Bacillus atrophaeus HAB-5 secretion metabolites preventing occurrence of systemic diseases in tobacco plant
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Finding synthetic pesticide alternatives for health and a healthy environment has become a crucial issue for scientific research. A number of studies have reported efficacy of Bacillus species on promoting plant development, as well as protecting plants against pathogen invasion, especially pathogenic fungi and bacteria. However, little was known about Bacillus species in controlling viral diseases. In this study, Bacillus atrophaeus strain HAB-5, isolated from cotton field, Xinjiang, China efficiently promoted the growth of tobacco plants. According to the results, the treatment with the strain HAB-5 increased the expression of NtEXP1 and NtEXP2. Then the Tobacco mosaic virus (TMV)/ Nicotiana tobacco system was employed to evaluate virus resistance induced by strain HAB-5. Tobacco leaves were treated with antimicrobial metabolites of strain HAB-5 (1 mg/mL), and 12 h later the treated leaves were challenged with TMV via rub-inoculation. The results showed that disease symptoms were obviously compromised by tobacco leaves treated with strain HAB-5, and the viral accumulation level was reduced extensively. Moreover, it was found that the signaling regulatory gene (NPR1), defense genes (PR-1a, PR-1b, Chia5), and hypertensive response related genes (Hsr203J, Hin1) were up-regulated in plants treated with the metabolites. Altogether, these accumulated results strongly support strain HAB-5 to be a biological controlling agent against TMV.
KeywordsBacillus atrophaeus TMV Biocontrol
Plant growth promoting rhizobacteria (PGPR), the beneficial bacteria having ability to enhance plant growth and control plant diseases by colonizing the roots have been well recognized for various beneficial effects in crop plants (Bodelier et al. 2000; Eric and Donohoe 2003; Nie et al. 2015; Ge et al. 2015). Their usage as biological control agent increases health and productivity of different plant species under various edaphic conditions (Kloepper et al. 2009; Chaudhry et al. 2012; Bharti et al. 2016; Devendra et al. 2016; Bharti et al. 2016); PGPR often resists phytopathogenic negative effects by inhibiting them (Leeman et al. 1996; Wang et al. 2009; Khan et al. 2012).Various studies has been carried out focusing on the identification of Bacillus spp. for their ability to promote plant growth and disease control (Emmert and Handelsman 1999; Kloepper et al. 2004; Niu et al. 2011; Piromyou et al. 2011).Various strains of B. amyloliquefaciens, B. subtilis, B. pumilus, elicit significant reductions in the incidence severity of various diseases on a diversity of hosts (Zhang et al. 2004; Han et al. 2006; Raj et al. 2008; Raza et al. 2016). Therefore, several PGPR based products are recently available on commercial basis mostly containing Bacillus spp. Strains (Emmert and Handelsman 1999).
These PGPRs, commonly known biocontrol agents, are famous for producing variety of metabolites that can improve plant resistance against different crop pathogens, including lipopeptides, lytic enzymes, etc. (Worasatit et al. 1994; Ongena et al. 2007). It is interesting to note that different studies focused on harvesting these secreted compounds from culture suspension, cell filtrate and crude extract have been applied directly on plant as foliar treatment. For example, culture suspension of Trichoderma. harzianum sprayed on leaves successfully reduced gray mold on fruits and foliage (Lo et al. 1997; Luca et al. 2016). Leaves sprayed with the spore suspension of Bacillus subtilis reduced different crop disease (Batchimeg and Dondov 2015). Zhang et al reported the efficacy of cell free supernatant of B. cereus on plant leaves to control Sclerotinia sclerotiorum (Zhang and Xue 2010). The foliar spray treatment of Streptomyces spp. was reported efficiently to control rice sheath blight pathogen (Yang et al. 2017). The crude extract metabolites from different strains of B. amyloliquefaciens were capable of reducing the severity of different diseases on plant (Ji et al. 2013).
Despite the successful use of Bacillus species, reports about Bacillus atrophaeus application are rare. We have reported that B. atrophaeus strain HAB-5 can control a broad spectrum of crop disease-causing agents (Rajaofera et al. 2017, 2019). B. atrophaeus are diverse in terms of plant protection playing important role in crop yield reducing risks. Furthermore, keeping in view the growing concerns over global food safety and environmental quality, efforts in increasing practical use of natural products need a better understanding and research.
The current investigation is an effort to elucidate the concept of B. atrophaeus strain HAB-5 as a biocontrol resource. We have investigated strain HAB-5’s ability to promote plant growth and evaluate the potential of crude metabolites extracts to control TMV on tobacco plant.
Materials and methods
The isolate designed as strain HAB-5 was obtained from a healthy cotton field in Xinjiang province, P.R China. After isolation, strain HAB-5 was examined for morphological, physiological and biochemical characteristics (Rajaofera et al. 2017, 2019).
Plant material, growth condition and TMV culture
Tobacco seeds were surface sterilized (2 min 70% ethanol, 5 min 5% calcium hypochlorite), rinsed several times with sterile water, and placed on Petri dishes containing half strength Murashige–Skoog (MS) medium containing 0.8% agar and 1.5% sucrose. The seeds were vernalized in dark for 72 h at 4 °C to break dormancy, and then germinated to pot culture containing autoclaved soil (vermiculite and Pindstrup substrate, in the ratio of 1:1). TMV was maintained by sap inoculations on N. tabacum cv. Samsun NN plants.
Assessment of plant growth promotion and plant inoculation
The ability of strain HAB-5 to promote plant growth was assessed. Tobacco seeds were surface sterilized as described above. The seedlings were grown till 3–4 leaves stage. Biological replicates of each treatment were used to minimize the influence of an individual plant results. Seedlings were transplanted individually into pots (with the same soil on which tobacco seeds were germinated) and grown.
Ten days after transplantation, bacterial strain was suspended in culture medium and incubated at 28 °C, 180 rpm for 24 h. After incubation, bacterial cells were harvested by centrifugation and re-suspended in distilled water, adjusted to 1 × 106–107 CFU/mL to maintain uniform cell density. The cell suspension (50 mL) was drenched in soil around each plant twice at weekly intervals. Plants only drenched with water were used as control. Experiments were carried out in three replicates and every replication contained 12 plants.
The experiment was terminated 60 days after inoculation. Plant length, fresh, dry weight of roots, shoots and chlorophyll content were measured from all harvested plants. Percent decrease or increase in each parameter was calculated with respective controls. The data obtained was analyzed statistically for standard error.
Quantification of HAB-5 strain colonization in N. tabacum rhizosphere
The resistant of the strain HAB-5 to antibiotics was first determined. The strain was streaked onto LB agar plate containing different antibiotics including 50 mg/L of streptomycin, 100 mg/L of riphamycine, 100 mg/L of ampicyline and 200 mg/L of kanamycin. The antibiotic which had the positive result was used for further experimentation.
The abundance of the PGPR rhizosphere was then quantified. Total root systems and tightly-adhering soil were harvested at five time points 0, 15, 30, 45 and 60 dpt. The samples were thoroughly mixed and 1 g of mixture was suspended in 9 ml of sterile 0.85% NaCl and vortexed for 5 min. The suspensions of each sample were plated on LB agar medium with 50 mg/L of streptomycin and 200 mg/L of kanamycin according to a previous method (Zhou et al. 2015). In order to inhibit the fungal growth, 100 mg/L of cycloheximide was also used. Colony Forming Units (CFUs) counting (based on morphological shape of HAB-5) was carried out after incubation at 28 °C for 24 h.
Biological control of tobacco mosaic virus by HAB-5 treatment
First, the antimicrobial metabolite from strain HAB-5 was extracted. The organism was cultured in LB for 48 h at 28 °C in an incubator shaker at 180 rpm. The fermentation broth was centrifuged at 10.000 rpm for 10 min at 4 °C. petroleum ether were added into the cell free supernatant in 1:1 ratio and shacked vigorously. The organic phase was concentrated to dryness under vacuum using rotary evaporator. The crude extract was lyophilized and re-suspended with distilled water (1 mg/mL). The substance obtained from the non-inoculated culture medium with same procedure was used as a control.
The evaluation of strain HAB-5 efficacy against TMV reduction and infection ratio was investigated. Two different set of experiment were carried out in order to evaluate strain HAB-5 effect against TMV. In one set of experiment, plants drenched with the bacteria root inoculation were subjected of TMV inoculation. In the other set of experiment, the crude extract antimicrobial metabolites from the strain were applied for foliar treatment and 12 h later the treated leaves were challenged with TMV via rub-inoculation. The effective treatment was used for further experiments.
For disease severity evaluation, plant treatments were assigned into three groups included HAB-5 + TMV group (sprayed with the antimicrobial metabolite crude extract from the strain 12 h before inoculation with TMV), mock+ TMV group (sprayed with the extract products from LB 12 h before inoculation with TMV) and control group (only sprayed with water, and PBS was used in the place of TMV). All experiments were carried out with three replicates.
The virus inoculum was prepared in the tobacco juvenile. Leaf tissue was macerated in 10 mL of 50 mM potassium phosphate buffer (pH 7.0). The suspension was sprayed on 3–4 leaf stage tobacco seedlings. Disease severity symptoms (mosaic, leaf deformation and stunting) were observed at 7 days post inoculation (dpi) according previous method (Manidipa et al. 2013). The rating scale were 0 = no symptoms observed; 1 = light mottling and a few thin yellow veins; 2 = mottling and vein clearing unevenly distributed on the leaf; 3 = mottling, leaf distortion, and stunting; and 4 = severe mottling, leaf curling, and stunting.
Enzyme-linked immunosorbent assay (ELISA)
ELISA analysis was carried out to determine the relative level of coat protein (CP) in leaves. Samples were ground in a 50 mM carbonate buffer, loaded onto ELISA 96-well plates, coated with plastic wrap, and incubated at 40 °C overnight. After washing with a washing buffer (0.5% Tween 20 in PBS), the plates were blocked with 3% nonfat dry milk in PBS at 37 °C for 1 h, and washed five times. Next, the primary antibody rabbit anti-TMV immunoglobulin (Ig) (1.0 μg/mL) was incubated in the 96-well plates at 37 °C for 1 h and rinsed. The secondary antibody goat anti-rabbit IgG-HRP (1/10.000) was added to each well, then incubated at 37 °C for 1 h and washed. Finally 3, 3′, 5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide were used as the substrate for developing at 37 °C for 15 min. The reaction was stopped by the addition of 50 μg of 1 M H2SO4. The plates were read in micro plate photometer.
Detection of hydrogen peroxide accumulation
To detect accumulation of hydrogen peroxide, leaf tissues were stained with 1 mg of diaminobenzidine (DAB, pH 3.8). Leaves were washed in distilled water and cleared with 95% (v/v) for 24 h. DAB staining visualizes hydrogen peroxide as dark brown precipitate under the light microscope.
Measurement of cell death
Experiment was carried out as describe earlier (Singh and Upadhyay 2014). Leaves incubated with TMV for 7 days were soaked in 0.05% Evans blue for 24 h, rinsed, dried, and then boiled in anhydrous alcohol and glycerol (9:1 V/V) for 30 min to remove the chlorophyll. A blue area represents cell death. The amount of cell death in tobacco leaves was also quantified. Stained leaves were added to 4 mL 1% SDS water solution for 3 days to extract Evans blue. The supernatant was collected after centrifugation at 10.000 rpm for 5 min and the absorbance was measured at 600 nm.
Nitric oxide production
According the previous method (Jia et al. 2016), the epidermis was peeled neatly from the surface of rosette leaves and then incubated in MES/KCl buffer (10 mM MES/KOH, 50 mM KCl, 100 μM CaCl2, pH 6.5) for 30 min in the light. Then, incubated in Tris/KCl buffer (10 mM Tris, 50 mM KCl, pH 7.2) containing 5 μmol/LDAF-2DA (sigma) for 30 min in the dark at room temperature. Excess DAF-2DA was washed three times with fresh Tris/KCl buffer, the epidermis was placed in Tris/KCl buffer containing strain HAB-5 for 15 min, Tris/KCl buffer as contrast. NO production was photographed under fluorescence microscopy. The NO content in leaves was determined using Griess reagent (Beyotime Biotech) according to the manufacturer’s instructions. Rosette leaves were sprayed with the antimicrobial metabolites from strain HAB-5 or products from inoculated LB, incubated for 15 min, weighed and quickly frozen in liquid nitrogen for subsequent NO content determination. The experiments were repeated at least three times for each treatment.
Evaluation of biophysical and biochemical parameters
The ion leakage of systemic leaf tissue was measured as described as earlier (Fan et al. 1997). Leaves were immersed in 10 mL and 0.4 mL mannitol solution at room temperature with 200 rpm shaking for 3 h, and the conductivity was measured. The total conductivity was determined after boiling leaves for 10 min. The conductivity due to leakage was expressed as the percentage of the initial conductivity versus the total conductivity.
The amount of chlorophyll present in the tobacco leaf was estimated. Fresh tissue (300 mg) was ground with 10 ml of 80% acetone. The homogenate was centrifuged at 3000 rpm for 15 min. Absorbance was measured at 645 nm and 663 nm. The chlorophyll content was determined according the previous method (Bashan et al. 2006).
Total sugar determined by improved procedure (Gerchacov and Hatcher 1972). 0.2 g of fresh tobacco leaf tissue was crushed in 80% methanol and centrifuged at 10.000 rpm for 10 min. The supernatant was incubated in water bath at 70 °C. Equal volume of 5% phenol and 5 ml of H2SO4 were added and absorbance was measured at 640 nm. A standard curve was prepared using different concentration of standard glucose solution. The estimation of total sugar content was done on background of the graph.
Total proline content was quantified by the acid-ninhydrin method (Bates et al. 1973). Leaf tissue (500 mg) was ground in 10 ml 3% sulfosalicylic acid. 2 mL of supernatant was mixed with equal volumes of acid-ninhydrin and acetic acid. The mixture was incubated at 100 °C for 1 h. Reaction was finished in an ice bath and extracted with 4 ml of toluene by using a vortex mixer for 15–20 s. Absorbance was measured at 520 nm.
RNA extraction, cDNA synthesis and qRT- PCR analysis
List of primers used in this study
Sequences (5′ to 3′)
Amplicon size (bp)
The data were subjected to an analysis of variance using SPSS software (SPSS Inc., Illinois, U.S.A.). When a significant F test was obtained at p < 0.05, the separation of the treatment means was accomplished by Fisher’s protected LSD.
The strain HAB-5 promotes growth of tobacco plants
To be an effective PGPR, bacteria must be able to colonize roots because they need to establish themselves in an adequate environment to produce beneficial effects. It was found that the strain HAB-5 was resistant to streptomycin and kanamycin. The colonization of strain HAB-5 of the tobacco plant soil was measured at different time points (Fig. 1d). The result showed that the amount of strain HAB-5 in soil was increased from 105 to 107 CFU per gram (0 h to 60 h) of rhizosphere soil in inoculated plants. In contrast, no HAB-5 strain was observed in non-inoculated soil. This indicates that strain HAB-5 successfully colonized the tobacco rhizosphere.
Metabolites of HAB-5 alleviate the virulence of TMV on tobacco plant
Two different applications were evaluated. Plants were amended with the bacteria suspension before virus inoculation. It was found at few days after virus inoculation; the bacteria non-inoculated plant was weak, faded and dead. The bacteria inoculated plant had vitality and did not display any disease symptom. Four weeks later, the bacteria inoculated plant showed disease symptom. Another set of experiment consist the application of crude extract metabolites from the strain as a foliar spray treatment 12 h before TMV inoculation. The antimicrobial-pretreated plant did not show any disease symptom except when the leaves were rubbed with the virus. The secretion metabolites from strain HAB-5 were found to inhibit systematic disease on tobacco plants.
Both ROS accumulation and cell death are greatly attenuated in tobacco leaves treated with metabolites of HAB-5/TMV
Metabolites of HAB-5 induced nitric oxide production in tobacco leaves
Gene expression on tobacco leaves and ELISA analysis
ELISA analysis was performed to quantify the amount of TMV CP. The healthy control plants did not show any virus accumulation; while virus accumulation in TMV mock treated plants was found to be approximately 3-fold higher than antimicrobial metabolite treated plants (Table 2). Based on the plant symptoms and virus accumulation, this result confirms that strain HAB-5 reduced the severity of TMV disease.
Enzyme linked immune sorbent assay (ELISA) detection of TMV accumulation in rub-inoculated leaves at 7 dpt
TMV disease severity
ELIZA TMV detection (450nm)
TMV disease severity
ELIZA TMV detection (450nm)
HAB-5 + TMV
Mock + TMV
In this study, the mechanism used by Bacillus spp. to stimulate plant growth is still elusive (Schmidt and Delaney 2010; Lyngwi et al. 2016). Strain HAB-5 cell suspension supported growth up to 60 days post inoculation under greenhouse conditions and increased tobacco plant growth parameters. Gene expression investigations on the interaction with Bacillus sp. still provide an avenue for understanding the molecular mechanisms convoluted in enhancing plant growth and increasing yield.
Biological control by using PGPR mechanisms emerged long time ago but unfortunately chemical control has proved to be irreplaceable. One of the major limitations to their success is because they are adapted for the soil ecological conditions and the mechanisms for disease control agents are found to be associated with plant roots. It is known that a wide range of soil microbial species secrete different kinds of antimicrobial compounds. Among these, surfactin and fengycin lipopeptides of Bacillus subtilis were reported as elicitors of induced systemic resistance in plants (Ongena et al. 2007, 2008). Research has shown that lipopeptides antibiotics of Bacillus spp. are involved in the suppression of Fusarium wilt in tomato (Abdallah et al. 2017).
As previously reported, except for root drenching, foliar application is one of the efficient methods for foliar disease control. Studies have focused on harvesting these compounds from the culture suspension, culture supernatant and crude extract sprayed on plants for disease control (Lo et al. 1997; Ji et al. 2013; Yang et al. 2017). The present study provides evidence that crude extract metabolites of efficacy from the PGPR strain HAB-5 in reducing the virulence of TMV on tobacco plant. Based on visible symptoms, the crude extract metabolites extracted from the strain protect plant from TMV. It was found that the crude extract metabolites can inhibit the systemic disease on tobacco plant. ELISA analysis also showed the ability of the strain to decrease (by approximately 3-fold) virus accumulation in pre-treated leaves. These data confirm successful protection by the strain HAB-5 against TMV and consolidate findings that crude extract can be used for diseases control (Ji et al. 2013).
To further clarify antimicrobial-metabolite mechanisms which will benefit in controlling plant diseases, physiological, biochemical, anatomical changes between the treatments were assessed. First, chlorophyll is important in photosynthesis for plant bioproductivity. In the present study, significant increases in chlorophyll content were observed in strain HAB-5 treatment compared to the control. Similar to other biocontrol methods (Mou et al. 2003), foliar treatment helped plants to maintain chlorophyll during TMV infection. Then, the content of MDA and ion leakage were reduced in pretreated leaves. It has been reported that MDA was induced during stress on plants leading to enhanced membrane peroxidation disease tolerance mechanisms (Bashan et al. 2006; Li et al. 2013). In addition, the molecular mechanisms of sugar in disease remain largely unknown. We found that the sugar content was increased in treated plants. The possible explanation is high sugar content helps to reduce disease in plants. This finding suggested the ex vivo treatment maneuvered the ion leakage sugar, proline, chlorophyll, and malondialdehyde to protect plant cells integrity leading to induced TMV tolerance.
The hypersensitive response is a mechanism used by plants to prevent the propagation of pathogen infection, and commonly results in the formation of necrotic lesions (Qiu et al. 2008). It is characterized by the appearance of localized cell death (Pontier et al. 2004; Wang et al. 2012). In this study, cell death in leaves were correlated with the amount of virus present in leaves (Fig. 6). The antimicrobial pretreated leaves were able to maintain the viability of host cells against TMV. In addition, reduction of H2O2 triggered by strain HAB-5 treatment may play a role in the enhancement of plant resistance. This suggests that the low level of H2O2 accumulation in tobacco leaves may insufficiently cause cell death. This finding provide new information on incompatible interactions between TMV infection and plant hosts.
Depressed proline and higher ROS accumulation in plants usually contributes to the ability of the bacteria to maintain ROS balance in accordance with plant requirements (Elsayed et al. 2013). The need for proline in maintaining the elevated ROS level has been reported earlier (Apostol and Heinstein 1989; Kumar et al. 2016). Interestingly, our results showed lower proline and ROS accumulation in pretreated plants. It suggests that the antimicrobial-metabolite induced systemic resistance via proline and ROS in epidermal plant cells was activated in an independent manner.
NO is an important second messenger involved in plant immunity, acting as a signaling molecule in plant/pathogen interactions (Savicka and Škute 2010). It was observed that NO content in rosette leaves of tobacco increased following strain HAB-5 antimicrobial treatment (Fig. 7). This finding suggests that NO may contribute to the induction of defense response in plant leaves.
The expression of plant defense-related genes usually occurs quickly after plant treatments. PR proteins were found in genotypes of tobacco infected with tobacco mosaic virus (Hare and Cress 1997; Singh et al. 2017). NPR1 is known to regulate SAR (Systemic acquired resistance) and Induced systemic resistance (ISR) resistance pathways (Pontier et al. 2004). We pretreated the leaves with antimicrobial metabolites 12 h before TMV inoculation, and we noticed that plant immune associated genes were stimulated. Hsr203J and Hin1 reported as HR-related genes exhibit rapid, high level, specific activation in response to HR-inducing bacteria (Pontier et al. 1994; Delledonne et al. 1998). Unexpectedly, evidence was not presented in the present study that expressions of those genes were noticed at late time point (96 hpt). The use of different pathogen species, different plant cultivar, different bacteria, different treatments from a different genetic background, or plant encoded functions can modulate their action. Similarly, recent reports also indicate the induction of Hsr203J in response to hrp mutants of Pseudomonas. syringae pv. tabaci WF4 and P. syringae pv. syringae at late time points (Park and Kloepper 2000; Raupach et al. 1996; Pontier et al. 1994).
Overall, results conclude that effective biological control not only depends on the organisms but also on the control strategy. We found that foliar application of a crude extract for controlling diseases was effective for foliar treatment and proved to be potential biocontrol treatment against TMV used as a bio-pesticide. This aspect will be a new instrument in improving the application of bioactive natural products for plant protection.
This study was supported in part by the National Natural Science Foundation of China (31160359, 31360029), China Agriculture Research System (No.CARS-33-BC1), China Agriculture Research System (No.CARS-34-BC1) and the Hainan Province Natural Science Foundation of China (No.20153131). The funder had no role in data collection and interpretation, or the decision to submit the work for publication.
Compliance with ethical standards
Conflict of interest
No potential conflict of interest was reported by the authors.
Human participants and/or animals
This article does not contain any studies with human or animals subjects performed by any of the authors.
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