新疆农业科学, 2022, 59(5): 1216-1222 DOI: 10.6048/j.issn.1001-4330.2022.05.021

植物保护·微生物·贮藏保鲜加工

枯草芽孢杆菌KXZ-33与化学药剂协同防控棉花枯萎病的效果评价

赵莹莹,1, 张军高2, 李进2, 梁晶2, 高翔宇1, 顾爱星,2, 雷斌,2

1.新疆农业大学农学院,乌鲁木齐 830052

2.新疆农业科学院核技术生物技术研究所/新疆作物化控调控工程技术研究中心,乌鲁木齐 830091

Preliminary Study on the Synergistic Effects of Bacillus subtilis KXZ-33 and Chemical Pesticides against Cotton Fusarium wilt

ZHAO Yingying,1, ZHANG Jungao2, LI Jin2, LIANG Jing2, GAO Xiangyu1, GU Aixing,2, LIE Bin,2

1. College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China

2. Xinjiang Crop Chemical Control and Regulatory Engineering Technology Research Center / Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

通讯作者: 顾爱星(1973-),女,新疆乌鲁木齐人,教授,硕士生导师,研究方向为棉花黄枯萎病,(E-mail)594903711@qq.com;雷斌(1973-),男,四川南江人,研究员,博士,硕士生导师,研究方向为作物根腐类病害防控及绿色农药,(E-mail)leib668@xaas.ac.cn

收稿日期: 2021-12-3  

基金资助: 自治区科技援疆项目“棉花抗逆抗病性状关键基因高通量分子鉴定和优异种质创新与利用”(2021E02003)
天山雪松计划“小麦、棉花提质增效关键技术及产品研发”(2020XS16)

Corresponding authors: GU Aixing(1973 -),female, Urumqi, Xinjing, Professor, Research direction: Fusarium wilt and Verticillium wilt of cotton,(E-mail)59490371@qq.com; LEI Bin (1973 -), male, Bazhong County, Sichuan, Researcher, Ph. D., Research direction: pesticide research and crop chemical regulation technology, (E-mail)leib668@xaas.ac.cn

Received: 2021-12-3  

Fund supported: The S&T Assisting Xinjiang Project "High-throughput Molecular Identification of Key Genes for Resistance to Stress and Disease in Cotton and Innovation and Utilization of Excellent Germplasm"(2021E02003)
Tianshan Cedar Program Project(2020XS16)

作者简介 About authors

赵莹莹(1997-),女,山东菏泽人,硕士,研究方向为棉花枯萎病生物防治,(E-mail)1208380386@qq.com

摘要

【目的】分析生防细菌与化学杀菌剂复配效果,筛选出有效防治棉花枯萎病的配方组合处理种子,为棉花病害绿色防控提供技术支撑。【方法】以棉花枯萎病主要致病菌尖孢镰刀菌为研究对象,采用室内平板对峙试验筛选拮抗细菌,含毒介质法对化学杀菌剂进行毒力测定,相容性测定筛选出与生防细菌协同使用的化学杀菌剂;评价盆栽试验防病效果。【结果】6种生防细菌对尖孢镰刀菌均有抑制作用,抑制率在56.69%~79.53%,其中枯草芽孢杆菌KXZ-33对其抑制率最好,为79.53%。化学药剂甲基硫菌灵、多菌灵、福美双抑菌效果较好,EC50分别为15.840 1、18.018 0、12.794 0 mg/L。KXZ-33与20 mg/L的甲基硫菌灵的相容性最高为90.46%,活菌数为2.18×107 cfu/mL。KXZ-33与10 mg/L多菌灵在7 d防治效果最高为90.5%,28 d下降为27.0%;KXZ-33与10 mg/L甲基硫菌灵协同防治棉花枯萎病7 d防效为87.7%,28 d防病效果为43.7%。【结论】枯草芽孢杆菌KXZ-33种子处理与喷施50%甲基硫菌灵10 mg/L防治棉花枯萎病可以起到提高防效,弥补生防细菌在农业生产中的防效不稳定的同时可以减少化学农药的使用量。

关键词： 棉花; 枯萎病; 生物防治; 化学防治; 协同防治

Abstract

【Objective】 Through the combination of biocontrol bacteria and chemical fungicides, the formula combination that can effectively control cotton Fusarium wilt was screened out, and the seed treatment was carried out with a view to providing technical support for the green prevention and control of cotton disease. 【Methods】 Fusarium oxysporum, the main pathogen of Fusarium wilt, was selected by indoor plate confrontation test. The virulence of chemical fungicides was tested by the method of toxic media, and the chemical fungicides used in coordination with biocontrol bacteria were screened by compatibility test. The control effect was evaluated by pot experiment. 【Results】 All the six biocontrol bacteria had inhibitory effects on Fusarium oxysporum, with the inhibition rates ranging from 56.69% to 79.53%, and Bacillus subtilis KXZ-33 had the best inhibition rate of 79.53%. The EC50 of Thiobacillam-methyl, Carbendazim and Formetrix were 15.840,1, 18.018,0 and 12.794,0 mg/L, respectively. The maximum compatibility of KXZ-33 with 20 mg/L Thiobacillam-methyl was 90.46%, and the viable bacteria count was 2.18×107 cfu/mL.The control effect of KXZ-33 and 10 mg/L Carbendazim on 7 d was 90.5%, and decreased to 27.0% on 28 d. KXZ-33 combined with 10 mg/L Thiobacillam-methyl was 87.7% for 7 days and 43.7% for 28 days. 【Conclusion】 Bacillus subtilis KXZ-33 seed treatment and spraying 50% methyl Thiobacillam-methyl 10 mg/L to control cotton Fusarium wilt disease can improve the control effect, make up for biocontrol bacteria in agricultural production of unstable control effect and at the same time reduce the use of chemical pesticides, thus further developing service production.

Keywords： cotton; Fusarium wilt; biological control; chemical control; collaborative prevention and control

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本文引用格式

赵莹莹, 张军高, 李进, 梁晶, 高翔宇, 顾爱星, 雷斌. 枯草芽孢杆菌KXZ-33与化学药剂协同防控棉花枯萎病的效果评价[J]. 新疆农业科学, 2022, 59(5): 1216-1222 DOI:10.6048/j.issn.1001-4330.2022.05.021

ZHAO Yingying, ZHANG Jungao, LI Jin, LIANG Jing, GAO Xiangyu, GU Aixing, LIE Bin. Preliminary Study on the Synergistic Effects of Bacillus subtilis KXZ-33 and Chemical Pesticides against Cotton Fusarium wilt[J]. Xinjiang Agricultural Sciences, 2022, 59(5): 1216-1222 DOI:10.6048/j.issn.1001-4330.2022.05.021

0 引言

【研究意义】棉花枯萎病是由尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f.sp.vasinfectum)通过侵染棉花纤维束而造成的系统性病害,是世界性土传性病害[1],在棉花的全生育期均可发生[2-3]。针对棉花枯萎病主要的防治有化学农药、种植抗病品种、与其他作物轮作等措施。化学农药的过度使用会造成一系列的环境问题,[4-5],生物防治是采用真菌、细菌、放线菌,通过调节土壤环境、分泌次生代谢物、竞争作用等拮抗机制达到病害防治的目的[6]。芽孢杆菌属对多种植物病害的发生都有拮抗作用,在自然界中广泛存在,且在不利环境下可以形成稳定的芽孢[7]。【前人研究进展】芽孢杆菌可以在低剂量的化学杀菌剂正常生长,将芽孢杆菌与化学杀菌剂相结合,在防治棉花枯萎病等真菌性病害时可以弥补两者的缺陷,在起到防治效果的同时又可以减少化学农药的使用[8]。此策略在植物保护领域广泛探讨,并取得成效[9]。将枯草芽孢杆菌属、多粘芽孢杆菌属、贝莱斯芽孢杆菌[10]、蜡状芽孢杆菌属、解淀粉芽孢杆菌属[11]、地衣芽孢杆菌[12]、甲基营养性芽孢杆菌[13]等与杀菌剂成功复配。贺惠文等[12]利用地衣芽孢杆菌W10与多菌灵按照100∶1的复配比例下对桃树褐斑病的防治效果有大幅度提升。【本研究切入点】因生防菌受环境、气候、定植能力影响较大,实际大田防治效果并不理想。需开展从棉花枯萎病发病严重病田中,分离出有筛选出拮抗效果的生防细菌。【拟解决的关键问题】以棉花枯萎病主要致病菌尖孢镰刀菌为研究对象,采用室内平板对峙试验筛选拮抗细菌,含毒介质法对化学杀菌剂进行毒力测定,筛选出与生防细菌与化学药剂协同防治棉花枯萎病有稳定防效的组合方式,弥补生防细菌在实际运用中的防效,减少化学农药的使用,减缓植物病害抗病性的增加,为农药减施和绿色生产提供依据。

1 材料与方法

1.1 材料

供试棉花品种为979,新疆金丰源种业股份有限公司提供;供试病原菌为棉花枯萎病萎蔫专化型中等致病力的7号生理小种st89(F.oxysporum),由新疆农业大学植物病理实验室提供。供试枯草芽孢杆菌(Bacillus subtilis)KXZ-33、KXZ-4由新疆农业大学植物病理实验室中保存,莫海威芽孢杆菌(Bacillus mojavensis)Hwt-34、Twt-34,解淀粉芽孢杆菌(Bacillus velezensis)UB7-1和A57由新疆农业学院核技术生物技术研究所保存。供试杀菌剂:50%甲基硫菌灵可湿性粉剂(四川润尔科技有限公司)、80%代森锰锌可湿性粉剂(山东邹平农药有限公司)、80%乙蒜素乳油(寿光禾大壮生物科技有限公司)、50%福美双可湿性粉剂(山东百农思达生物科技有限公司)、30%噁霉灵可湿性乳油(四川润尔科技有限公司)、40%多菌灵悬浮剂(深圳诺普信农化股份有限公司)、43%氟菌肟菌酯乳油(德国拜耳股份公司生产)。均为市售。

1.2 方法

1.2.1 拮抗细菌的筛选与鉴定

将实验室保存的拮抗细菌从4℃冰箱取出,在NA培养基上活化24 h备用。将尖孢镰刀菌st89从-20℃甘油取出,在PDA培养基上活化7 d备用。将活化后的棉花枯萎病病原菌利用打孔器(Φ:6 mm)接到PDA培养基的中心,并将6种拮抗细菌利用牙签(已灭菌)涂到距离病原菌左、右1 cm处,并涂等量蒸馏水做CK,每个处理重复3次,待CK长到满皿时采用平板对峙法[14],测量每株拮抗细菌的抑菌宽度,计算抑菌率[15]。

菌丝生长抑制速率(%)=

(对照菌落直径-处理菌落直径)(对照菌落直径-6mm)×100。

1.2.2 室内化学杀菌剂的毒力测定

选取7种化学药剂利用菌丝抑制生长速率法[16]测定毒力大小。将2 g化学药剂溶解到100 mL无菌水中,配制成药剂含量为20 g/L的母液备用。按照不同比例稀释后,加入经灭菌后、冷却到60℃的PDA培养基中,将50%甲基硫菌灵可湿性粉剂、40%多菌灵悬浮剂、43%氟菌肟菌酯乳油、80%代森锰锌可湿性粉剂、80%乙蒜素乳油、30%噁霉灵可湿性乳油、50%福美双可湿性粉剂制备成6.15、12.5、25、50、100 mg/L(制剂浓度)的含药PDA培养基,加入等量的蒸馏水做空白对照。利用打孔器(Φ:6 mm)将长势良好且生长状态一致的病原菌制成菌饼后,接到不同含药浓度的PDA培养基中,每组试验重复3次。待7 d后观察菌丝生长情况,测定菌丝生长宽度,并计算菌丝生长抑制速率,同上。表1

表 1   棉花根基细菌对尖孢镰刀菌的抑制率

Table 1  The inhibition rate of bacteria from cotton roots against F.oxysporum

处理
Treatment		抑菌率
Inhibition rate(%)
KXZ-3379.53±10.20a
KX-458.66±8.66b
A5768.50±7.39ab
UB17-175.51±9.23a
Twt3471.61±6.17ab
Hwt3456.69±3.02c

注:表中数值为“平均值±标准差”, 不同小写字母表示处理间在 0.05 水平上差异显著(P<0.05)。下同

Note: The values in the table are "average ± standard deviation", Different lowercase letters in the table indicate that there is significant difference between treatments at the level of 0.05 (P< 0.05).The same as below

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1.2.3 KXZ-33与化学药剂的相容性检测
1.2.3.1 种子液制备

将活化好的KXZ-33利用接种环在无菌条件下接入装有NA液体培养基的三角瓶中,30℃、24 h摇床活化,再接入NA液体培养基中30℃、24 h制成种子液备用[17]。

1.2.3.2 相容性测定

将生防细菌KXZ-33种子液接到含有效成分含量为20、50 mg/L甲基硫菌灵、多菌灵、福美双的改良NYBD液体培养基中,30℃、180 r/min、恒温摇床上培养24 h后利用紫外分光光度计测量OD600。将培养液梯度稀释10-6、10-7、10-8进行平板计数法统计活菌数,确定与KXZ-33相容性最好的化学药剂。

1.2.4 KXZ-33与化学药剂协同防治棉花枯萎病盆栽试验

将籽粒饱满均匀的棉种用75%乙醇消毒30 s后晾干,将棉种放入活菌数为1×107 cfu/mL的KXZ-33发酵液中浸种30 min,蒸馏水浸种30 min作为CK。将棉种播种到装有营养土中,花盆长×宽×高为120 mm×120 mm×100 mm,每盆12粒。用待棉花长至2片真叶时进行利用伤根接菌法进行接菌,接菌3 d后喷施化学杀菌剂。试验共设置6个处理,每组4个重复,其中A:KXZ-33浸种;B:喷施30 mg/L的甲基硫菌灵10 mL;C: KXZ-33浸种+喷施15 mg/L甲基硫菌灵10 mL;D:喷施20 mg/L多菌灵10 mL;E:KXZ-33浸种+喷施10 mg/L的多菌灵10 mL;F:CK。分别在药剂处理后7、14 、21 、28 d分别调查棉花的病情指数,并计算防病效果。苗期棉花病情分级标准参照石磊岩5级分类标准进行调查[18]。

病情指数=Σ(各级病株数×对应病级值)调查总株数×最高级值;

防治效果(%)=

空白对照病株率(病情指数)-处理病株率(病情指数)空白对照病株率(病情指数)×100。

1.3 数据处理

用Microsoft Excel 2010进行数据计算,SPSS 25.0软件进行方差分析(P<0.05),Duncan法进行多重比较检验,GraphPad Prism 7.0进行绘图。

2 结果与分析

2.1 棉花根基细菌对尖孢镰刀菌的抑制率

研究表明,3株对棉花枯萎病病原菌的抑制率达到70.00%以上,其中KXZ-33菌株对尖刀镰孢菌的抑菌率最高为79.53%,Hwt34的抑菌率最低为56.69%。UB17-1对棉花枯萎病的抑菌率可达到75.51%,Twt34的抑菌率为71.65%。KXZ-33、UB17-1和Twt343种生防细菌对棉花枯萎病的防治均有较大的生防潜力。表1

2.2 室内化学杀菌剂的毒力测定

研究表明,50%甲基硫菌灵可湿性粉剂、40%多菌灵悬浮剂、43%氟菌肟菌酯乳油、80%代森锰锌可湿性粉剂、80%乙蒜素乳油、80%噁霉灵乳油、50%福美双可湿性粉剂的EC50分别为15.840 1、18.018 0、188.827 7、1 987.788 6、125.748 0、89.221 3、12.794 0 mg/L。其中50%福美双可湿性粉剂对病原菌的毒力最高,50%甲基硫菌灵可湿性粉剂、40%多菌灵悬浮剂次之,且三者EC50值都小于20 mg/L。40%多菌灵悬浮剂的灵敏度最高。毒力较差的分为是80%乙蒜素乳油、80%代森锰锌可湿性粉剂,其中80%代森锰锌可湿性粉剂最差为1 987.788 6 mg/L。表2

表 2   化学杀菌剂对尖刀镰孢菌的室内毒力测定

Table 2  Laboratory toxicity test of chemical pesticides to F.oxysporum

化学杀菌剂
Fungicide		回归方程
Regression equation		EC50
(mg/L)		R2
Correlation
coefficient
50%甲基硫菌灵可湿性粉剂
Thiophanate-methyl		y=0.609 8x+3.315 415.840 10.997 9
40%多菌灵悬浮剂
Carbendazim		y=1.221 6x+1.467 918.018 00.995 1
43%氟菌肟菌酯乳油
Fluomycetin		y=0.177 3x+4.070 8188.827 70.951 2
80%代森锰锌可湿性粉剂
Mancozeb		y=0.258 2x+3.040 21 987.788 60.972 8
80%乙蒜素乳油
Ethylicin		y=0.668 6x+1.767 8125.748 00.969 2
30%噁霉灵可湿性粉剂
Hymexazo		y=1.047 4x+0.533 089.221 30.997 3
50%福美双可湿性粉剂
Formetrix		y=0.550 3+3.597 312.794 00.978 6

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2.3 KXZ-33与化学药剂的相容性

研究表明,在含20 mg/L KXZ-33在含有不同浓度的化学杀菌剂NYBD液体培养基中培养24 h后,CK活菌数为2.41×107 cfu/mL,含有20 mg/L的甲基硫菌灵的改良NYBD液体培养基中的活菌数为2.18×107 cfu/mL的相容性最佳,与生防细菌的相容性达到90.46%。含有50 mg/L的甲基硫菌灵改良NYBD液体培养基中的活菌数为1.76×107 cfu/mL,KXZ-33的相容性为73.03%,与20 mg/L的甲基硫菌灵相比下降了17.43%。20 mg/L的多菌灵活菌数为1.45×107 cfu/mL,与KXZ-33的相容性为60.17%。KXZ-33与福美双的相容性最低,50 mg/L福美双下无法正常生长。图1

图1

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图1   KXZ-33与化学药剂的相容性

Fig.1   Compatibility analysis of KXZ-33 with chemical agents


2.4 化学杀菌剂与KXZ-33协同防控棉花枯萎病效果

研究表明,KXZ-33在与化学杀菌剂协同使用时,50%甲基硫菌灵可湿性粉剂与40%多菌灵悬浮剂在防效上表现出较大的差异。施药7 d时,E:组KXZ-33浸种+10 mg/L多菌灵的防治效果最好为90.5%,A组:KXZ-33浸种的防效为72.4%,C组:KXZ-33浸种+10 mg/L甲基硫菌灵的防治效果为87.7%。施药后14 d防治效果均有所下降,E组:KXZ-33浸种+10 mg/L多菌灵下降最为明显,C组:KXZ-33浸种+10 mg/L甲基硫菌灵防效最高为57.7%。E防效持续下降为35.2%,且与D组防效相当。施药后21 d,C组:KXZ-33浸种+10 mg/L甲基硫菌灵的防效最高为56.3%,优于单独使用化学药剂、KXZ-33浸种的试验组。施药28 d时C组的防效最高为43.7%,相较于其他试验组稳定性最强,E组防治效果为27.0%。KXZ-33与化学杀菌剂进行协同防治的过程中虽然整体防效表现为下降趋势,但10 mg/L甲基硫菌灵与生防菌KXZ-33的协同防治效果优于10 mg/L多菌灵与生防菌KXZ-33的防治效果。在28 d时棉花枯萎病的防病效果KXZ-33浸种+10 mg/L甲基硫菌灵>20 mg/L甲基硫菌灵>KXZ-33浸种+10 mg/L多菌灵>KXZ-33浸种>20 mg/L多菌灵。表4

表4   不同化学杀菌剂与KXZ-33协同防控棉花枯萎病的防病效果

Table 4  Synergistic control effect of different chemical fungicides and KXZ-33 on Fusarium wilt of cotton

处理
Treatment		7 d14 d21 d28 d
病情指数
Condition
index		防病效果
Control
effect(%)		病情指数
Condition
index		防病效果
Control
effect(%)		病情指数
Condition
index		防病效果
Control
effect(%)		病情指数
Condition
index		防病效果
Control
effect
(%)
KXZ-33
107cfu/mL		0.11±0.08c72.4±7.3ab0.36±0.09bc45.2±4.2ab0.46±0.04bcd37.0±4.3ab0.62±0.04b20.2±5.5bc
50%甲基硫菌灵
20 mg/L		0.31±0.07ab21.0±0.1b0.35±0.09bc47.1±8.0ab0.38±0.03cd48.9±4.7a0.50±0.02c35.6±0.3ab
KXZ-33
107cfu/mL+20 mg/L
甲基硫菌灵		0.05±0.04c87.7±0.1a0.28±0.09b57.7±3.7a0.32±0.08d56.3±5.8a0.44±0.06c43.7±8.0a
20 mg/L多菌灵0.20±0.06bc50.0±0.1ab0.40±0.03bc38.4±5.6ab0.58±0.03ab20.5±3.4b0.64±0.04b17.8±6.4cd
KXZ-33
107 cfu/mL+10 mg/L
多菌灵		0.04±0.03c90.5±0.1a0.42±0.09ab35.2±2.0ab0.56±0.14abc24.3±3.1b0.57±0.123a27.0±4.6bc
CK0.40±0.04a-0.66±0.06a-0.73±0.08a-0.78±0.06a-

注:-表示无次项值

Note: The blanks represent no such item

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3 讨论

刘邮洲等[19]通过利用1×1010 cfu/mL可湿性枯草芽孢杆菌与17%咪鲜胺锰盐进行协同防治番茄枯萎病在田间70 d的防效可以达到79.5%,实现对番茄枯萎病的有效防治。棉花初期使用拮抗菌KXZ-33浸种后的实验组的棉花枯萎病的防效较未使用KXZ-33的实验组防病效果高,但随着时间的延长KXZ-33在土壤的复杂环境中的生防细菌的防效从72.4%逐渐降低到20.2%。

崔凯等[20]探究甲基硫菌灵与其主要代谢产物多菌灵对棉花枯萎病的根基细菌多样性与相对丰富度表明,高剂量的化学农药会降低根际土壤细菌多样性与相对丰富度的降低。KXZ-33与甲基硫菌灵具有很好的相融性,并通过种子处理与喷施相结合方式在室内进行了评价,而种子包衣是一项先进的轻简化种子处理技术,具有高效、低毒、环保等优点,也是农药减施、绿色生产的有效措施。

4 结论

7种化学杀菌剂对病原菌的生长有不同程度影响显示。50%福美双可湿性粉剂对尖孢镰刀菌(F.oxysporum)的毒力最强,50%甲基硫菌灵可湿性粉剂、40%多菌灵悬浮剂次之,80%代森锰锌可湿性粉剂的毒力最差。20 mg/L甲基硫菌灵的OD600与CK差距最小,且较高浓度50 mg/L的甲基硫菌灵也对生防细菌有较高的相容性,20 mg/L多菌灵次之。无论是低浓度20 mg/L福美双,还是高浓度50 mg/L的福美双与生防细菌KXZ-33的相容性都较差。

甲基硫菌灵与KXZ-33的联合使用中可以有效的提高棉花枯萎病的防治效果,在生防细菌随时间推移的防治效果下降的情况下,合理的使用化学杀菌剂可以达到提高的防效作用。

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