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

Lee, Young Hee; Jang, Su Jeong; Han, Joon-Hee; Bae, Jin Su; Shin, Hyunsuk; Park, Hee Jin; Sang, Mee Kyung; Han, Song Hee; Kim, Kyoung Su; Han, Sang-Wook; Hong, Jeum Kyu

doi:10.5423/ppj.oa.07.2018.0130

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

The plant pathology journal, v.34 no.6, 2018년, pp.555 - 566

Lee, Young Hee (Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech)) , Jang, Su Jeong (Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech)) , Han, Joon-Hee (Division of Bioresource Sciences, Kangwon National University) , Bae, Jin Su (Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech)) , Shin, Hyunsuk (Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech)) , Park, Hee Jin (Institute of Glocal Disease Control, Konkuk University) , Sang, Mee Kyung (National Institute of Agricultural Science, Rural Development Administration) , Han, Song Hee (Hyunnong Co., Ltd) , Kim, Kyoung Su (Division of Bioresource Sciences, Kangwon National University) , Han, Sang-Wook (Department of Integrative Plant Science, Chung-Ang University) , Hong, Jeum Kyu (Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech))

Abstract ▼ AI-Helper

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 $50^{\circ}C$). 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.

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표/그림 (5)

그림 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 (℃) 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.
그림 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℃) 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.
그림 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-weekold 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.
그림 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℃ 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.
그림 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.

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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 inde-pendent experimental replications.

제안 방법

In this study, roles of B. aryabhattai H26-2 and B. siamensis H30-3 in protecting Chinese cabbage plant against high temperature and/or drought stresses were investigated. In vitro antifungal activities and crop protection efficacies of the B.
No significant difference was found in growth of five-week-old two Chinese cabbage cultivars under growing conditions (data not shown). To investigate growth of the two cultivars under high temperature condition, the two cultivars planted at the four different sowing dates (4 Jul, 4 Aug, 5 Sep and 5 Oct) were compared in a plastic house (Fig. 1A). Sowing at 5 Sep resulted in most efficient growth in both cultivars, and followed by sowing at 4 Aug.
To investigate growth of two Chinese cabbage cultivars under different ambient temperatures, seeds of two cultivars were sown in the pots at four different planting dates (4 Jul, 4 Aug, 5 Sep and 5 Oct) and grown for five weeks in the greenhouse. Thereafter, fresh weight (FW) of the seedlings was measured.

대상 데이터

Bacillus aryabhattai H26-2 and Bacillus siamensis H30-3 were isolated from rhizosphere of tomato plants grown in Sacheon, Gyeong-nam Province. After being identified using physiological and molecular procedures, they were deposited in Korean Agricultural Culture Collection as KACC 92165P and KACC 92187P, respectively. They were subcultured on nutrient agar (NA) media at 30℃.

데이터처리

Asterisks indicate significant differences as determined by Student’s t-test (P < 0.05).

후속연구

higginsianum infections. The current study will provide more insights on the rhizobacteria-mediated crop protection against abiotic and biotic stresses in Chinese cabbage plants.

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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