甜瓜黑斑病菌的拮抗细菌筛选、鉴定及发酵条件优化

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

摘要/Abstract

摘要：

从健康甜瓜植株的根、茎、叶和果实分离内生细菌,明确优良拮抗菌株的分类地位及其最优发酵条件。利用平板稀释法分离内生细菌,平板对峙法筛选黑斑病菌（Alternaria tenuissima）的优良拮抗细菌,通过形态学和分子生物学鉴定,并使用单因素和正交试验方法优化发酵条件。分离共得到50株内生细菌,21株对细极链格孢具有拮抗作用,11株的抑制率达到70%以上,其中菌株G2的抑菌率最高,达76.41%,室内离体防效达44.90%,经形态特征和基因序列分析鉴定为枯草芽孢杆菌（Bacillus subtilis）,其最优发酵培养基为葡萄糖20 g、硝酸钠30 g、酵母膏10 g和水1 000 mL,最优发酵条件为摇床转速150 r/min,温度32℃,150 mL摇瓶装液50 mL,最佳接种量为0.2％,培养基初始pH为7.5,其最佳发酵时间为18 h。该结果为甜瓜黑斑病生物防治提供了菌种资源,也为甜瓜内生细菌G2的产业化开发提供了依据。

关键词: 内生拮抗细菌, 筛选, 鉴定, 发酵条件, 优化

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

施兆荣, 孙述俊, 张广荣, 梅大海, 刘彦超, 杨成德. 甜瓜黑斑病菌的拮抗细菌筛选、鉴定及发酵条件优化[J]. 生物技术通报, 2022, 38(1): 115-124.

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.

图/表 20

表1 L9（33）正交试验设计

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

表2 甜瓜黑斑病菌拮抗菌的筛选

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

图1 菌株G2对甜瓜黑斑病菌的拮抗效果

Fig.1 Effect of strain G2 against melon black spot

表3 拮抗菌株对甜瓜黑斑病的防效

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

图2 菌株G2的单菌落（A）和革兰氏染色（B）

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

图3 菌株G2基于16S rDNA和gyrB序列的系统发育树

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

图4 基础培养基对菌株G2生长的影响不同字母表示差异显著性（P<0.05）,下同

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

图5 碳源对菌株G2生长的影响 1：蔗糖;2：玉米淀粉;3：麦芽糖;4：甘露醇;5：D-果糖;6：CK;7：葡萄糖

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

图6 氮源对菌株G2生长的影响 1：尿素;2：硫酸铵;3：硝酸钾;4：氯化铵;5：硝酸钠;6：CK;7：蛋白胨

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

图7 无机盐对菌株G2生长的影响

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

图8 葡萄糖浓度对菌株G2生长的影响

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

图9 NaNO3浓度对菌株G2生长的影响

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

图10 NaCl浓度对菌株G2生长的影响

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

表4 正交试验优化

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	-

图11 转速对菌株G2生长的影响

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

图12 温度对菌株G2生长的影响

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

图13 装液量对菌株G2生长的影响

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

图14 接种量对菌株G2生长的影响

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

图15 不同pH对菌株G2生长的影响

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

图16 发酵时间对菌株G2生长的影响

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

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