Enhanced Tolerance of Chinese Cabbage Seedlings Mediated by Bacillus aryabhattai H26-2 and B. siamensis H30-3 against High Temperature Stress and Fungal Infections

Abstract

Two rhizobacteria Bacillus aryabhattai H26-2 and B. siamensis H30-3 were evaluated whether they are involved in stress tolerance against drought and high temperature as well as fungal infections in Chinese cabbage plants. Chinese cabbage seedlings cv. Ryeokgwang (spring cultivar) has shown better growth compared to cv. Buram-3-ho (autumn cultivar) under high temperature conditions in a greenhouse, whilst there was no difference in drought stress tolerance of the two cultivars. In vitro growth of B. aryabhattai H26-2 and B. siamensis H30-3 were differentially regulated under PEG 6000-induced drought stress at different growing temperatures (30, 40 and 50°C). Pretreatment with B. aryabhattai H26-2 and B. siamensis H30-3 enhanced the tolerance of Chinese cabbage seedlings to high temperature, but not to drought stress. It turns out that only B. siamensis H30-3 showed in vitro antifungal activities and in planta crop protection against two fungal pathogens Alternaria brassicicola and Colletotrichum higginsianum causing black spots and anthracnose on Chinese cabbage plants cv. Ryeokgwang, respectively. B. siamensis H30-3 brings several genes involved in production of cyclic lipopeptides in its genome and secreted hydrolytic enzymes like chitinase, protease and cellulase. B. siamensis H30-3 was found to produce siderophore, a high affinity iron-chelating compound. Expressions of BrChi1 and BrGST1 genes were up-regulated in Chinese cabbage leaves by B. siamensis H30-3. These findings suggest that integration of B. aryabhattai H26-2 and B. siamensis H30-3 in Chinese cabbage production system may increase productivity through improved plant growth under high temperature and crop protection against fungal pathogens.

Keywords: Bacillus; Chinese cabbage; antifungal; biocontrol; high temperature stress.

Fig. 1

Different sensitivity of two Chinese cabbage seedlings (cvs. Ryeokgwang and Buram-3-ho) to high temperature and/or drought stress under greenhouse conditions. (A) Plant growth of two cultivars grown for five weeks after planting at four different planting dates (4 Jul, 4 Aug, 5 Sep and 5 Oct) under a plastic greenhouse condition. Changes in average maximum (T_max), average mean (T_mean) and average minimal (T_min) temperatures (°C) for five weeks under the greenhouse conditions were demonstrated. (B) Plant growth of two Chinese cabbage cultivars by stopping irrigations. Three-week-old seedlings were undergone different irrigation regimes for additional two-weeks. Error bars represent the standard errors of the means of the four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test.

Fig. 2

Bacterial tolerance to dehydration at different growing temperatures during in vitro liquid cultures. Two Bacillus species (B. aryabhattai H26-2 and B. siamensis H30-3) and two phytopathogenic bacteria Pectobacterium carotovorum subsp. carotovorum (Pcc) strain PCC21 and Xanthomonas campestris pv. campestris (Xcc) strain 8004 were cultured with increasing concentration (0, 4, 12 and 20%) of polyethylene glycol (PEG) 6000 at different temperatures (30, 40 and 50°C) for 48 h. Bacterial numbers were initially inoculated with 10⁵ cfu/ml and indirectly measured using a spectrophotometer with optical density at 600 nm. Error bars represent the standard errors of the means of the four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test.

Fig. 3

Effect of pretreatment with rhizobacteria on growth of Chinese cabbage seedling (cvs. Ryeokgwang and Buram-3-ho) under drought stress at two different temperatures. Two-week-old seedlings were treated with Bacillus aryabhattai H26-2 and B. siamensis H30-3 for one week and then undergone different concentrations (0, 2 and 4%) of polyethylene glycol (PEG) 6000-mediated drought stresses under two growth temperature regimes. The seedlings were sub-irrigated with water as mocks. Fresh weight (g) of the seedlings was measured after 12 days and 5 days for normal and high temperature conditions, respectively. Error bars represent the standard errors of the means of the four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test.

Fig. 4

Protective effects of Bacillus siamensis H30-3 against fungal pathogens on Chinese cabbage plants. (A) Dual culture assay for in vitro inhibition of mycelial growth of Alternaria brassicicola and Colletotrichum higginsianum by B. siamensis H30-3. The fungal pathogens were co-cultured with the bacterial strain H30-3 for 15 and 12 days at 25°C for A. brassicicola and C. higginsianum, respectively. (B) Inhibitory mycelial growth measured by half of the fungal colony diameter after co-culture. Error bars represent the standard errors of the means of the six independent experimental replications. Asterisks indicate significant differences as determined by Student’s t-test (P < 0.05). (C) Reduced black spot and anthracnose disease severities on Chinese cabbage plants by the antagonistic B. siamensis H30-3. Bacterial suspension (10⁹ cfu/ml) of B. siamensis H30-3 was foliar sprayed at 1 day prior to challenge inoculations of the fungal pathogens. Disease severities were evaluated at 4 days after fungal inoculation based on 0–5 scales. Error bars represent the standard errors of the means of the four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test.

Fig. 5

Antifungal metabolites production and induced defence response of Chinese cabbage plants by Bacillus siamensis H30-3. (A) PCR-based detection of bacterial genes encoding antimicrobial lipopeptides from B. aryabhattai H26-2 and B. siamensis H30-3 genomes. M, DNA size marker. bacD, bacilysin; bmyA, bacillomycin; fenD, fengycin; ituA, iturin A; srfA, surfactin; zwiA, zwittermicin A. (B) Characteristics of hydrolytic enzyme secretion, siderophore production and phosphate (P)-solubilisation originated from B. siamensis H30-3. In vitro productions of chitinase, protease and cellulase by B. siamensis H30-3 were examined on different agar media described in Materials and methods. (C) Expression of defence-related genes in Chinese cabbage leaves treated with B. siamensis H30-3. Expression of basic glucanase 2 gene BrBGL2, chitinase 1 gene BrChi1, glutathione-S-transferase 1 gene BrGST1 and ascorbate peroxidase 1 gene BrAPX1 was analyzed by semi-quantitative RT-PCR technique. BrActin7 was used as an internal control. The number of PCR cycles of each result is indicated within the right parenthesis.