Optimization of Culture Conditionsand Analysis of Fermentation Process for Plant Vaccine-producing Bacterium, FJAT-1458
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摘要: 对番茄青枯病植物疫苗菌株FJAT-1458进行培养条件优化和发酵过程状态参数研究。采用摇瓶培养和单因素试验研究菌株FJAT-1458的培养条件，确定最适培养基为SPA（蛋白胨5 g·L-1，蔗糖20 g·L-1，KH2PO4 0.5 g·L-1，MgSO4 0.25 g·L-1），最适培养温度为30℃，250 mL三角瓶最适装液量为35 mL。用50 L发酵罐发酵FJAT-1458，发酵过程中菌体数量呈现先升后降的变化趋势，FJAT-1458的生长可分为4个时期：0～6 h为发酵适应期、6～36 h为对数生长期、36～60 h为稳定生长期，72 h后为消亡期。发酵过程中，菌体的致病性指标-弱化指数变化小，介于0.832～0.855，方差分析表明其差异不显著，P>0.05；发酵液pH值从7.23上升至8.78；菌体细胞为短杆状，大小为（1.0～1.5）μm×（0.5～0.8）μm，随着发酵进程，细胞颜色变深，发酵后期细胞开始变形、裂解。Abstract: Conditions to culture FJAT-1458, a bacterium that produces plant vaccine against the tomato bacterial wilt caused by Ralstonia solanacearum, were optimized by asingle factor experiment. They were determined to be the use of an SPA medium containing peptone 5 g·L-1, sugar 20 g·L-1, KH2PO40.5 g·L-1, and MgSO40.25 g·L-1 at 30℃ with 35 mL of broth in a 250 mL shaking flask. Subsequently, in a 50 L fermentation tank, the fermentation was carried out and the process monitored. During the fermentation, the cell count on FJAT-1458 increased initially and followed by a gradual decline with a 4-stage growth period that included the adaptive (0-6 h), logarithmic (6-36 h), stationary (36-60 h), and decline phases (after 72 h). In addition, it was observed that(a) the pathogenicity indicator, attenuation index, of FJAT-1458 ranged from 0.832 to 0.855 showing no significant difference (P>0.05) throughout the entire process; (b) the pH of the fermentation broth increased from 7.23 to 8.78; (c) morphologically, the bacteria appeared to be(1.0-1.5)μm×(0.5-0.8)μm rods with a darkening color as the process proceeded; (d) the cellular secretion from the bacteria increased with time; and, (e) some of the bacterial cells deformed and lysed near the end of fermentation.
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图 1 菌株FJAT-1458发酵过程菌体形态变化
Figure 1. Morphological changes on FJAT-1458 during fermentation process
表 1 植物疫苗菌株FJAT-1458在不同培养条件下的生长量
Table 1. Growth of avirulent strain, FJAT-1458, under varied culture conditions
测定项目 FJAT-1458菌体浓度/
(×108cfu·mL-1)培养基 SPA 1.56±0.12 a NA 0.93±0.05 c LB 1.39±0.11 a 温度 20℃ 1.14±0.06 bc 25℃ 1.27±0.12 b 30℃ 1.77±0.10 a 35℃ 0.91±0.06 c 40℃ 0 装液量 15mL 1.82±0.07 a 25 mL 1.75±0.07 a 35 mL 1.87±0.11a 45 mL 1.77±0.04 a 55 mL 1.71±0.06a 注：同例不同小写字母表示差异达显著水平(P < 0.05)。表 2同。 
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表 2 植物疫苗菌株FJAT-1458发酵过程各参数变化
Table 2. Changes on parameters during fermentationprocess of avirulent strain, FJAT-1458
时间
/hFJAT-1458菌体浓度
/(×108cfu·mL-1)菌株弱
化指数pH值 0 1.12±0.07 a 0.84±0.03 a 7.23±0.01 h 6 1.37±0.15 a 0.84±0.01 a 7.51±0.01 g 12 3.05±1.56 b 0.84±0.03 a 8.27±0.01 f 24 9.37±1.02 c 0.85±0.01 a 8.53±0.03 d 36 12.48±2.55 d 0.85±0.01 a 8.38±0.02 e 48 11.72±1.82 e 0.85±0.01 a 8.72±0.01 b 60 11.05±1.22 f 0.85±0.02 a 8.67±0.01 c 72 8.32±0.43 g 0.83±0.03 a 8.78±0.03 a
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[1] HAYWARD A C. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum [J]. Annual Review of Phytopathology, 1991, 29: 65-87. doi: 10.1146/annurev.py.29.090191.000433 [2] VANITHA S C, NIRANJANASR, UMESHA S. Role of phenylalanine ammonia lyase and polyphenol oxidase in host resistance to bacterial wilt of tomato [J]. Journal of Phytopathology, 2009, 157(9): 552-557. doi: 10.1111/jph.2009.157.issue-9 [3] 黄真池, 刘媛, 曾富华, 等.青枯菌致病机理及作物抗青枯病研究进展[J].热带亚热带植物学报, 2008, 16(5): 491-496. http://www.cnki.com.cn/Article/CJFDTOTAL-RYZB200805016.htm [4] 袁立和, 苏清岚, 易由华.番茄青枯病防治技术研究[J].中国蔬菜, 1992, 18(5): 4-6. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGSC199205001.htm [5] JONES J D G, DANGL J L. The plant immune system[J]. Nature, 2006, 444(16): 323-329. http://cn.bing.com/academic/profile?id=2162515800&encoded=0&v=paper_preview&mkt=zh-cn [6] CHANG Y H, YAN H Z, LIOU R F. A novel elicitor protein from phytophthora parasitica induces plant basa immunity and systemic acquired resistance [J]. Molecular Plant Pathology, 2015, 16(2):123-136. doi: 10.1111/mpp.2015.16.issue-2 [7] DALE W, ADRIAN N, GARY L. Induced resistance for plant defence [M]. Oxford: Blackwell Publishing, 2007: 31-81. [8] 邱德文.植物免疫与植物疫苗研究与实践[M].北京:科学出版社, 2008. [9] NORMAN D J, ZAPATA M, GABRIEL D W, et al. Genetic diversity and host range variation of Ralstonia solancearum strains entering North America [J]. Phytopathology, 2009, 99(9): 1070-1077. doi: 10.1094/PHYTO-99-9-1070 [10] CELLIER G, PRIOR P. Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato [J]. Phytopatholy, 2010, 100: 1250-1261. doi: 10.1094/PHYTO-02-10-0059 [11] SUGA Y, HORITA M, UMEKITA M, et al. Pathogenic characters of Japanese potato strains of Ralstonia solanacearum [J]. Journal of General Plant Pathology, 2013, 79: 110-114. doi: 10.1007/s10327-013-0429-7 [12] 刘波, 蓝江林, 朱育菁, 等.植物免疫系统的研究与应用[J].中国农学通报, 2007, (23): 163-172. http://wiki.cnki.com.cn/HotWord/6090486.htm [13] 郑雪芳, 刘波, 林乃铨, 等.青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析[J].植物病理学报, 2013, 43(5): 518-531. http://www.cnki.com.cn/Article/CJFDTOTAL-ZWBL201305010.htm [14] KELMAN A. The relationship of pathogenicity in P. solanacearum to colony appearance on a tetrazolium medium [J]. Phytopathology, 1954, 44: 693-695. https://www.cabdirect.org/cabdirect/abstract/19551101405 [15] 刘波, 林营志, 朱育菁, 等.生防菌对青枯雷尔氏菌的致弱特性[J].农业生物技术学报. 2004, 12(3): 322-329. http://www.cnki.com.cn/Article/CJFDTOTAL-NYSB200403015.htm [16] 陈燕萍, 刘波, 葛慈斌, 等.作物青枯病植物疫苗工程菌FJAT-T8发酵过程生长适合度研究[J].福建农业学报, 2012, 27(3): 287-293. http://www.fjnyxb.cn/CN/abstract/abstract1874.shtml [17] 周青, 王笑, 苏婷, 等.青枯病和根肿病生防细菌BS2004的发酵培养基和培养条件优化[J].中国生物防治学报, 2012, 28(4): 537-544. http://www.cnki.com.cn/Article/CJFDTOTAL-ZSWF201204014.htm [18] 马耀华, 谭小艳, 黄思良, 等.石榴干腐病生防菌株Z2的鉴定及其培养条件的优化[J].植物病理学报, 2015, 45(4): 425-437. http://www.cnki.com.cn/Article/CJFDTOTAL-ZWBL201504012.htm [19] 巫建新, 席刚俊, 杨卫君, 等.抑制杨木变色生防细菌的筛选及其发酵培养条件优化研究[J].西部林业科学, 2013, 42(2): 99-102. http://www.cnki.com.cn/Article/CJFDTOTAL-YNLK201302018.htm [20] 车建美, 蓝江林, 刘波.转绿色荧光蛋白基因的青枯雷尔氏菌生物学特性[J].中国农业科学, 2008, 41(11): 3626-3635. http://www.cnki.com.cn/Article/CJFDTOTAL-ZNYK200811026.htm [21] EINSTONE J G, HENNESSY J, WILSON J K, et al Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts. EPPO Bull, 1996, 26: 674-678. http://cn.bing.com/academic/profile?id=2006967948&encoded=0&v=paper_preview&mkt=zh-cn -
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