Exogenous Application of Sodium Hydrosulde Suppresses Bacterial Wilt And Regulates The Soil Microecology

The role of hydrogen sulde (H 2 S) in regulating the pathogenic bacteria has been well documented. However, whether exogenous H 2 S addition inhibits the pathogens in soil is not understood, and whether H 2 S can suppress the plant disease caused by pathogen R. Solanacearum is not clear. In the present study, different concentrations of H 2 S donor NaHS were applied to the tobacco eld to explore the interrelation among NaHS, tobacco baterial wilt, soil physicochemical properties and microbial community. In order to decipher the disease suppression mechanism from the perspective of soil microecology. Application of NaHS signicantly reduced the disease incidence and disease index of TBW, increased soil pH, alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), available phosphorus (AP) and organic matter (OM). NaHS addition also changed soil microbial community composition and structure. Furthermore, NaHS addition signicantly reduced the abundance of Ralstonia and Fusarium, and increas pathogenic ed benecial microorganisms Solirubrobacter, Rhodococcus, Rhizobium, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium, Bacillus, Trichoderma and Aspergillus at the genus level. Our ndings suggested that exogenous application of NaHS signicantly suppressed TBW caused by R. Solanacearum through regulated soil microecology. This study revealed the potential of NaHS in control of bacterial wilt.


Introduction
Bacterial wilt is a bacterial soil-borne disease caused by R. Solanacearum 1 . The disease have become one of the potential threats to agriculture, and causing huge losses to yields of Solanaceous crops 2 . Lots of researches on the control of bacterial wilt focus on resistant varieties, chemical control, biological control and agricultural control. However, these traditional control methods may have limited e cacy and many problems, such as lack of resistant varieties, poor control e cacy, pathogen resistant, environmental pollution, and so on 3,4 . Therefore, more effective and environmentally friendly approaches need to be developed to control bacterial wilt.
Hydrogen sul de (H 2 S) was thought as the third newest gaseous signal molecular, which was not only applied in animal and human physiological processes (such as dermatological diseases, cell behaviors, vascular system, neuronal disease, digestive systems, COVID-19 and so on) [5][6][7][8][9][10][11][12] , but also applied in agriculture 13 . A large number of studies in the eld of plants have shown that H 2 S can directly or indirectly involve in a wide range of plant physiological processes including stomatal movement 14 , photosynthesis 15 , seed germination 16 , root growth 17 , fruit ripening 18 , as well as plant senescence 19 . H 2 S can participate in enhance plant tolerance to drought, salinity, high-temperature and heavy metal stress by initiating plant redox signal, antioxidant capacity and speci c components of cellular defence [20][21][22][23] .
Exogenous application of H 2 S induces plant cross-adaptation to multiple abiotic stresses 24 . Therefore, H 2 S as a sulfur-containing defence compound, plays an important role in plant resistance to biotic and abiotic stresses 25 . Fu et al. 26 found that H 2 S has antifungal role on the postharvest pathogens Aspergillus niger and Penicillium italicum. They also demonstrated that the antifungal effect of H 2 S might be associated with the increased accumulation of reactive oxygen species (ROS) in H 2 S-treated fungi 26 . Previous studies showed that H 2 S both positively and negatively impacts on the bacterial growth [27][28][29] . Some reports revealed that H 2 S also regulate the pathogenic bacteria 30,31 . However, It is still unknown whether H 2 S can suppress the plant disease caused by soil-born pathogen R. Solanacearum.
Tobacco is an important economic plants of Solanaceous, which is easily infected with serious soil-borne disease by R. Solanacearum 32 . In this study, sodium hydrosul de (NaHS), a H 2 S donor, was applied to the tobacco eld. We aimed to investigate the control e cacy of NaHS application on tobacco bacterial wilt (TBW), as well as the effects on soil physicochemical properties and microbial community. The application of NaHS was proposed as a new approach to control TBW.

Results
Disease severity index of TBW. Disease incidence (I), disease index (DI) and control e cacy with ve treatments were calculated at 100 d post-transplantation ( Table 1). The I and DI of the control (CK) were signi cantly higher than those of NaHS treatments. As the concentration of NaHS increased from 200 mg/L to 800 mg/L, the I and DI continuously decreased from 44.22-15.21%, and 13.21 to 4.26, respectively. With the increase of NaHS concentration, the control e cacy increased gradually. All the results suggested that the application of NaHS reduce the disease incidence and disease index of TBW, and the control e cacy of TBW is as high as 89.49%. Table 1 The occurrence of tobacco bacterial wilt in different concentration of NaHS. Effects of NaHS application on soil physicochemical properties. Seven physicochemical properties of the rhizosphere soil were analyzed ( Table 2). The value of pH, alkali-hydrolyzed nitrogen (AN), available phosphorous (AP) and organic matter (OM) were increased as the concentration of NaHS increased from 200 mg/L to 800 mg/L. There was no signi cant difference in available potassium (AK) and exchangeable calcium (Ca) between NaHS treatments and CK. The results showed that the application of NaHS could increase the soil pH, AN, AP and OM. What's more, pH, AN, AP and OM showed signi cantly negative (p < 0.01) correlation with the incidence of TBW (Table S1). These results indicated that the application of NaHS may reduce the incidence of TBW by changing soil physicochemical properties. Effects of NaHS application on bacterial diversity and community. In total 679,451 high-quality raw sequences with the average length of 252 bps for bacteria were obtained from rhizospherial soil samples after quality ltering. The OTUs, Chao1 and Shannon index were used to evaluate and compare the richness and diversity of bacterial community among different treatments (Table S2). The OTUs, Chao1 and Shannon index were lower in the rhizophere soil of NaHS treatments than the CK. With the increase of NaHS concentration from 0 mg/L to 600 mg/L, the OTUs, Chao1 and Shannon index decreased gradually. Comparing with NaHS600 treatment, the OTUs, Chao1 and Shannon index in NaHS800 treatment was slightly higher (Table S2). While, the OTUs, Chao1 and Shannon index in NaHS800 treatment signi cantly lower than the CK. This result suggested that NaHS treatments could change the richness and diversity of soil bacterial community.
The relative abundance of Proteobacteria in NaHS800 treatment was lower than that in other treatments, while the relative abundance of Verrucomicrobia and Bacteroidetes were higher than that in other treatments (Fig. 1B). The Heatmap analysis of the top 40 genera with hierarchical clusters was used to identify the different composition of bacterial community structure. There were distinctions of bacterial community structures among different treatments in the Heatmap. The application of NaHS signi cantly increased the abundances of Streptomyces, Microvirga, Rhodococcus, Haliangium, Paenibacillus, Chthonomonas, Bacillus, Solirubrobacter, Gaiella, Lysobacter, Pseudolabrys, Pseudomonas, Granulicella, Stenotrophomonas, Flavobacterium and Rhizobium. In contrast, NaHS signi cantly decreased the abundances of Massilia, Acidibacter and Ralstonia (pathogen of bacterial wilt) (Fig. 1C). These results suggested that NaHS application play impact on the the structure of bacterial community.
Further analyses were carried out at the genus level, and the different distributions of the top forty abundant bacterial genera among the ve treatments were illustrated in Fig. 2. Twelve varied among the ve treatments were signi cantly different, including Solirubrobacter, Rhodococcus, Rhizobium, Ralstonia, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium and Bacillus. The genus Solirubrobacter which was dominant in NaHS treatments, and occupied low percentage in CK (Fig. 2). The trends in change in the genera Rhodococcus, Rhizobium, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium and Bacillus were the same as that in Solirubrobacter. In contrast, Ralstonia was dominant in CK, and decreased signi cantly in NaHS treatments (Fig. 2).
Effects of NaHS application on fungal diversity and community. The difference of the OTUs, Chao1 and Shannon index of fungal community among different treatments were also analyzed (Table S2). There was no signi cant difference in OTUs, Shannon and Chao1 indexes between CK and NaHS 200. With increase of NaHS concentration from 400 mg/L to 800 mg/L, the OTUs, Chao1 and Shannon index reduced signi cantly. The results showed that NaHS application could impact the diversity and richness of soil fungi.
According to PCoA analysis, PC1 and PC2 explained 23.47% and 13.75% of the total fungal community variations respectively (Fig. 3A). The distribution of fungi among different treatments was relatively discrete, indicating that there were obvious differences in the fungal community structure among different treatments. 6 main known fungal phyla were identi ed from all soil samples, including Ascomycota (43.56 ~ 73.45%), followed by Basidiomycota (7.98 ~ 28.21%), Chytridiomycota (6.84 ~ 30.15%), Glomeromycota (1.00 ~ 8.16%), Neocallimastigomycota (0.05 ~ 6.19%) and Zygomycota (0.66 9.31%) (Fig. 3B). The relative abundance of Ascomycota decreased as the concentration of NaHS increased from 200 mg/L to 600 mg/L, and slightly increased when the concentration of NaHS was 800 mg/L. However, the relative abundance of Ascomycota was lower in NaHS800 than in CK. The relative abundance of Zygomycota increased as the concentration of NaHS increased from 200 mg/L to 600 mg/L, and signi cantly decreased when the concentration of NaHS was 800 mg/L. The relative abundance of Basidiomycota, Glomeromycota, Chytridiomycota and Zygomycota also varies with the concentration of NaHS. These results indicated that NaHS altered the fungal community composition, which was associated with NaHS concentration. (Fig. 3B). In the Heatmap for fungal community, The relative abundance of Xanthoria, Monograpella, Candida, Paludomyces, Microidium and Sakaguchia in CK were signi cantly higher than in NaHS treatment. NaHS800 signi cantly enriched the relative abundance of Batrachochytrium, Gorgonomyces, Populocrescentia, Cladosporium, Rhodosporidium, Aspergillus, Tomentella, Lycogalopsis, Trichoderma, Pseudocamarosporium, Russula, Byssochlamys and Paecilomyces (Fig. 3C).
The different distributions of the top forty abundant fungal at genus level among the ve treatments were analyzed (Fig. 4). Three were signi cantly different among the ve treatments, including Trichoderma, Fusarium and Aspergillus. The genus Trichoderma and Aspergillus were dominant in NaHS, and occupied low percentage in CK (Fig. 4). In contrast, Fusarium was dominant in CK, and decreased signi cantly in NaHS treatments (Fig. 4).
The relationship between rhizosphere soil physicochemical properties and microbial community. The relationship between rhizosphere soil physicochemical properties and microbial community structure were analysed by redundancy analysis (RDA). The results showed that 64.27% and 59.47% of bacterial and fungal community variation, respectively (Fig. 5). The bacterial Rhodococcus, Solirubrobacter, Paenibacillus, Haliangium, Bacillus, Lysobacter, Pseudomonas, Flavobacterium and Granulicella were positively correlated with pH, AN, AP and OM. While, Ralstonia presented contrasting behavior that was negatively correlated with pH, AN, AP and OM (Fig. 5A). The fungal Trichoderma and Aspergillus were positively correlated with AN, Ca, AK, AP and pH. While, Fusarium showed negatively correlated with AN, Ca, AK, AP and pH (Fig. 5B). The redundancy analysis revealed that rhizosphere soil AN, Ca, AK, AP and pH had great in uence on microbial community. Discussion H 2 S has long been considered as a phytotoxin. But in recent years, it has been found that it play an important role in plant physiological process as gas signal molecule [14][15][16][17][18][19] . Application of exogenous H 2 S to plants can provide additional protection against stresses, such as drought, salinity and heavy metals are mainly induced antioxidant system to reduce oxidative cell damage 13 . H 2 S has also been found to inhibited the growth of pathogens Aspergillus niger, Penicillium italicum, Rhizopus oryzae, Candida albicans, and so on 26-30 . However, all above evidence focused on the interaction between plant and H 2 S, whether exogenous H 2 S addition directly inhibits the pathogens in soil is not clear, and whether H 2 S can suppress the plant disease caused by pathogen R. Solanacearum is still unknown. In the present study, different concentrations of H 2 S donor NaHS were applied to the tobacco eld, we found that the exogenous NaHS signi cantly suppressed TBW caused by R. Solanacearum through changing the soil physicochemical properties and microbial community.
Since researchers indicated that the exogenous application of H 2 S can affect plant growth by altering soil nutrient content 33,34 . In our study, the exogenous application of NaHS increased the the soil pH, AN, AP and OM (Table 2). Increase pH is important for inhibited the survival of R. Solanacearum, and increase OM, N and P meet the need of plant growth 35,36 . In addition, the higher soil carbon and phosphorus could increase activity of bene cial microorganisms against pathogen 36 . This study demonstrated that the application of NaHS signi cantly reduce the disease incidence and disease index of TBW, and pH, AN, AP and OM were signi cantly negative correlation with the incidence of TBW. It was speculated that application of NaHS changed soil physicochemical properties that indirectly suppressed R. Solanacearum growth by promoting antagonistic microorganisms.
The rhizosphere soil microbial community structure in uences the plant immunity and quality, and the microbial community are considered to be a key mechanism that can suppress soil-born pathogens 37,38 . Our ndings explored that application of NaHS altered the diversity and richness of soil microbial (Table   S2), which was consistent with the ndings of Fang et al. 34 In this study, microbial analysis revealed a different pattern among treatments ( Fig. 1A; Fig. 3A). NaHS application signi cantly in uenced the composition of the soil microbial community ( Fig. 1;Fig. 3). NaHS treatments reduced the relative abundance of Proteobacteria (Fig. 1B), which was similar to the results by Li et al. 38 The phylum Proteobacteria included the pathogen R. solanacearum is less abundant in healthy soils than in the bacterial wilt soil 38 . NaHS treatments also reduced the relative abundance of Acidobacteria (Fig. 1B). Acidobacteria was mainly driven by soil pH, and the low pH was more suitable for the survival of Acidobacteria 39 . Our results also showed that the relative abundance of Bacteroidetes were increased in NaHS treatments (Fig. 1B), which could promote plant growth, and improve the resistance of plants to environmental stress 40 . The Heatmap based on signi cant changes indicated that NaHS application signi cantly increased the abundances of some bacteria and fungi (such as Paenibacillus, Bacillus, Lysobacter, Aspergillus and Trichoderma etc.) ( Fig. 1C; Fig. 3C). The function of these increased genera may be related to soil physicochemical properties and pants, which can accelerate the cycling of elements and promote plant growth and environment adaption.
In the present investigation, certain genera of microorganisms of Solirubrobacter, Rhodococcus, Rhizobium, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium, Bacillus, Trichoderma and Aspergillus were signi cantly high in NaHS treatments. However, Ralstonia and Fusarium were signi cantly low in NaHS treatments ( Fig. 2; Fig. 4). The genus Ralstonia includes many soil-borne pathogens, which only infects root via wounds caused by microbe and insect 41 . Pathogenic Fusarium can infect the root by penetration hyphae, causing more wounds to the root, and thus increasing the infection of the root by pathogenic Ralstonia 42 . The application of NaHS may inhibited soil-borne diseases caused by pathogenic Ralstonia and Fusarium. Some species in Solirubrobacter and Granulicella have positive effects on the transition of the organic carbon in the soil 43 . Rhodococcus and Bacillus were reported as phosphate-mobilizing bacteria, which have the ability to solubilize organic and inorganic phosphate 44 . Furthermore, some species of the genus Bacillus can affect the growth and virulence traits of Ralstonia by producing volatile organic compounds 45 . The genus Pseudomonas is known for its ability to promote plant growth, inhibit pathogens, and induce the systemic resistance to diseases in many plants 46 . Microvirga is nitrogen xing bacteria, which can involved in nitrogen cycling 44 . Previous studies have documented that Pseudomonas, Rhizobium, Paenibacillus, Lysobacter, Haliangium, Flavobacterium and Bacillus as antagonistic bacteria can mitigate many soil-borne diseases and promote plant growth and health 45,[47][48][49] . Trichoderma and Aspergillus have been reported as antagonistic fungal. They directly interact with roots to produce bioactive substances, which can improve plant growth, and resist abiotic and biotic stress 50,51 . The application of NaHS may provide a suitable environment for promoting the growth of these bene cial microorganisms, increasing the relative abundance of these bene cial microorganisms, and reducing the incidence of TBW. In this study, the redundancy analysis (RDA) revealed that these bene cial microorganisms were positively correlated with pH, AN and AP, whereas The genus Ralstonia and Fusarium were negatively correlated with pH, AN and AP (Fig. 5). In sum, the soil microecology changes induced by NaHS are strong related to the suppression of soil-borne diseases.

Conclusion
Our study demonstrated that exogenous NaHS signi cantly reduced the disease incidence and disease index of TBW. The application of NaHS increased soil pH and improved soil nutrient status, and some soil physicochemical properties had a positive relationship with the abundance of the soil microbial community. NaHS addition changed soil microbial community composition and structure at the phylum and genus levels. Taken together, exogenous application of NaHS shifting the soil microecology to suppresses TBW, which may provide a new perspective to control TBW. (2) 200 mg/L NaHS (NaHS200); (3) 400 mg/L NaHS (NaHS400); (4) 600 mg/L NaHS (NaHS600); (5) 800 mg/L NaHS (NaHS800). 50 mL NaHS of different concentrations was applied to each tobacco root when transplantation. The planting density of all treatments were the same.

Materials And Methods
Soil sample collection and physicochemical properties analysis. Rhizosphere soil were collected by vespot-sampling method at 100 d post-transplantation when recording the disease occurrence. Then the soil samples from the ve separate sites were mixed to one soil sample, partitioned into two subsamples, ones were immediately transported on ice to the laboratory and stored at − 80°C for genomic DNA extraction, and the other subsamples were air-dried for physicochemical properties analysis. The analysis of soil pH, alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), available potassium (AK), organic matter (OM), exchangeable calcium (Ca) and exchangeable magnesium (Mg) was performed according to Hu et al. 52 Bacterial wilt recording. Symptoms of TBW were monitored at ve different sites in each plot at 100 d post-transplantation. The TBW disease index (DI) based on severity scale of 0-9 was described in a previous study 52 . Brie y, "0" represents the plants without visible symptoms; "1" represents the presence of occasional chlorotic spots on stems, or less than half of the leaves wilted on unilateral stems; "3" represents the presence of a black streak less than half the height of the stem, or between half to twothirds of the leaves wilted on unilateral stems; "5" represents the presence of a black streak over half the length of the stem, but not reaching the top of the stem, or more than two-thirds of the leaves wilted on unilateral stems; "7" represents the presence of a black streak reaching the top of the stem, or all leaves wilted; and "9" represents the dead plant. Based on the number of plants in each rating scale, disease incidence (I) and disease index (DI) of TBW were calculated as I = n′/ N × 100% and DI = ∑(r × n)/(N × 9) × 100, where n′ is the total number of infected tobacco plants, r is the rating scale of disease severity, n is the number of infected tobacco plants with a rating of r, and N is the total number of plants. The extracted DNA from each soil sample was used as a template for ampli cation. The V4 region of the bacterial 16S rRNA genes and the ITS1 regions of the fungal rRNA genes were ampli ed. Each the DNA sample was ampli ed separately using the primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') 53 for bacterial, ITS5-1737F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′-GCTGCGTTCTTCATCGATGC-3′) 54 for fungi.
Sequences processing and analysis. All PCR reactions were performed on Illumina HiSeq platforms (Illumina Inc., USA) at Novogene Bioinformatics Technology Co., Ltd (Beijing, China). The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scienti c) and Agilent Bioanalyzer 2100 system. The sequence quality was statistically analyzed by CASAVA1.8. The raw sequence data was preliminarily ltrate using the FASTX Toolkit 0.0.13 software package, removing the low mass base at the tail of the sequence (Q value less than 20) and the sequences with lengths less than 35 bp. nally, the length of the valid reads was approximately 250 bp. All effective tags of all samples were clustered using Uparse software (V7.0.1001, http://drive5.com/uparse/). Sequences with ≥ 99.5% identity for 16S rDNA and sequences with ≥ 97% identity for ITS were assigned to the same OTUs (operational taxonomic units).