Screening,Identification and Fermentation Condition Optimization of an Antagonistic Bacterium for Melon Black Spot

doi:10.13560/j.cnki.biotech.bull.1985.2021-0118

Abstract

Abstract:

Endophytic bacterial strains were isolated from the roots,stems,leaves and fruits of healthy melon plants,and the taxonomic status and optimal fermentation conditions of the excellent antagonistic strains were clarified. The dilution plate coating was applied to isolate endophytic bacteria,and the plate confrontation was used to screen the excellent antagonistic bacteria for Alternaria tenuissima. Identification was conducted via morphology and molecular biology,and the fermentation conditions were optimized through single-factor and orthogonal experiment. The results showed that total 50 strains of endophytic bacteria were isolated,21 strains of them had antagonistic effects against A. tenuissima,and the inhibition rate by 11 strains reached > 70%. Among them,strain G2 had the highest inhibition rate,reaching 76.41%,indoor in vitro prevention effect reached 44.90%. According to the results of morphological characteristics and gene sequence analysis,strain G2 was identified as Bacillus subtilis. The optimal culture conditions of strain G2 were with a medium included 20 g glucose,30 g sodium nitrate,10 g yeast extract in 1 000 mL water. The optimal fermentation conditions were as followed：shaking 50 mL liquid（the best inoculum size was 0.2%）in an 150 mL flask at 150 r/min at 32℃,with an initial pH value of 7.5,and the optimal fermentation time was 18 h. The results may supply new strain resource for the biological control of melon black spot and also provide a basis for the industrial development of melon endophytic bacterium strain G2.

Key words: endophytic antagonistic bacteria, screening, identification, fermentation condition, optimization

SHI Zhao-rong, SUN Shu-jun, ZHANG Guang-rong, MEI Da-hai, LIU Yan-chao, YANG Cheng-de. Screening,Identification and Fermentation Condition Optimization of an Antagonistic Bacterium for Melon Black Spot[J]. Biotechnology Bulletin, 2022, 38(1): 115-124.

Figures/Tables 20

Table 1 Design of L9（33）orthogonal experiment

水平 Level	因素Factor
水平 Level	碳源 Carbon source	氮源 Nitrogen source	无机盐 Inorganic salt
1	A1	B1	C1
2	A2	B2	C2
3	A3	B3	C3

Table 2 Screening of antagonistic bacteria against melon black spot

拮抗菌Antago- nistic bacteria	抑菌率Inhibition rate/%	拮抗菌Antago- nistic bacteria	抑菌率 Inhibition rate/%
G2	76.41±0.55 a	J3	68.68±1.81 cde
G16	74.63±1.96 ab	F9	67.43±1.78 def
S19	73.70±0.36 ab	Y1	66.18±1.30 ef
G4	73.28±1.46 ab	F12	65.76±2.57 ef
S2	73.17±0.38 ab	F8	65.66±0.38 ef
F7	71.82±0.79 bc	G5	65.55±0.36 ef
S23	71.50±0.18 bcd	F5	64.51±1.71 f
G3	70.98±2.11 bcd	F3	63.47±0.42 f
S7	70.88±0.79 bcd	F4	57.62±1.50 f
S21	70.77±2.61 bcd	G1	48.02±1.58 f
S18	70.67±1.09 bcd

Fig.1 Effect of strain G2 against melon black spot

Table 3 Control effects of antagonistic strains on melon black spot

处理 Treatment	病斑直径 Lesion diameter/cm	防效 Control effect/%
ck	0.82 ± 0.12 a	-
G2	0.45 ± 0.03 c	44.90
G16	0.73 ± 0.02 ab	10.20
S19	0.58 ± 0.02 bc	28.57
G4	0.57 ±0.02 c	30.61
S2	0.55 ± 0.03 c	32.65

Fig.2 Colony（A）and Gram stain（B）of strain G2

Fig.3 Phylogenetic tree of strain G2 based on 16S rDNA and gyrB sequence

Fig.4 Effect of basal medium on the growth of strain G2 Different letters refer to significant difference（P<0.05）,the same below

Fig.5 Effects of carbon sources on the growth of strain G2 1：Sucrose. 2：Corn starch. 3：Maltose. 4：Mannitol. 5：D-fructose. 6：CK. 7：Glucose

Fig.6 Effects of nitrogen sources on the growth of strain G2 1：Urea. 2：（NH4）2SO4. 3：KNO3. 4：NH4Cl. 5：NaNO3. 6：CK. 7：Peptone

Fig.7 Effects of inorganic salts on the growth of strain G2 1：KH2PO4;2：CuSO4;3：CaCl2;4：NaCl;5：ZnSO4;6：CaCO3;7：CK

Fig.8 Effects of glucose concentration on the growth of strain G2

Fig.9 Effects of sodium nitrate on the growth of strain G2

Fig.10 Effects of sodium chloride on the growth of strain G2

Table 4 Orthogonal experiment

试验号 No.	因子 Factor
试验号 No.	NaNO₃含量 NaNO₃ content/g	葡萄糖含量 Glucose concentration/g	NaCl含量 NaCl content/g	OD₆₀₀
1	20	15	0	1.617 d
2	25	15	5	2.385 ab
3	30	15	10	2.527 a
4	20	20	5	2.384 ab
5	25	20	10	2.543 a
6	30	20	0	2.586 a
7	20	25	10	2.468 ab
8	25	25	0	2.473 a
9	30	25	5	2.139 c
ck	25	20	5	2.227 bc
T1	2.176	2.156	2.225	-
T2	2.504	2.467	2.303	-
T3	2.360	2.417	2.513	-

Fig.11 Effects of rotation speed on the growth of strain G2

Fig.12 Effects of temperature on the growth of strain G2

Fig.13 Effects of liquid volumes on the growth of strain G2

Fig.14 Effects of inoculation size onthe growth of strain G2

Fig.15 Effects of different pH values on the growth of strain G2

Fig.16 Effects of fermentation time on the growth of strain G2

References 24

[1]	王毓洪, 高天一, 张华峰, 等. 设施西甜瓜连作障碍综合防控技术[J]. 中国蔬菜, 2018, (11):81-84
	Wang YH, Gao TY, Zhang HF, et al. Comprehensive prevention and control technology for continuous cropping obstacles of western melon[J]. China Vegetables, 2018, (11):81-84.
[2]	赵娟, 吴学宏. 甜瓜真菌性叶斑病研究进展[J]. 中国植保导刊, 2014, 34(11):24-27+14.
	Zhao J, Wu XH. Research progress on muskmelon fungal leaf spot[J]. China Plant Protection, 2014, 34(11):24-27+14.
[3]	李怡, 彭文涛, 李勤奋, 等. 一株耐铝甜瓜枯萎病拮抗细菌的分离鉴定及其耐铝特性研究[J]. 热带作物学报, 2018, 39(6):1176-1182.
	Li Y, Peng WT, Li QF, et al. Isolation, identification of an aluminum-tolerant bacteria strain antagonizing Fusarium wilt of muskmelon and its Al-tolerant characteristics[J]. Chinese Journal of Tropical Crops, 2018, 39(6):1176-1182.
[4]	杨良, 史应武, 娄恺. 甜瓜细菌性斑点病拮抗菌P-13产抑菌物质培养条件的优化[J]. 西北农业学报, 2012, 21(4):181-186.
	Yang L, Shi YW, Lou K, Culture conditions optimization for antimicrobial substances production by antagonistic strain P-13 against bacterial spot diseases of muskmelon[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2012, 21(4):181-186.
[5]	马英元, 栾非时, 马鸿艳, 等. 甜瓜白粉病内生拮抗细菌的筛选鉴定及其防治效果的研究[J]. 植物保护, 2011, 37(2):25-30.
	Ma YY, Luan FS, Ma HY, et al. Screening, identification and control efficacy of melon endophytic antagonistic bacteria against Sphaerotheca fuliginea[J]. Plant Protection, 2011, 37(2):25-30.
[6]	刘建利, 曹君迈, 马文平. 宁夏乌拉尔甘草内生菌鉴定及甜瓜采后病害病原拮抗菌的筛选[J]. 干旱地区农业研究, 2011, 29(1):252-256.
	Liu JL, Cao JM, Ma WP. Identification of endophytes from glycyrrhiza uralensis in Ningxia and screening of antagonism against pathogens of post-harvest muskmelon[J]. Agricultural Research in the Arid Areas, 2011, 29(1):252-256.
[7]	李刚, 尹志刚, 陈振, 郭太忠. 拮抗细菌EBS05生长曲线及粗提物抑菌活性测定[J]. 浙江农业科学, 2015, 56(3):369-372.
	Li G, Yin ZG, Chen Z, et al. EBS05antagonistic bacteria growth curve and crude extracts antifungal activity[J]. Journal of Zhejiang Agricultural Sciences, 2015, 56(3):369-372.
[8]	贾钧辉, 郑宇, 杨青娟, 等. 生防枯草芽孢杆菌B579补料分批发酵工艺优化[J]. 山东农业大学学报:自然科学版, 2013, 44(1):35-39.
	Jia JH, Zheng Y, Yang QJ, et al. Optimization of fed-batch fermentation process of biocontrol bacillus subtilis B579[J]. Journal of Shandong Agricultural University(Natural Science Edition), 2013, 44(1):35-39.
[9]	陈力, 王中康, 黄冠军, 等. 柑橘溃疡病生防菌株CQBS03的鉴定及其培养特性研究[J]. 中国农业科学, 2008(8):2537-2545.
	Chen L, Wang ZK, Huang GJ, et al. Evaluation of Bacillus subtilis strain CQBS03against Xanthomonas axonopodis pv. citri. Scientia Agricultura Sinica, 2008, 41(8):2537-2545
[10]	赵海霞, 王晨, 孔双, 等. 哈密瓜中内生细菌的分离与鉴定[J]. 中国酿造, 2015, 34(9):26-29.
	Zhao HX, Wang C, Kong S, et al. Isolation and identification of endophytic bacteria from Hami melon[J]. China Brewing, 2015, 34(9):26-29.
[11]	方中达. 植病研究方法[M]. 北京: 中国农业出版社, 2007, 46-47.
	Fang ZD. Method of Plant Pathology[M]. Beijing: China Agriculture Press, 2007, 46-47.
[12]	周德庆. 微生物学实验手册[M]. 上海: 上海科技出版社, 1986.
	Zhou DQ. Microbiology Experiment Manual[M]. Shanghai: Shanghai Science and Technology Press, 1986.
[13]	Caňamás T P, Viňas I, Torres R, et al. Field applications of improved formulations of Candida sake CPA-1 for control of Botrytis cinerea in grapes[J]. Biological Control, 2011, 56(2):150-158 doi: 10.1016/j.biocontrol.2010.11.007 URL
[14]	Hassnai MA, Dur NP, Hacquard S. Microbial interactions within the plant holobiont[J]. Microbiome, 2018, 6:58. doi: 10.1186/s40168-018-0445-0 URL
[15]	高增贵, 李天来, 赵王月, 等. 甜瓜枯萎病拮抗内生细菌筛选[J]. 西北农林科技大学学报:自然科学版, 2005(S1):85-87.
	Gao ZG, Li TL, Zhao WY, et al. Selection of endophytic bacteria for suppression of melon wilt caused by Fusarium oxysporium[J]. Journal of Northwest A & F University:Natural Science Edition, 2005(S1):85-87.
[16]	兰琴英, 殷小雯, 鲁卓越, 等. 2株南方红豆杉内生细菌的分离鉴定及其抑菌活性[J]. 贵州农业科学, 2014, 42(2):123-127.
	Lan QY, Yin XW, Lu ZY, et al. Isolation and identification of two endophytic bacteria isolated from Taxus chinensis var. mairei and their antifungal effects[J]. Guizhou Agricultural Sciences, 2014, 42(2):123-127.
[17]	Habib M, Bhat M, Dar BN, et al. Sweet cherries from farm to table:a review[J]. Critical Reviews in Food Science and Nutrition, 2017, 57(8):1638-1649. doi: 10.1080/10408398.2015.1005831 URL
[18]	崔欣, 李伟, 朱建兰. 葡萄灰霉病优势拮抗细菌的筛选与鉴定[J]. 中国植保导刊, 2018, 38(1):12-16.
	Cui X, Li W, Zhu JL. Selection and identification of antagonistic strain to Botrytis cinerea[J]. China Plant Protection, 2018, 38(1):12-16.
[19]	穆香轶, 张璇, 胡有贞, 等. 库尔勒香梨黑头病拮抗菌的筛选和鉴定[J]. 微生物学通报, 2020, 47(6):1795-1806.
	Mu XY, Zang X, Hu YZ, et al. Screening and identification of an antagonistic bacterium against blackhead disease of Korla fragrant pear[J]. Microbiology China, 2020, 47(6):1795-1806.
[20]	陈小洁, 王其, 张欣悦, 等. 杜仲内生细菌拮抗小麦赤霉病菌研究[J]. 浙江农业学报, 2019, 31(5):766-776.
	Chen XJ, Wang Q, Zhang XY, et al. Study on endophytic bacteria isolated from Eucommia ulmoides with antimicrobial activity against Fusarium graminearum[J]. Acta Agriculturae Zhejiangensis, 2019, 31(5):766-776.
[21]	张新杰, 刘彦策, 钱欣雨, 等. 弯曲平脐蠕孢拮抗细菌的筛选、鉴定及拮抗作用[J]. 微生物学通报, 2020, 47(8):2417-2424.
	Zhang XJ, Liu YC, Qian XY, et al. Isolation, identification and characterization of an antagonistic bacterium against Bipolaris papendorfii[J]. Microbiology China, 2020, 47(8):2417-2424.
[22]	赖家豪, 宋水林, 刘冰. 柑桔炭疽病生防内生细菌的筛选、鉴定及摇瓶发酵条件优化[J]. 中国南方果树, 2020, 49(6):11-16, 19.
	Lai JH, Song Sl, Liu B. Screening, identification and optimization of fermentation conditions for shaking flask culture of endophytic bacterial strain GN222 with the potential of control citrus anthracnose[J]. South China Fruits, 2020, 49(6):11-16, 19.
[23]	陈莉, 汪立平, 赵勇, 等. 枯草芽孢杆菌K-6-9发酵条件优化及放大[J]. 食品工业科技, 2014, 35(3):172-176.
	Chen L, Wang LP, Zhao Y, et al. Optimization\sof cultural conditions and amplification of Bacillus subtilis K-6-9[J]. Science and Technology of Food Industry, 2014, 35(3):172-176.
[24]	张慧, 林陈强, 吴大华, 等. 枯草芽孢杆菌(Bacillus subtilis)CS27液体发酵条件优化[J]. 热带作物学报, 2019, 40(5):995-1001.
	Zhang H, Lin CQ, Wu DH, et al. Optimization of liquid fermentation conditions of Bacillus subtilis CS27[J]. Chinese Journal of Tropical Crops, 2019, 40(5):995-1001.