in Sanmenxia. Diseased plants exhibited leaf chlorosis and purplish to brown vascular discoloration of stem, taproot and lateral roots. The disease incidence ranged from 15% to 40% in 11 surveyed fields, 36.7 ha in total. Twenty five diseased tissues were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Fifteen single-spore isolates were obtained from 25 diseased tissue samples. All cultures growing on PDA had white colonies with abundant aerial mycelia initially, turning into yellow to orange in the center and produced red pigmentation after seven days of growth. The 7-day-old cultures grown on carnation leaf agar (CLA) produced macroconidia that were curved with 3-5 septa, had wide central cells, slightly pointy apex, and measured 17.0-45.9 μm long×3.0-4.6 μm wide (n=50). The microconidia formed on CLA were slightly curved, ovoid with zero to two septa, measuring 5.4-15.5 μm long×2.0-3.2 μm wide (n=50). Spherical chlamydospores (7.58-13.52 μm; n=50) were terminal or intercalary, single or in chains. Such characteristics were typical of Fuarium brachygibbosum (Tirado-Ramírez et al. 2018). DNA from one representative single-spore isolate (MC1) was extracted, and the translation elongation factor 1-alpha (EF1-α), RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes were amplified with primers EF1/EF2, F5/G2R and RPB2F/R respectively (O’Donnell et al. 1998, 2010), and sequenced. Sequences were submitted to GenBank under accession numbers MT947796 (EF1-α), MW679536 (RPB1) and MW430664 (RPB2). The consensus sequences showed 99.70%, 99.94% and 100% identity to the sequences of F. brachygibbosum strain NRRL 34033 (accession no. GQ505418.1, HM347172.1 and GQ505482.1, Wang et al 2021). Morphological and molecular results confirmed this species as F. brachygibbosum (Al-Mahmooli, et al., 2013, Rentería -Martínez, et al., 2018). Pathogenicity tests were performed on tobacco seedlings grown on autoclaved tobacco specific substrate (Tobacco specific matrix, Ainong Biotechnology Co. Ltd, China). Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with MC1 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7-14 days after inoculation. The pathogen was reisolated from the necrotic tissue from all inoculated seedlings and was identified by sequencing partial EF1-α and RPB2 genes. Control plants remained asymptomatic and no pathogen was recovered from the control plants. Fusarium brachygibbosum is known as a pathogen of grains and cash crops in China (Shan, et al., 2017, Xia, et al., 2018). To our knowledge, this is the first report of F. brachygibbosum causing root rot on tobacco. We believe that our results will help to better understand rhizome fungal diseases affecting tobacco production in China. Acknowledgements: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010). References: Al-Mahmooli, I. H., et al. 2013. Plant Dis. 97:687; https://doi.org/10.1094/PDIS-09-12-0828-PDN Berbeć A. K. and Matyka M. 2020. Agric. 10(11), 551; https://doi.org/10.3390/agriculture10110551 O’Donnell, K., et al. 1998. P. Natl. Acad. Sci. USA. 95(5):2044-2049; https://doi.org/10.1073/pnas.95.5.2044 O’Donnell, K., et al. 2010. J. Clin. Microbiol. 48(10)3708-3718; https://doi.org/10.1128/JCM.00989-10 Rentería -Martínez M.E., et al. 2018. Mex. J. of Phytopathol. 36(2):1-23; https://doi.org/10.18781/R.MEX.FIT.1710-1 Shan, L. Y., et al. 2017. Plant Dis. 101:837; https://doi.org/10.1094/PDIS-10-16-1465-PDN Tirado-Ramírez, M. A., et al. 2018. Plant Dis. 103; https://doi.org/10.1094/PDIS-04-18-0710-PDN Wang, S., et al. 2021. Plant Dis. 2021 Jan 6. doi: 10.1094/PDIS-05-20-0941-PDN. Epub ahead of print. PMID: 33406862. Xia, B., et al. 2018. Plant Dis. 102(11):2372; https://doi.org/10.1094/PDIS-12-17-1939-PDN The author(s) declare no conflict of interest.

Improving Drought Tolerance in Tobacco By Application of Salicylic Acid

Drought causes not only the decrease of tobacco yield and quality, but also the lowering of net photosynthetic rate, leading to reactive oxygen species accumulation and even the death of plants. Salicylic Acid is involved in regulating many plant physiological processes and has increasingly been applied to improve tolerance in plants exposed to drought stress. To explore the regulating mechanism of SA, flue-cured tobacco K326 was used in the hydroponic experiments to design PEG drought stress. The photosynthetic characteristics, antioxidant enzymes activities and osmotic regulatory substances contents of tobacco seedlings under drought stress were investigated after 0.3 mmol L-1 SA treatment. Transcriptome sequencing and GO/KEGG analysis were also performed. The main results showed that SA-applied greatly increased the activities of SOD, POD, CAT activity, Pn, proline and soluble protein by 44.27%, 50.18%, 26.23%, 45.74%, 34.67% and 24.91% while reduced the MDA content by 23.89%. GO and KEGG analysis showed that SA treatment was able to up-regulate the genes involved in photosynthesis, carbon metabolism, porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins. The conclusion is that SA application would effectively improve the ability of pigment biosynthesis and photosystem repair of tobacco under drought conditions, thus enhance the photosynthesis, reduce the accumulation of ROS and increase drought resistance, which would provide a measure for alleviating the damage of tobacco caused by drought stress.

First Report of Fusarium Root Rot of Tobacco Caused by Fusarium sinensis in Henan Province China

Tobacco (Nicotiana tabacum L.) is an economically important crop in China, with an estimated production of 2.2 million tons every year. In June 2018, tobacco plants within the municipality of Sanmenxia (Henan, China) showed symptoms of wilting with leaf yellowing and stunting. Diseased plants exhibited severe necrosis that advanced through the main root (Figure 1 A). The symptoms were observed in nineteen surveyed tobacco fields, 60 ha in total, and approximately 25% of the plants were symptomatic. The disease resulted in a severe loss in tobacco leaf production. Five symptomatic tobacco plants were sampled. Diseased tissues from roots were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Eighteen of the 25 diseased tissues had cultures growing from them, and all the cultures were white colonies with abundant aerial mycelium produced scarlet pigmentation on PDA. One pure culture was obtained by single-spore culturing (SL1). A 10-day-old culture grown on CLA (carnation leaf agar) produced macroconidia that were falcate, straight or slightly curved, 3-septate, 25-35×3.5-4.5 μm (average 26.8×3.7 μm) (n=50). Two types of microconidia (napiform and fusiform) were formed on CLA that were hyaline, with one to two cells. Napiform conidia were 4.5-9.3×3.8-5.9 (average 7.3×5.0 μm) (n=50); fusiform conidia were 6.9-15.8×1.8-3.1 (average 9.9×2.5 μm). Spherical chlamydospores (7-12.5 μm) (n=50) were terminal or intercalary and produced in clumps or in chains (Figure1 B-D). Morphological characteristics of the isolate were similar to the features of Fusarium sinensis previously described by Zhao and Lu (2008). Molecular identification was performed using partial sequences of EF1-α gene (primers EF1/EF2, O’Donnell et al. 1998). Maximum parsimony and maximum likelihood-based methods were fitted using MEGA 7 (Moreira et al. 2019，Figure 2). The isolate was also sequenced for β-tubulin (primers T1/Bt-2b, O’Donnell & Cigelnik 1997)，ribosomal RNA gene (LSU, LROR/LR5 primers, Vu et al. 2019) and rDNA-ITS (ITS 1/ ITS 4 primers, White et al. 1990). Sequences were deposited in GenBank under accession numbers MT947797 (EF1-α), MW484999 (β-tubulin), MW486649 (LSU) and MT907471 (ITS). The obtained EF1-α sequence was 98.10% identity with those of F. sinensis (MG670388.1) in the GenBank database, whereas the β-tubulin, LSU and ITS sequences showed 100% identities to the corresponding DNA sequences in F. sinensis (GenBank Acc. Nos. KX880370.1, NG_067454.1 and MH863232.1, respectively). Morphological and molecular results confirmed this species as F. sinensis (Zhao and Lu 2008). Pathogenicity tests were performed on tobacco seedlings grown on an autoclaved matrix (YC/T310-2009). Healthy 6-leaf stage tobacco seedlings were inoculated by pouring a 20 mL conidial suspension (1×106 conidia/mL-1) around the stem base of each plant, 30 plant were inoculated. Thirty control seedlings received sterilized water. All treatments were maintained for 30 days under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Root rot and foliage chlorosis similar to the ones observed on infected plants in the field were observed on the inoculated tobacco seedlings, whereas the control seedlings remained asymptomatic after 30 days (Figure1 E). The pathogen isolated from the inoculated plant exhibited morphological characteristics identical to F. sinensis and was identified by a partial EF1-α gene sequence. This disease has previously been reported as the causal agent of root and crown rot of wheat in China (Zhao and Lu 2008; Xu et al. 2018). To our knowledge, this is the first report of F. sinensis causing root rot on tobacco in China. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010). References: Moreira, G.M., et al. 2019 Plant Dis. O’Donnell, K., et al. 1998. Proc. Natl. Acad. Sci. USA 95:2011. O'Donnell, K., et al. 2008. J. Clin. Microbiol. 46:2477. Xu, F., et al. 2018. Front Microbiol. 9:1054. Zhao, Z.H., and Lu, G. Z., 2008. Mycologia, 100:746. The author(s) declare no conflict of interest. Keywords: tobacco root rot, Henan Province, Fusarium sinensis

Exogenous 1′,4′-trans-Diol-ABA Induces Stress Tolerance by Affecting the Level of Gene Expression in Tobacco (Nicotiana tabacum L.)

1′,4′-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1′,4′-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1′,4′-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1′,4′-trans-diol-ABA. These findings indicated that treatment with 1′,4′-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.