Salinity amelioration and plant growth promotion by Paenibacillus castaneae in tomato

Abiotic stressors such as salt, drought, and temperature have all had a negative impact on agriculture. These are important impediments to global sustainable agriculture [1]. The major abiotic stress conditions of concern are light, temperature, drought, salinity, soil, air, and water contaminants. Salinity is a key abiotic stress and harsh environmental element that has a negative impact on crop productivity and quality. Salinity (salt stress) affects approximately 20% of the total cultivated area and 33% of irrigated agricultural lands. Agricultural production is greatly reduced on soils with high salt concentrations due to poor plant nutrition, osmotic imbalance, and drought stress [2]. Salinity stress induced by NaCl is one of the most prevalent abiotic stresses that impact plant productivity through morphological (e.g., decrease in leaf number and fruit output) and metabolic disorders (e.g., imbalance in stomatal conductance, poor photosynthetic activity)[3].

In recent years lot of research has been used to alleviate the problem of soil salinity and acidity. Alternative approaches for reclaiming salt-affected soils include phytoremediation and bioremediation. Plant growth-promoting rhizobacteria (PGPR) are a diverse group of bacteria renowned for their beneficial properties [3]. The acquisition of nutrients, the solubilization of phosphorus (P), the production of siderophores, the fixation of atmospheric nitrogen (N), the production of hydrocyanic acid (HCN), the regulation of plant hormones, and the defense against biotic pathogens are just a few of the mechanisms used by PGPR to promote plant growth. Plant development is promoted by N₂ fixation, P availability, and hormonal response; other systems indirectly support plant growth [4].

The most common bacteria that tolerate elevated concentrations of NaCl (1-15%) are Ochrobactrum intermedium, Bacillus subtilis, Pseudomonas fluorescence, Kocuria rhizophila, , Bacillus amyloliquefaciens, Bacillus firmus, Azotobacter chrooccocum, P. stutzeri, Azospirillum brasiliense, A.lipoferum, P. putida, Curtobacterium flaccumfaciens, Arthrobacter sp., and Paenibacillus. These strains were discovered to promote plant growth and sodium chloride tolerance in maize, wheat, barley, rice, soybean, sunflower, and tomato [5, 6].

The current work focuses on evaluating Paenibacillus castaneae for plant growth-promoting properties in saline soil conditions under in vitro and field conditions in tomatoes.

Materials and methods

Microorganisms and Growth Conditions

Paenibacillus castaneae was obtained from, the Culture collection centre of the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.

Growth analysis Paenibacillus castaneae in salt stress

The salt stress tolerance potential of P. castaneae was evaluated. The Luria Bertani-broth was made and supplemented with various salinity stress concentrations (0, 100, 200, and 300 mM NaCl). To ascertain the tolerance capability, the bacterium P. Castaneae was inoculated in LB-broth and cultured for 8 days at 150 rpm and 32 °C in a shaking incubator. To assess how well each strain of bacteria could withstand salinity stress, the optical density of each strain was measured at 600 nm every day for seven days. A salt-tolerant bacterial strain was regarded as one with an optical density (OD) of 0.5 in the face of salinity stress. In LB broth and media, the P. castaneae proliferated rapidly and exhibited tolerance to salinity stress up to 300 mM NaCl.

In vitro evaluation of Paenibacillus castaneae under induced salt stress

A loopful bacterial culture that was 24 hours old was maintained at 10⁸ CFU mL/L and aseptically dissolved in phosphate buffer saline (pH 7.4). The cell pellet was re-suspended in a 1% phosphate buffer, which had undergone sterilization. In order to surface-sterilize tomato seeds (PKM 1), 1.0% NaOCl was applied for 1 minute, followed by 70% ethanol for 3 minutes, and finally three rinses with sterile distilled water. The tomato seeds were immersed in a bacterial suspension for priming. The primed seeds were incubated for 24 hours at 28̊  C. After incubation, seeds were planted in sterile soil in earthen pots [7[.

There were four treatment groups: (1) the control group, (2) Soil with 200 Mm (NaCl) group, (3)Paenibacillu scastaneae inoculation group, and (4) Paenibacillus castaneae + 200Mm (NaCl). Three replications of each treatment were present under controlled circumstances throughout the whole experiment, which used a randomized block design. In each of the treatments, a salt treatment was administered seven days after germination by irrigation with 50 mM NaCl for four days. At 21 days, seedlings were taken and their growth characteristics were assessed.

Physiological Responses of Plants to Salt Stress

The Bates et al. method was used to measure the proline content. At 520 nm, the toluene that included a chromophore was measured ([8]. From a standard curve, the amount of proline was calculated in g/g fresh weight (FW). Chlorophyll estimation was performed by the method of Moran and Porath [9]. The relative water content (RWC) was measured in plant leaves according to the protocol of Sade et al. [10].

Antioxidant Enzyme Activity Assays

Antioxidant enzyme activity was observed in tomato leaf tissues. The extraction of the crude protein followed the procedure described by Qureshi et al. [11]. One gram of tissue was homogenized in 4 mL of a 100 mM potassium phosphate buffer (pH 7.4) that also contained 2% polyvinyl pyrrolidone (PVP), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM ethylenediaminetetraacetate (EDTA). The supernatant from the centrifugation of the crude extract at 15,000 g for 20 min at 4 °C was utilized as an enzyme extract.

Determination of peroxidase (POD) enzyme activity was performed as per the method of Kumari et al. [12]. Polyphenol oxidase (PPO) activity was quantified as per the method of Weisany et al. [13], and activity was expressed in U/mg protein.

Field experiment

The field experiment was conducted at Horticultural College and Research Institute for Women at Trichy district of Tamil Nadu during the Rabi season of 2022-23. The experiment was designed with six treatments and four replications in a randomized block design. Standard cultural practices were followed as recommended by Tamil Nadu Agricultural University, Coimbatore, Tamilnadu.   T₁– Control, T₂ – 75% recommended N + 75% recommended P, T₃ – 100% N, P and K, T₄ – 75% recommended N + 75% recommended P + Azospirillum + Phosphobacteria (Seed treatment + Soil application), T₅ – 75% recommended N + 75% recommended P + Paenibacillus strain (Seed treatment + Soil application), T₆ – 75% recommended N + 75% recommended P + Azospirillum + Phosphobacteria + Paenibacillus strain.

Observations

The following observations viz., plant growth parameters and yield parameters were recorded .

Statistical analysis

            The results of the experiments were subjected to statistical scrutiny as per the methods detailed by Panse and Sukhatme [14]. All the treatments were compared at p £ 0.01 and 0.05 levels of significance using the Critical Difference (CD) test which was performed by Excel-2000. The Analysis of Variance (ANOVA), Standard Error (SE), and Critical Difference (CD) for dependent parameters were tabulated and the level of significance was reported.

Result and discussion

Growth curve analysis of P.castaneae (VPB1)

It was investigated that P.castaneae can endure salinity stress and showed healthy growth in salt-amended LB media. Paenibacillus castaneae can resist NaCl concentrations of up to 3 M (Figure 1). As the salinity increased, the bacterial population continued to decline in number. Hence, salinity stress and bacterial growth showed an inverse relation as compared to the control which is similar to that in a previous report of Ali et al.[15].

Effect of P.castaneae on Plant Growth Parameters Under Salt Stress

Plant growth characteristics were significantly impacted negatively by salt stress. In comparison to the control condition, salt stress resulted in a reduction in all growth metrics. P.castaneae, however, was able to protect plants from serious harm brought on by salt toxicity. In comparison to T₂ treatment, plants inoculated with P.castaneae (T₃) showed considerably longer shoot and root lengths (15.36 cm and 7.6 cm). Similar to this, during salt stress, bacterial inoculation dramatically increased plant biomass compared to un-inoculated plants. Under salt stress, plants treated with T₄ had a germination percentage and vigour index than plants treated with T₂, the uninoculated control (Table 1). Both treatments (T₁ and T₃) displayed comparable trends in plant growth parameters under non-salt environments.

Additionally, P.castaneae demonstrated abilities to boost plant development, suggesting the possibility that the Paenibacillus castaneae isolate may support plant growth in nutrient-restricted environments. According to various studies Sphingobacterium spp. exhibit advantageous PGP features with the capacity to promote plant growth under various stress circumstances [16-19]. Additionally, it was discovered that the isolate BHU-AV3 could withstand salt stress up to 4% NaCl concentration([7].

Effect of Paenibacillus castaneae on physiological and antioxidant enzymes response to salt stress

Under salt stress, a decrese in chlorophyll concentration was seen. However, compared to uninoculated plants (T₂), P. Castaneae (VPB1)inoculated plants (T₃) showed a much higher chlorophyll content (80.12 g/g). Under saltwater circumstances, the relative water content of tomato plants was considerably decreased. Nevertheless, compared to salt treatment plants, microbe inoculated plants accumulated more water content (89%) (Table 2).

Under salt stress, the leaves of P. Castaneae (VPB1)-inoculated plants collected more proline than the non-inoculated plants, suggesting that proline plays a role in maintaining the osmotic equilibrium within the root [19,20]. Furthermore, more proline shields membrane enzymes and proteins from oxidative bursts [21].

A comparative study of antioxidant enzyme activities was performed in leaf tissues with different treatments. Salt stress generally stimulates the antioxidant system throughout the plant. P. castaneae strain-inoculated leaves (T₃) showed higher POD , SOD and PPO as compared to T₂-treated plant roots. In leaf tissue, un-inoculated plants. (T₂) displayed significant increases in all enzymatic activities compared to T₄ plants. Under non-salt conditions, both treatments (T₁ and T₃) maintained the same level of POD, SOD, and PPO activities.

Field evaluation of P.castaneae in tomato

The influence of biofertilizers on plant growth parameters viz., plant height, 50% flowering, No of branches, was recorded at 30 and 60 DAS under saline soil. The yield parameters like number of leaves (57 Nos), plant height (72 cm), days to 50 % flowering (34 days) and first flowering (30 days), and no of branches (10 nos) were highest in 75% recommended N + 75% recommended P + Azospirillum + Phosphobacteria + Paenibacillus strain followed by other treatments (Table 3 and 4). According to Kamanga et al. (22), salinity adaptation lessens the detrimental effects of salt stress, whereas Meza et al. (23) found that moderate salt stress increases tomato fruit quality without affecting yield. By enhancing antioxidant capacity, ion homeostasis, and PA metabolism, lowering ABA and ET levels, and suppressing transpiration, elevated CO₂ provides tomato tolerance to progressively greater soil salinity and secondary soil salinization [24].

Conclusion

The Paenibacillus castaneae (VPB1)- enhances tomato plant growth and yield in the presence of 200 mM NaCl, as well as alleviates the harmful impact caused by salt stress under pot culture conditions. The tomato plant inoculated with bacteria showed more severe changes in accumulating, proline, and antioxidant enzymatic activities compared to the control. Enhanced activities of these parameters in plants resulted in a decrease in oxidative stress in tomato plants, concerning ROS content. The field study results confirmed that 75% recommended N + 75% recommended P + Azospirillum + Phosphobacteria + Paenibacillus strain exhibited grateful results in improving tomato plant growth and yield under salinity stress.

References

[1]. Kapadia, C, Sayyed, R. Z., Enshasy, H. E., Vaidya, H., Sharma, D., Patel, V., Malek, R.A., Syed, A., Elgorban, A.M, Ahmad, K and Zuan, A.T. 2021.”Halotolerant microbial consortia for sustainable mitigation of salinity stress, growth promotion, and mineral uptake in tomato plant and soil nutrient enrichment”. Sustainability 13: 8369.

[2].Egamberdieva, D, Davranov, K., Wirth, S., Hashem, A. and Allah, EFA. 2017. “Impact of soil salinity on the plant-growth – promoting and biological control abilities of root associated bacteria”. Saudi Journal of Biological Science. 24:1601–1608. 

[3].Kumar, A., Patel, J. S., Meena, V. S. and Ramteke, P. W. 2019. “Plant growth-promoting rhizobacteria: Strategies to improve abiotic stresses under sustainable agriculture”. Journal of Plant Nutrition. 42(11–12): 1402–1415.

[4].Li, X., Sun, P., Zhang, Y., Jin, C. and Guan, C. 2020. “A novel PGPR strain Kocuria rhizophila Y1 enhances salt stress tolerance in maize by regulating phytohormone levels, nutrient acquisition, redox potential, ion homeostasis, photosynthetic capacity and stress-responsive genes expression”. Environmental and Experimental botany,174: 104023.

[5].  Sukweenadhi, J., Balusamy, S.R., Kim, Y-J., Lee, C.H, Kim, Y-J., Koh, S.C and Yang,D.C2018.“AGrowth-Promoting Bacteria, Paenibacillus yonginensis DCY84^T Enhanced Salt Stress Tolerance by Activating Defense-Related Systems in Panax ginseng”. Frontiers in Plant Science, 9: 813. 

[6]. Shilev, S. 2020. “Plant-Growth-Promoting Bacteria Mitigating Soil Salinity Stress in Plants”. Applied Science. 10: 7326.

[7].Vaishnav, A., Singh, J., Singh, P., Rajput, R.S., Singh, H.B. and Sarma, B.K. 2020. “Sphingobacterium sp. BHU-AV3 Induces Salt Tolerance in Tomato by Enhancing Antioxidant Activities and Energy Metabolism”. Frontiers in Microbiology, 11: 443.

[8].Bates, L. S., Waldren, R. P. and Teare, I. D. 1973. “Rapid determination of free proline for water-stress studies”. Plant and Soil. 39: 205–207.

[9]. Moran, R. and Porath, D. 1980. “Chlorophyll determination in intact tissues using N, N-dimethylformamide”. Plant Physiology. 65: 478–479.

[10].Sade, N., Galkin, E, and Moshelion, M. 2015. “Measuring arabidopsis, tomato and barley leaf relative water content (RWC)”. Bio protocol. 5:1451

[11].Qureshi, M. I., Abdin, M. Z., Ahmad, J., and Iqbal, M. 2013. “Effect of long-term salinity on cellular antioxidants, compatible solute and fatty acid profile of Sweet Annie (Artemisia annua L.)”. Phytochem. 95: 215–223.

[12].Kumari, S., Vaishnav, A., Jain, S., Varma, A., and Choudhary, D. K. 2015. “Bacterial-mediated induction of systemic tolerance to salinity with expression of stress alleviating enzymes in soybean (Glycine max L. Merrill)”. Journal of Plant growth regulation, 34: 558–573.

[13].Weisany, W., Sohrabi, Y., Heidari, G., Siosemardeh, A., and Ghassemi-Golezani, K. 2012. “Changes in antioxidant enzymes activity and plant performance by salinity stress and zinc application in soybean (Glycine max L.)”. Plant Omics, 5:60

[14].Pansey and Sukhatme.  1985.  Statistical methods for Agricultural Workers (UIV edn).  Indian Council for Agricultural Research, New Delhi.   

[15].Ali, S., Charles, T. C., and Glick, B. R. 2014. “Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase”. Plant Physiology and Biochemistry. 80: 160–167.

[16].Ahmed, I., Ehsan, M., Sin, Y., Paek, J., Khalid, N.,  and Hayat, R., 2014. “Sphingobacteriumpakistanensis sp. nov., a novel plant growth promoting rhizobacteria isolated from rhizosphere of Vigna mungo”. Anton Van Leeuwenhoek, 105: 325–333.

[17].Cardinale, M., Ratering, S., Suarez, C., Montoya, A. M. Z., Geissler-Plaum, R., and Schnell, S. 2015. “Paradox of plant growth promotion potential of rhizobacteria and their actual promotion effect on growth of barley (Hordeum vulgare L.) under salt stress”. Microbiological Research.181: 22–32.

[18].Rolli, E., Marasco, R., Vigani, G., Ettoumi, B., Mapelli, F and Deangelis, M. L., 2015. “Improved plant resistance to drought is promoted by the root−associated microbiome as a water stress−dependent trait”. Environmental Microbiology. 17: 316–331.

[19].Claussen, W. 2005. “Proline as a measure of stress in tomato plants”. Plant Science. 168: 241–248.

[20].Zhu, D., Hou, L., Xiao, P., Guo, Y., Deyholos, M. K., and Liu, X. 2019. “VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress”. Plant Science,280: 132–142.

[21].Szabados, L., and Savouré, A. 2010. “Proline: a multifunctional amino acid”. Trends in Plant Science. 15: 89–97.

[22].Kamanga, R. M., Echigo, K., Yodoya, K., Mekawy, A. M. M., and Ueda, A. 2020. “Salinity acclimation ameliorates salt stress in tomato (Solanum lycopersicum L.) seedlings by triggering a cascade of physiological processes in the leaves”. Scientific Horticulture. 270:109434.

[23].Meza, S. L., Egea, I., Massaretto, I. L., Morales, B., Purgatto, E., Egea-Fernández, J. M., MC. Bolarinand   Flores, F B. 2020. “Traditional tomato varieties improve fruit quality without affecting fruit yield under moderate salt stress”. Frontier in  Plant Science 11:1717.

[24]. Zhang, Y., Yao, Q., Shi, Y., Li, X., Hou, L., Xing, G.,  and Ahammed G.J .2020. “Elevated CO₂ improves antioxidant capacity, ion homeostasis, and polyamine metabolism in tomato seedlings under Ca(NO₃)₂-induced salt stress”.Scientia Horticulturae, 273:109644.