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Effect of Bacillus aryabhattai H26-2 and B. siamensis H30-3 on Growth Promotion and Alleviation of Heat and Drought Stresses in Chinese Cabbage
Plant Pathol. J. 2019;35:178-187
Published online April 1, 2019
© 2019 The Korean Society of Plant Pathology.

Da Jeong Shin1, Sung-Je Yoo1,2, Jeum Kyu Hong3, Hang-Yeon Weon1, Jaekyeong Song1, and Mee Kyung Sang1,*

1Division of Agricultural Microbiology, National Institute of Agricultural Science, Rural Development Administration, Wanju 55365, Korea, 2Department of Agbiotechnology and Natural Resources, Gyeongsang National University, Jinju 52828, Korea, 3Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech), 33 Dongjin-ro, Jinju 52725, Korea
Correspondence to: *Phone) +82-63-238-3055, FAX) +82-63-238-3834, E-mail) mksang@korea.kr
Received August 9, 2018; Revised December 4, 2018; Accepted December 18, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Plants are exposed to biotic stresses caused by pathogen attack and complex abiotic stresses including heat and drought by dynamic climate changes. To alleviate these stresses, we investigated two bacterial stains, H26-2 and H30-3 in two cultivars (‘Ryeokkwang’ and ‘Buram-3-ho’) of Chinese cabbage in plastic pots in a greenhouse. We evaluated effects of bacterial strains on plant growth-promotion and mitigation of heat and drought stresses; the role of exopolysaccharides as one of bacterial determinants on alleviating stresses; biocontrol activity against soft rot caused by Pectobacterium carotovorum subsp. carotovorum PCC21. Strains H26-2 and H30-3 significantly increased fresh weights compared to a MgSO4 solution; reduced leaf wilting and promoted recovery after re-watering under heat and drought stresses. Chinese cabbages treated with H26-2 and H30-3 increased leaf abscisic acid (ABA) content and reduced stomatal opening after stresses treatments, in addition, these strains stably colonized and maintained their populations in rhizosphere during heat and drought stresses. As well as tested bacterial cells, exopolysaccharides (EPS) of H30-3 could be one of bacterial determinants for alleviation of tested stresses in Chinese cabbages, however, the effects were different to cultivars of Chinese cabbages. In addition to bacterial activity to abiotic stresses, H30-3 could suppress incidence (%) of soft rot in ‘Buram-3-ho’. The tested strains were identified as Bacillus aryabhattai H26-2 and B. siamensis H30-3 based on 16S rRNA gene sequence analysis. Taken together, H26-2 and H30-3 could be candidates for both plant growth promotion and mitigation of heat and drought stresses in Chinese cabbage.

Keywords : abiotic stress, exopolysaccharide, soft rot
Supplementary Information
Acknowledgment

This work was supported by National Institute of Agricultural Science (Project No. PJ012517) of Rural Development Administration, Republic of Korea.

Figures
Fig. 1. (A) Growth promotion by Bacillus aryabhattai H26-2 and Bacillus siamensis H30-3 compared to 10 mM MgSO4 solution (control) in Chinese cabbage (‘Ryeokkwang’ and ‘Buram- 3-ho’) under non-stress condition; (B) Leaf wilting score (0–5) under heat and drought conditions; (C) fresh weight after rewatering of Chinese cabbages (‘Ryeokkwang’ and ‘Buram-3-ho’). Plants were treated with bacterial strains H26-2 and H30-3, or 10 mM MgSO4 solution (untreated control), one week later, heat and drought stresses were treated; Twenty-four h after end of stress treatment, fresh weight were assessed. Error bars indicate the standard errors of 20 replications from two experiments. An asterisk on bars with standard errors indicate significant (P < 0.05) differences between treatments according to the least significant difference test.
Fig. 2. (A) Abscisic acid (ABA) contents and (B) stomatal opening (%) in leaves of Chinese cabbages (‘Ryeokkwang’ and ‘Buram-3-ho’) at 24 h after heat and drought stresses treatment. Plants were drenched with strain H26-2 and H30-3, and 10mM MgSO4 solution as untreated control, one week later, heat and drought stresses were performed. Error bars indicate the standard errors of eight (for ABA contents) or 16 (for stomatal opening) replications from two experiments. An asterisk on bars with error bars indicate significant (P < 0.05) differences between treatments according to the least significant difference test.
Fig. 3. A phylogenetic tree performed by the neighbor-joining method showing the relationship of Bacillus aryabhattai H26-2 and Bacillus siamensis H30-3 with other species of the genus Bacillus based on 16S rRNA sequences analysis. Numbers at the blanching points are the bootstrap values (> 50%) for 1,000 replicates. A scale bar indicates 5 nucleotide substitutions per 1000 nucleotides of the sequence. Strains H26-2 and H30-3 are shown in bold type. T, type strain.
Tables

Bacterial colonization in rhizosphere soils of Chinese cabbages treated with spontaneous rifampicin-resistant mutants, H26-2 Rif and H30-3 Rif under normal and stressed conditions

CultivarsDASaBacterial colonization [Log(cfu/g of soils)b

H26-2 RifH30-3 Rif


Non-stressStressesNon-stressStresses
Ryeokkwang14.83 ± 0.19 ac5.33 ± 0.11 A*4.26 ± 0.22 a4.00 ± 0.01 A
34.85 ± 0.20 a4.96 ± 0.13 A3.66 ± 0.08 a4.01 ± 0.08 A*
54.74 ± 0.19 a4.98 ± 0.25 A4.21 ± 0.21 a3.83 ± 0.21 A
Buram-3-ho15.17 ± 0.13 a*4.62 ± 0.20 A3.88 ± 0.18 a4.04 ± 0.28 A
35.35 ± 0.09 a5.06 ± 0.28 A3.83 ± 0.03 a3.83 ± 0.10 A
54.72 ± 0.08 b4.56 ± 0.20 A4.05 ± 0.13 a4.04 ± 0.10 A

aDAS; days after stress treatment

bBacterial colonization was determined by counting colony-forming unit (cfu) in pot soils at 1, 3, 5 d after stress treatment on Chinese cabbage plants.

cMean ± standard errors with same letters are not significantly different between DAS at each non-stress or stresses in cultivars according to the least significantly difference test at P < 0.05. An asterisk indicates significant difference between non-stress vs. stresses at each DAS according to the t-test. Experiments were conducted twice with four replicates each.


Effect of exopolysaccharides of H26-2 and H30-3 on cotyledon greening (%) and number of lateral roots after germination in plate assay, and fresh weight and leaf wilting score in Chinese cabbage under non-stressed or heat and drought stressed conditions

Cultivars, treatmentsIn plate assayaIn plantab

(MS media amended with 400 mM mannitol)Non-stressStresses



Cotyledon greening (%)Number of lateral rootFresh weight (g)Leaf wilting score (0–5)Fresh weight (g)
CultivarsF value = 1.37,F value = 2.50,F value = 1.13F value = 25.30F value = 13.53
P value = 0.2485P value = 0.1191P value = 0.2937P value < 0.0001P value = 0.0006
Ryeokkwang67.5 ± 4.8 a7.5 ± 0.4 a8.8 ± 0.8 a4.2 ± 0.1 a1.0 ± 0.1 b
Buram-3-ho75.4 ± 4.7 a6.8 ± 0.3 a7.8 ± 0.6 a2.9 ± 0.2 b3.3 ± 0.6 a
Ryeokkwang
TreatmentsF value = 0.58,F value = 0.79,F value = 0.32F value = 0.18F value = 3.49
P value = 0.5732P value = 0.4644P value = 0.7326P value = 0.8370P value = 0.0491
Control67.5 ± 5.3 a6.5 ± 0.3 a9.6 ± 1.5 a4.1 ± 0.1 a0.8 ± 0.1 b
EPS of H26-275.0 ± 11.2 a6.6 ± 0.5 a8.9 ± 1.3 a4.1 ± 0.1 a1.0 ± 0.1 ab
EPS of H30-360.0 ± 12.7 a7.3 ± 0.6 a8.1 ± 1.3 a4.2 ± 0.1 a1.3 ± 0.2 a
Buram-3-ho
TreatmentsF value = 4.53,F value = 2.95,F value = 0.01F value = 4.00F value = 5.03
P value = 0.0231P value = 0.0494P value = 0.9860P value = 0.0337P value = 0.0164
Control65.0 ± 5.6 b6.5 ± 0.4 a8.0 ± 1.0 a3.8 ± 0.2 a1.1 ± 0.4 b
EPS of H26-290.0 ± 10.0 a7.6 ± 0.5 ab7.8 ± 0.9 a2.8 ± 0.4 ab4.0 ± 1.0 a
EPS of H30-390.0 ± 5.6 a8.4 ± 0.7 b7.7 ± 1.2 a2.3 ± 0.5 b4.8 ± 1.1 a

aThe numbers of seedlings with open and green leaves after seed germination were recorded at 3 and 4 d after seeding in repeated experiments; number of lateral root was evaluated at 17 d after seeding. Values ± standard errors of each cultivar and treatment in a column followed by same letters are not significantly different according to the least significant difference test at P < 0.05. Experiment were conducted twice with ten replicates each.

bPlants were treated with EPS (10 μg/ml) of strains H26-2 and H30-3, and 10 mM MgSO4 solution (control), one week later, heat and drought stress were treated; 24 h after end of stress treatment, leaf wilting score was assessed, and fresh weight after re-watering was assessed. Values ± standard errors of each treatment in a column followed by same letters are not significantly different according to the least significant difference test at P < 0.05. Experiments were conducted twice with eight replicates each.


Biocontrol activity of tested bacterial strains against soft rot caused by Pectobacterium carotovorum subsp. carotovorum PCC21 in two cultivars of Chinese cabbages

TreatmentDisease incidence (%)
RyeokkwangBuram-3-ho
Control60.0 ± 4.7 ab72.0 ± 6.8 a
BTH46.0 ± 8.5 b46.0 ± 7.9 b
H26-262.0 ± 8.7 ab60.0 ± 8.4 ab
H30-370.0 ± 7.5 a40.0 ± 7.9 b

aValues ± standard errors of treatments in a column followed by same letters are not significantly different according to the least significant difference test at P < 0.05. Experiment was conducted twice with three replicates with 15 seedling each.

References
  1. Alami Y, Achouak W, Marol C, and Heulin T. 2000. Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots. Appl Environ Microbiol 66: 3393-3398.
    Pubmed KoreaMed CrossRef
  2. Barnawal D, Bharti N, Maji D, Chanotiya CS, and Kalra A. 2014. ACC deaminase-containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization in Pisum sativum. J Plant Physiol 171: 884-894.
    Pubmed CrossRef
  3. Barrs HD, and Weatherley PE. 1962. A re-examination of the relative turgidity technique for estimating water deficit in leaves. Aust J Biol Sci 15: 413-428.
    CrossRef
  4. Bashan Y, and Holguin G. 1997. Azospirillum-plant relationships: environmental and physiological advances. Can J Microbiol 43: 103-121.
    CrossRef
  5. Belimov AA, Dodd IC, Hontzeas N, Theobald JC, Safronova VI, and Davies WJ. 2009. Rhizosphere bacteria containing 1-aminocyclopropane-1-carboxylate deaminase increase yield of plants grown in drying soil via both local and systemic hormone signalling. New Phytol 181: 413-423.
    Pubmed CrossRef
  6. Bresson J, Varoquaux F, Bontpart T, Touraine B, and Vile D. 2013. The PGPR strain Phyllobacterium brassicacearum STM196 induces a reproductive delay and physiological changes that result in improved drought tolerance in Arabidopsis. New Phytol 200: 558-569.
    Pubmed CrossRef
  7. Bric JM, Bostock RM, and Silverstone SE. 1991. Rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57: 535-538.
    Pubmed KoreaMed
  8. Castric KF, and Castric PA. 1983. Method for rapid detection of cyanogenic bacteria. Appl Environ Microbiol 45: 701-702.
    Pubmed KoreaMed
  9. Chenu C, and Robersin EB. 1996. Diffusion of glucose in microbial extracellular polysaccharide as affected by water potential. Soil Biol Biochem 28: 877-884.
    CrossRef
  10. Dimkpa C, Weinand T, and Asch F. 2009. Plant-rhizobacteria interactions alleviate abiotic stress conditions. Plant Cell Environ 32: 1682-1694.
    Pubmed CrossRef
  11. Dobra J, Motyka V, Dobrev P, Malbeck J, Prasil IT, Haisel D, Gaudinova A, Havlova M, Gubis J, and Vankova R. 2010. Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content. J Plant Physiol 167: 1360-1370.
    Pubmed CrossRef
  12. Ghosh D, Sen S, and Mohapatra S. 2017. Modulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions by a drought-mitigating Pseudomonas putida strain. Ann Microbiol 67: 655-668.
    CrossRef
  13. Jung SS, Jeong HH, and Kim KS. 2000. Effects of uniconazole treatment on the growth and flowering of potted Chrysanthemum indicum L. Korean J Hortic Sci Technol 18: 28-32.
  14. Lee SH, and Cha JS. 2001. Efficient induction of bacterial soft rot using mineral oil. Phytopathology 91: S53-S54.
  15. Levene H. 1960. Robust tests for equality of variances. In: Contributions to prorobability and statistics: Essays in honor of harold hotelling, eds. by I. Olkin, SG. Ghurye, W. Hoeffding, WG. Madow, and HB. Mann , pp. 278-292. Stanford University Press, Stanford, California, USA.
  16. Kang S-M, Radhakrishnan R, Khan AL, Kim M-J, Park J-M, Kim B-R, Shin D-H, and Lee I-J. 2014. Gibberellin secreting rhizobacterium, Pseudomonas putida H-2-3 modulates the hormonal and stress physiology of soybean to improve the plant growth under saline and drought conditions. Plant Physiol Biochem 84: 115-124.
    Pubmed CrossRef
  17. Kaushal M, and Wani SP. 2016. Plant-growth-promoting rhizobacteria: drought stress alleviators to ameliorate crop production in drylands. Ann Microbiol 66: 35-42.
    CrossRef
  18. Khandelwal A, and Sindhu SS. 2013. ACC deaminase containing rhizobacteria enhance nodulation and plant growth in clusterbean (Cyamopsis tetragonoloba L.). J Microbiol Res 3: 117-123.
  19. Kim C-G, Lee S-M, Jeong H-K, Jang J-K, Kim Y-H, and Lee C-K. 2010. Impacts of climate change on Korean agriculture and its counterstrategies . Korea Rural Economic Institute, Seoul, Korea. 305 pp.
  20. Kim H-S, Sang MK, Myung IS, Chun SC, and Kim KD. 2009. Characterization of Bacillus luciferensis strain KJ2C12 from pepper root, a biocontrol agent of Phytophthora blight of pepper. Plant Pathol J 25: 62-69.
    CrossRef
  21. Kumar A, Kumar A, Devi S, Patil S, Payal C, and Negi S. 2012. Isolation, screening and characterization of bacteria from rhizospheric soils for different plant growth promotion (PGP) activities: an in vitro study. Recent Res Sci Technol 4: 1-5.
  22. Milagres AMF, Machuca A, and Napoleão D. 1999. Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J Microbiol Methods 37: 1-6.
    Pubmed CrossRef
  23. Penrose DM, and Glick BR. 2003. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118: 10-15.
    Pubmed CrossRef
  24. Pikovskaya RI. 1948. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiology 17: 362-370.
  25. Sandhya V, Shaik ZA, Grover M, Reddy G, and Venkateswarlu B. 2009. Alleviation of drought stress effects in sunflower seedlings by the exopolysaccharides producing Pseudomonas putida strain GAP-P45. Biol Fert Soils 46: 17-26.
    CrossRef
  26. Sang MK, and Kim KD. 2012. The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper. J Appl Microbiol 113: 383-398.
    Pubmed CrossRef
  27. Sang MK, Jeong J-J, Kim J, and Kim KD. 2018. Growth promotion and root colonization in pepper plants by phosphate-solubilising Chryseobacterium sp. strain ISE14 that suppresses Phytophthora blight. Ann Appl Biol 172: 208-223.
    CrossRef
  28. Santaella C, Schue M, Berge O, Heulin T, and Achouak W. 2008. The exopolysaccharide of Rhizobium sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus roots but contributes to root colonization. Environ Microbiol 10: 2150-2163.
    Pubmed KoreaMed CrossRef
  29. Sherameti I, Tripathi S, Varma A, and Oelmüller R. 2008. The root-colonizing endophyte Pirifomospora indica confers drought tolerance in Arabidopsis by stimulating the expression of drought stress–related genes in leaves. Mol Plant-Microbe Interact 21: 799-807.
    Pubmed CrossRef
  30. Singh RP, and Jha PN. 2016. The Multifarious PGPR Serratia marcescens CDP-13 augments induced systemic resistance and enhanced salinity tolerance of wheat (Triticum aestivum. L.). PLoS One 11: e0155026.
    Pubmed KoreaMed CrossRef
  31. Tallgren AH, Airaksinen U, Von Weissenberg R, Ojamo H, Kuusisto J, and Leisola M. 1999. Exopolysaccharide-producing bacteria from sugar beets. Appl Environ Microbiol 65: 862-864.
    Pubmed KoreaMed
  32. Timmusk S, Abd El-Daim IA, Copolovici L, Tanilas T, Kännaste A, Behers L, Nevo E, Seisenbaeva G, Stenström E, and Niinemets Ü. 2014. Drought-tolerance of wheat improved by rhizosphere bacteria from harsh environments: enhanced biomass production and reduced emissions of stress volatiles. PLoS One 9: e96086.
    Pubmed KoreaMed CrossRef
  33. Tiwari S, Prasad V, Chauhan PS, and Lata C. 2017. Bacillus amyloliquefaciens confers tolerance to various abiotic stresses and modulates plant response to phytohormones through osmoprotection and gene expression regulation in rice. Front Plant Sci 8: 1510.
    Pubmed KoreaMed CrossRef
  34. Vurukonda SS, Vardharajula S, Shrivastava M, and Shaik ZA. 2016. Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria. Microbiol Res 184: 13-24.
    Pubmed CrossRef
  35. Weisburg WG, Barns SM, Pelletier DA, and Lane DJ. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173: 697-703.
    Pubmed KoreaMed CrossRef
  36. Yang J, Kloepper JW, and Ryu CM. 2009. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci 14: 1-4.
    Pubmed CrossRef
  37. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, and Chun J. 2017. Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67: 1613-1617.
    Pubmed KoreaMed CrossRef
  38. Zhang H, Mao X, Wang C, and Jing R. 2010. Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis. PLoS One 5: e16041.
    Pubmed KoreaMed CrossRef
  39. Zhang J, Jia W, Yang J, and Ismail AM. 2006. Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Res 97: 111-119.
    CrossRef
  40. Zhu J-K. 2002. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247-273.
    Pubmed KoreaMed CrossRef