Unraveling the expression of differentially expressed proteins and enzymatic activity in response to Phytophthora nicotianae across different flue-cured tobacco cultivars.

BACKGROUND: Black shank disease caused by Phytophthora nicotianae is a serious threat to flue-cured tobacco production. Whole-plant resistance is characterized by the expression of a number of pathogenesis-related proteins, genes, and the activity of different defense-related enzymes. In this study, we investigated the activity of defense-related enzymes and expression of differentially expressed proteins through the iTRAQ technique across two flue-cured tobacco cultivars, i.e., K326 and Hongda, in response to the black shank pathogen. RESULTS: Results showed that the highest disease incidence was recorded in flue-cured tobacco cultivar Hongda compared with K326, which shows that Hongda is more susceptible to P. nicotianae than K326. A total of 4274 differentially expressed proteins were detected at 0 h and after 24 h, 72 h of post-inoculation with P. nicotianae. We found that 17 proteins induced after inoculation with P. nicotianae, including pathogenesis (5), photosynthesis (3), oxidative phosphorylation (6), tricarboxylic acid cycle (1), heat shock (1), and 14-3-3 (1) and were involved in the resistance of flue-cured tobacco against black shank disease. The expression of 5 pathogenesis-related proteins and the activities of defense-related enzymes (PPO, POD, SOD, and MDA) were significantly higher in the leaves of K326 than Hongda after inoculation with P. nicotianae. CONCLUSION: These results provide new molecular insights into flue-cured tobacco responses to P. nicotianae. It is concluded that differences in protein expressions and defense-related enzymes play an important role in developing resistance in flue-cured tobacco cultivars against black shank disease.

Dynamic changes in physiological and biochemical properties of flue-cured tobacco of different leaf ages during flue-curing and their effects on yield and quality.

BACKGROUND: The leaf age for harvesting flue-cured tobacco leaves is closely related to the quality of tobacco leaves, so an appropriate leaf age for harvesting is important for improving yield and quality of flue-cured tobacco, however, at present, there are few studies on effects of leaf age on physiological and biochemical changes during flue-curing and there is no clear standard of proper leaf ages for harvesting in production. RESULTS: In the Yunnan tobacco-growing area, an experiment was carried from 2016 to 2017 and different leaf ages were set. The results demonstrate that leaf age has a significant on tissue cell gap, leaf age and flue-curing stages exert significant effects on upper epidermis, palisade and spongy tissue, and leaf thickness of tobacco leaves. The thicknesses of upper and lower epidermis as well as palisade and spongy tissues at different ages show an approximately W-shaped change trend during flue-curing. With the advance of flue-curing stages, contents of starch, chlorophyll, carotenoid, and water in tobacco leaves at different leaf ages decrease, while polyphenol and malondialdehyde (MDA) contents increase. The older the leaf, the faster the chlorophyll, carotenoid, and water contents reduce, while the faster the polyphenol and MDA content rise during flue-curing. The flue-cured tobacco leaves at 116 DAT (days after transplanting) show the highest contents of total nitrogen and nicotine, followed by 123 DAT and those at 130 DAT are the lowest; however, the contents of total sugar and reducing sugar demonstrate a contrary tendency, and the starch content at 116 DAT is much lower than those in the other two treatments. The proportion of superior tobacco, average price, yield, and output value of upper tobacco leaves at different leaf ages are the highest at 123 DAT. The highest sensory evaluation score is found at 123 DAT, while that at 130 DAT is significantly lower in comparison with the other two treatments. CONCLUSIONS: Tobacco leaves harvested at 123 DAT are mature and exhibit a low degree of membrane lipid peroxidation, moderate chemical compositions, and high economic value. 123 DAT improves availability of tobacco leaves.

Exploring the effect of different application rates of biochar on the accumulation of nutrients and growth of flue-cured tobacco (Nicotiana tabacum).

Background: Biochar application has become one of the most potential tools to improve soil fertility and plant growth for sustainable and eco-friendly agriculture. However, both positive and negative effects of biochar application have been recorded on plant growth and soil fertility. Methods: This study investigated the impact of different application rates (0, 600, 900, 1200, and 1800 kg/ha) of biochar on the soil nutrient contents, accumulation of nutrients and dry matter in different plant parts, and growth of flue-cured tobacco plants under field conditions. Results: Results demonstrated that soil organic carbon pool and carbon/nitrogen ratio were increased proportionally with the increasing dosage of biochar, 25.54 g/kg and 14.07 g/kg compared with control 17 g/kg and 10.13 g/kg, respectively. The contents of soil total nitrogen were also significantly increased after biochar application in the middle (1.77 g/kg) and late-growth (1.54 g/kg) stages of flue-cured tobacco than in control (1.60 g/kg and 1.41 g/kg, respectively). The contents of soil nitrate nitrogen were also higher under low (600 and 900 kg/ha) application rates of biochar and reduced when higher (1200 and 1800 kg/ha) dosages of biochar were applied. However, it was observed that varying application rates of biochar had no impact on soil ammonium nitrogen content during the growth period of flue-cured tobacco plants. The nutrient accumulation (N, P, K) in different parts of flue-cured tobacco plants was significantly increased under a low application rate of biochar, which enhanced the soil and plant analyzer development values, effective leaves number, growth, dry matter accumulation, and leaf yield of flue-cured tobacco. In contrast, the high biochar application rate (1200 and 1800 kg/ha) negatively impacted nutrient accumulation and growth of flue-cured tobacco. Conclusion: Conclusively, the optimum application of biochar (600 and 900 kg/ha) is beneficial for plant growth, soil fertility, accumulation of nutrients, and dry matter in different plant parts. However, excessive biochar application (> 900 kg/ha) could inhibit flue-cured tobacco plant growth. This study provides a theoretical foundation for biochar application in tobacco and other crop production to obtain agricultural sustainability and economic stability.

Elucidating the impact of biochar with different carbon/nitrogen ratios on soil biochemical properties and rhizosphere bacterial communities of flue-cured tobacco plants.

Background and aims: In agriculture, biochar (BC) and nitrogen (N) fertilizers are commonly used for improving soil fertility and crop productivity. However, it remains unclear how different levels of BC and N fertilizer affect soil fertility and crop productivity. Methods: This study elucidates the impact of different application rates of BC (0, 600, and 1200 kg/ha) and N fertilizer (105 and 126 kg/ha) on biomass accumulation, soil microbial biomass of carbon (SMC) and nitrogen (SMN), and soil biochemical properties, including soil organic carbon (SOC), total nitrogen (TN), soil nitrate nitrogen (NO3--N), ammonium nitrogen (NH4+-N), urease (UE), acid phosphatase (ACP), catalase (CAT), and sucrase (SC) of tobacco plants. In addition, a high throughput amplicon sequencing technique was adopted to investigate the effect of different application rates of BC/N on rhizosphere bacterial communities of tobacco plants. Results: The results confirm that high dosages of BC and N fertilizer (B1200N126) significantly enhance dry matter accumulation by 31.56% and 23.97% compared with control B0N105 and B0N126 under field conditions and 23.94% and 24.52% under pot experiment, respectively. The soil biochemical properties, SMC, and SMN significantly improved under the high application rate of BC and N fertilizer (B1200N126), while it negatively influenced the soil carbon/nitrogen ratio. Analysis of rhizosphere bacteriome through amplicon sequencing of 16S rRNA revealed that the structure, diversity, and composition of rhizosphere bacterial communities dramatically changed under different BC/N ratios. Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Acidobacteria were highly abundant bacterial phyla in the rhizosphere of tobacco plants under different treatments. Co-occurrence network analysis displayed fewer negative correlations among rhizosphere bacterial communities under high dosages of biochar and nitrogen (B1200N126) than other treatments, which showed less competition for resources among microbes. In addition, a redundancy analysis further proved a significant positive correlation among SMC, SMN, soil biochemical properties, and high dosage of biochar and nitrogen (B1200N126). Conclusions: Thus, we conclude that a high dosage of BC (1200 kg/ha) under a high application rate of N fertilizer (126 kg/ha) enhances the biomass accumulation of tobacco plants by improving the soil biochemical properties and activities of rhizosphere bacterial communities.

Genome-Wide Identification of the RR Gene Family and Its Expression Analysis in Response to TDZ Induction in Rhododendron delavayi.

The cytokinin response regulator (RR) gene is essential for cytokinin signal transduction, which plays a crucial role in plant growth and development. Here, we applied bioinformatics to Rhododendron delavayi's genome to identify its RR gene family and systematically analyzed their gene characteristics, phylogenetic evolution, chromosomal localization, collinearity analysis, promoter cis-elements, and expression patterns. Overall, 33 RdRR genes were distinguished and classified into three types. All these genes harbored motif 5 (YEVTTVNSGLEALELLRENKB), the most conserved one, along with the plant-conserved domain (REC domain), and could be mapped to 10 chromosomes with four gene pairs of segmental replication events but no tandem replication events; 13 RdRR genes showed collinearity with Arabidopsis thaliana genes. Promoter analysis revealed multiple hormone-related cis-elements in the RR genes. After a TDZ (thidiazuron) treatment, 13 genes had higher expression levels than the control, whose magnitude of change depended on the developmental stage of leaves' adventitious buds. The expression levels of RdRR14, RdRR17, RdRR20, and RdRR24 agreed with the average number of adventitious buds post-TDZ treatment. We speculate that these four genes could figure prominently in bud regeneration from R. delavayi leaves in vitro. This study provides detailed knowledge of RdRRs for research on cytokinin signaling and RdRR functioning in R. delavayi.

Diterpenes from oriental tobacco Nicotiana tabacum 'YNOTBS1' and their bioactivities.

Seven diterpenes including two new seco-cembranoid basmanoids A (1) and B (2), and two new labdane basmadanes A (3) and B (4), and three known compounds (-)-dehydro-nor-ambreinolid (5), (13E)-8-hydroxy-13-labden-12-one (6), and 14,15,16-dinor-7-oxolabda-8-ene-13-oic acid (7) were isolated from oriental tobacco Nicotiana tabacum 'YNOTBS1'. Compounds 1-3 and 5 showed anti-tobacco mosaic virus (TMV) activity with inhibition rates in the range of 30-69% at the concentration of 1 mg/mL. However, none of them exhibited any inhibitory effects against nitric oxide (NO) production in LPS and IFN-γ-induced RAW 264.7 murine macrophages and anti-proliferative activities against cancer cell lines SMMC-7721 and A-549.

Polyethylene and poly (butyleneadipate-co-terephthalate)-based biodegradable microplastics modulate the bioavailability and speciation of Cd and As in soil: Insights into transformation mechanisms.

Microplastics (MPs) that enter the soil can alter the physicochemical and biochemical properties of soil and affect speciation of heavy metals (HMs), thereby perturbing the bioavailability of HMs. However, the mechanisms underlying these effects are not understood. Therefore, we investigated the effects of MPs from poly (butyleneadipate-co-terephthalate)-based biodegradable mulch (BM) and polyethylene mulch (PM) in Cd- or As-contaminated soil on soil properties and speciation of HMs. MPs were characterised using Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The addition of MPs reduced the bioavailability of HMs in soil and promoted the transformation of HMs into inert fractions. The mechanisms underlying the reduction of the bioavailability of HMs in soils could be as follows: (1) the entry of MPs into the soil changed its properties, which reduced the bioavailability of HMs; (2) FTIR and XPS analyses revealed that the hydroxyl and carboxyl groups and benzene ring present on the surface of aged MPs stabilized complexes (As(V)-O) with As(V) may have directly reduced the bioavailability of As(V) in soil; (3) aged BM exposed more amounts and types of reactive functional groups and was more effective in stabilising soil HMs than PM. Overall, this study provides new insights regarding the complexation mechanisms of soil HMs by MPs from different plastic mulch sources.

The regulation of Alternaria alternata resistance by LRR-RK4 through ERF109, defensin19 and phytoalexin scopoletin in Nicotiana attenuata.

Leucine-rich repeat receptor-like kinases (LRR-RKs), belonging to the largest subfamily of transmembrane receptor-like kinases in plants, are proposed to be involved in pathogen resistance. However, it is currently unknown whether LRR-RKs regulate Nicotiana attenuata resistance to Alternaria alternata, a notorious fungal pathogen causing tobacco brown disease. During transcriptome analysis, we identified a highly induced receptor kinase (NaLRR-RK4) in N. attenuata leaves after A. alternata inoculation. We speculated that this NaLRR-RK4 might be the resistance gene of tobacco to brown spot disease, and if so, what is its function and mechanism of action? Silencing of NaLRR-RK4 via virus-induced gene silencing (VIGS) lead to plants highly susceptible to A. alternata, and this result was further confirmed by two stable transformation lines (NaLRR-RK4-RNAi lines) generated by RNA interference technology. The susceptible of NaLRR-RK4-RNAi lines to A. alternata was associated with reduced levels of phytoalexin scopoletin and its key synthesis gene NaF6'H1. Further transcriptome analysis of leaves of WT and NaLRR-RK4-RNAi line after A. alternata inoculation revealed that NaLRR-RK4 regulated NaERF109 and NaDEF19. Silencing NaERF109 or NaDEF19 by VIGS lead to plants more susceptible to A.alternata, demonstrating their role in pathogen resistance. Interestingly, A.alternata-induced expression of NaF6'H1 and NaDEF19 were dramatically reduced in NaERF109-silenced VIGS plants. Taken all together, we identified LRR-RK4 as the first Leucine-rich repeat receptor-like kinases involved in A.alternata resistance in tobacco species, by regulating NaERF109, and subsequently NaDEF19 and NaF6'H1.

Microbial Cross-Talk: Dissecting the Core Microbiota Associated With Flue-Cured Tobacco (Nicotiana tabacum) Plants Under Healthy and Diseased State.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of flue-cured tobacco production which poses significant yield losses all around the world. In this study, we evaluated the rhizosphere microbiome of healthy and bacterial wilt-infected (diseased) flue-cured tobacco plants through amplification of V3-V4 and ITS1-5f variable regions of 16S and internal transcribed spacer (ITS) rRNA. The study was based on the location (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and sample types (healthy and diseased) to assess the diversity of bacterial and fungal communities. Bacterial and fungal communities present in roots primarily emanated from rhizosphere soil. Healthy flue-cured tobacco plants exhibit high microbial diversity compared to diseased plants. Among three variables, plant components significantly influence the diversity of microbial communities, whereas rhizosphere soil harbors higher microbial diversity than roots. Bacterial phyla Cyanobacteria and Proteobacteria were found in high relative abundance in roots and rhizosphere soil samples, respectively. As far as fungi is concerned, a high relative abundance of Ascomycota and Basidiomycota was found in both rhizosphere soil and root. Bacterial genera such as Bacillus, Bradyrhizobium, Ensifer, Neorhizobium, and Lysobacter related to plant growth promotion and disease suppressing abilities were dominant than fungal genera. Analysis of relative abundance at specie-level revealed that most fungal species are pathogenic to flue-cured tobacco and could provide a conducive environment for wilt infection. In conclusion, R. solanacearum significantly influences the microbial diversity of flue-cured tobacco plants and negatively affects the bacterial community composition. Altogether, our study demonstrates the complexity of bacterial and fungal communities that possibly interact with each other (microbe-microbe) and host (host-microbe). This cross-talk could be helpful for healthy flue-cured tobacco plant growth and to induce resistance against bacterial wilt disease.

Plant-Microbe Interaction: Mining the Impact of Native Bacillus amyloliquefaciens WS-10 on Tobacco Bacterial Wilt Disease and Rhizosphere Microbial Communities.

Ralstonia solanacearum, the causative agent of bacterial wilt disease, has been a major threat to tobacco production globally. Several control methods have failed. Thus, it is imperative to find effective management for this disease. The biocontrol agent Bacillus amyloliquefaciens WS-10 displayed a significant control effect due to biofilm formation, and secretion of hydrolytic enzymes and exopolysaccharides. In addition, strain WS-10 can produce antimicrobial compounds, which was confirmed by the presence of genes encoding antimicrobial lipopeptides (fengycin, iturin, surfactin, and bacillomycinD) and polyketides (difficidin, bacilysin, bacillibactin, and bacillaene). Strain WS-10 successfully colonized tobacco plant roots and rhizosphere soil and suppressed the incidence of bacterial wilt disease up to 72.02% by reducing the R. solanacearum population dynamic in rhizosphere soil. Plant-microbe interaction was considered a key driver of disease outcome. To further explore the impact of strain WS-10 on rhizosphere microbial communities, V3-V4 and ITS1 variable regions of 16S and ITS rRNA were amplified, respectively. Results revealed that strain WS-10 influences the rhizosphere microbial communities and dramatically changed the diversity and composition of rhizosphere microbial communities. Interestingly, the relative abundance of genus Ralstonia significantly decreased when treated with strain WS-10. A complex microbial co-occurrence network was present in a diseased state, and the introduction of strain WS-10 significantly changed the structure of rhizosphere microbiota. This study suggests that strain WS-10 can be used as a novel biocontrol agent to attain sustainability in disease management due to its intense antibacterial activity, efficient colonization in the host plant, and ability to transform the microbial community structure toward a healthy state. IMPORTANCE The plant rhizosphere acts as the first line of defense against the invasion of pathogens. The perturbation in the rhizosphere microbiome is directly related to plant health and disease development. The introduction of beneficial microorganisms in the soil shifted the rhizosphere microbiome, induced resistance in plants, and suppressed the incidence of soilborne disease. Bacillus sp. is widely used as a biocontrol agent against soilborne diseases due to its ability to produce broad-spectrum antimicrobial compounds and colonization with the host plant. In our study, we found that the application of native Bacillus amyloliquefaciens WS-10 significantly suppressed the incidence of tobacco bacterial wilt disease by shifting the rhizosphere microbiome and reducing the interaction between rhizosphere microorganisms and bacterial wilt pathogen.

[Difference of rhizosphere microbe quantity and functional diversity among three flue-cured tobacco cultivars with different resistance].

Field experiments were conducted in Shilin and Xundian respectively to study the diffe- rence of rhizosphere microbe quantity and functional diversity with plate culture method and Biolog technique among Hongda (high susceptibility, S), Yun87 (middle resistance, MR) and K326 (high resistance, R) , three flue-cured tobacco cultivars with different resistance to bacterial wilt and black shank. The results indicated that the amounts of bacteria, actinomycetes and the total number of microbes in tobacco plants' rhizosphere were positively correlated with the cultivar' s re- sistance, while it was opposite for the fungi. The consistent tendency was obtained not only at 35 d, 55 d and 75 d after transplanting, but also at two experimental sites. Cultivar and experimental con- ditions greatly affected the utilization of six types of carbon source by rhizospheric microbes, as well as the AWCD value. In Xundian site, rhizospheric microbes' utilization of carbohydrates, amino acids, carboxylic acids, polymers,. amines and the AWCD value were all higher at 55 d and 75 d after transplanting for the resistant cultivar than the susceptible one, but it was opposite at 75 d after transplanting for the phenolic acids. In Shilin, significant differences existed among the three culti- vars at 35 d, 55 d and 75 d after transplanting for the indices mentioned above, although they were not consistent with cultivars' resistance. Principal component (PC) analysis even showed that utili- zation of carbon sources by rhizosphere microorganisms differed significantly among the three culti- vars at the two sites, and it was better reflected by PC1 and PC2 at 55 d and 75 d after transplan- ting, respectively. In conclusion, rhizosphere microbial community structure and functional diversity were greatly affected not only by the cultivars' difference in resistance, but also by experimental conditions.

[Nutrient balance between N, P, and K in flue-cured tobacco production under different preceding crops planting].

A field experiment was conducted to study the nutrient balance between N, P, and K in flue-cured tobacco production in Shilin County of Yunnan Province under the effects of different preceding crops including rape, wheat, barley, and green manure planting. Overall, there existed significant differences in the soil nitrogen availability, tobacco plant dry matter accumulation, plant uptake of N, P and K, residual amounts of soil N, P, and K, apparent soil N loss, and apparent soil P- and K surplus during tobacco growth period under different preceding crops planting. Under preceding crop green mature planting, the soil mineral N content before tobacco plant transplanting, the soil N net mineralization rate during tobacco growth period, and the tobacco plant dry matter accumulation and N- and K uptake at maturation were all the highest, followed by under preceding rape planting, and under preceding barley or wheat planting. The P uptake by tobacco plant was also obviously higher under the preceding green manure or rape planting than under preceding barley or wheat planting. After the harvest of tobacco leaves, the soil residual mineral N content was the highest under the preceding green manure planting, followed by under preceding rape planting, and by preceding barley or wheat planting, while the soil available P and K contents were in adverse, being the highest under preceding barley or wheat planting and the lowest under preceding green manure planting. The apparent soil N loss during tobacco growth period was the highest under the preceding green manure planting, whereas the apparent soil P- and K surplus was obviously higher under preceding wheat or barley planting. It was suggested that an appropriate adjustment should be made on the fertilization rates of N, P, and K for tobacco production based on the preceding crops, i. e., lesser N application when the preceding crop was green manure or rape, and lesser application of P and K when the preceding crop was wheat or barley.