渔业科学进展  2023, Vol. 44 Issue (2): 186-195  DOI: 10.19663/j.issn2095-9869.20220426001
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引用本文 

翁佩文, 杨慧超, 李杰, 张文彬, 阎永伟, 莫照兰. 紫菜腐霉拮抗菌的筛选和鉴定[J]. 渔业科学进展, 2023, 44(2): 186-195. DOI: 10.19663/j.issn2095-9869.20220426001.
WENG Peiwen, YANG Huichao, LI Jie, ZHANG Wenbin, YAN Yongwei, MO Zhaolan. Screening and Identification of Antagonistic Bacteria Against Pythium Causing Red Rot Disease in Neopyropia[J]. Progress in Fishery Sciences, 2023, 44(2): 186-195. DOI: 10.19663/j.issn2095-9869.20220426001.

基金项目

财政部和农业农村部：国家现代农业产业技术体系和中央高校基本科研业务费专项(202064006)共同资助

作者简介

翁佩文，E-mail: wengpw912@163.com

通讯作者

莫照兰，研究员，E-mail: mzl@ouc.edu.cn

文章历史

收稿日期：2022-04-26
收修改稿日期：2022-05-10
Contents              Abstract              Full text              Figures/Tables              PDF
紫菜腐霉拮抗菌的筛选和鉴定
翁佩文 1,2, 杨慧超 1,3, 李杰 1, 张文彬 1,3, 阎永伟 1, 莫照兰 1,4     
1. 中国水产科学研究院黄海水产研究所 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 山东 青岛 266071;
2. 中国农业科学院研究生院 北京 100081;
3. 上海海洋大学 水产科学国家级实验教学示范中心 上海 201306;
4. 中国海洋大学三亚海洋研究院 海南省热带水产种质重点实验室 海南 三亚 572000
收稿日期：2022-04-26；收修改稿日期：2022-05-10
基金项目：财政部和农业农村部：国家现代农业产业技术体系和中央高校基本科研业务费专项(202064006)共同资助
作者简介：翁佩文，E-mail: wengpw912@163.com.
通讯作者：莫照兰，研究员，E-mail: mzl@ouc.edu.cn.
摘要：生物防治广泛用于农作物的病害防治，该方法在藻类病害防控方面尚未有相关的报道。腐霉(Pythium sp.)是引起紫菜(Neopyropia)赤腐病(red rot disease)的主要病原，本研究的目的是筛选和鉴定对紫菜腐霉有拮抗能力的细菌。从养殖藻类及其养殖环境中分离鉴定了385株细菌，通过平板对峙法筛选到9株对腐霉有拮抗作用的细菌，进一步通过含毒介质法筛选到3株胞外产物对腐霉具有抑制活性的细菌(P3、P6和P19)。3株拮抗菌对8株腐霉均有拮抗活性，对腐霉生长的抑制率分别为52.09%~97.95% (P3)、26.81%~78.04% (P6)、10.47%~41.91% (P19)。通过16S rRNA鉴定和多位点序列进化分析(16S rRNA-dnaA-dnaN-recA)，将P3和P6鉴定为杀鱼假交替单胞菌(Pseudoalteromonas piscicida)，P19鉴定为解肽假交替单胞菌(Pseudoalteromonas peptidolytica)。本研究筛选得到的拮抗菌为下一步建立紫菜赤腐病的生物防治方法奠定了基础。
关键词：腐霉    拮抗菌    假交替单胞菌    生物防治    
Screening and Identification of Antagonistic Bacteria Against Pythium Causing Red Rot Disease in Neopyropia
WENG Peiwen 1,2, YANG Huichao 1,3, LI Jie 1, ZHANG Wenbin 1,3, YAN Yongwei 1, MO Zhaolan 1,4     
1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao); Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao, Shandong 266071, China;
2. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China;
3. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;
4. Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanology Institute, Ocean University of China, Sanya, Hainan 572000, China
Corresponding author: MO Zhaolan, E-mail: mzl@ouc.edu.cn.
Abstract: China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis.
Key words: Pythium    Antagonistic bacteria    Pseudoalteromonas    Biocontrol    

紫菜(Neopyropia)是一类生长于潮间带的大型经济红藻，广泛分布于寒带至热带海域，具有较高的经济价值及生态价值(章守宇等, 2019; 江涛等, 2021)。我国是紫菜生产大国，紫菜的栽培规模及产量均居世界之首，年产值约13亿美元(http://www.fao.org/fishery/statistics/global-aquaculture production/zh)。然而，由细菌、卵菌和病毒等引起的病害频繁发生，给养殖户带来了巨大的经济损失(Arasaki, 1947; Migita, 1969; Saito et al, 1972; Kim et al, 2016)。其中，由卵菌病原腐霉(Pythium sp.)引起的赤腐病(red rot disease)是紫菜栽培期最主要的病害，常导致紫菜病烂、空帘而绝收(Arasaki, 1947; Takahashi, 1970; Lee et al, 2015)。目前，生产上经常采用干出、冷藏、酸洗的方法来应对紫菜赤腐病(Fujita et al, 1980; Sakaguch et al, 2001; Akizuki et al, 2007)。将患病紫菜干燥至含水量为30%~40%并在–20℃冷藏1~2周，可使紫菜腐霉菌丝大量死亡，但紫菜腐霉的卵孢子对干燥及冷藏具有较好的耐受性，存活率为10%~30% (Fujita et al, 1980)。用pH 2.0~5.0的无机酸或有机酸处理患病紫菜，在腐霉孢子浓度较低的情况下可有效抑制赤腐病，但在腐霉孢子浓度较高时处理效果不明显(Sakaguchi et al, 2001; Akizuku et al, 2007)。此外，这些物理或化学的处理方法还存在一系列问题，如冷藏设备及场地会大幅度提高栽培成本；酸性杀菌剂不仅污染栽培海区，长期使用还会提高腐霉的耐酸性而影响防治效果(Hwang et al, 2009)。多年来，研究人员一直尝试选育或培育紫菜抗病品系，到目前为止还未得到对赤腐病完全免疫的品系(Polne-Fuller et al, 1984; Park et al, 2014、2015)。

在陆地农作物生产中利用有益的拮抗微生物或其代谢产物进行病害防控是一种绿色、安全、有效的方法(王丁等, 2018)，这为经济海藻的病害防控提供了新思路。藻类及其生长环境存在多种多样的微生物，且处在动态变化中，这些微生物分泌的多种活性物质具有抗菌、抗病毒、抗寄生虫等活性(Singh et al, 2014、2015; 阎永伟等, 2022)，是筛选生防微生物的良好来源。本研究的目的是从海藻及其生长环境中筛选和鉴定出对紫菜腐霉具有拮抗作用的细菌，为建立紫菜赤腐病的生物防治奠定基础。

1 材料与方法 1.1 供试菌株及培养基

紫菜赤腐病病原为腐霉Pythium porphyrae和Pyt. chondricola。本研究所用的Pyt. porphyrae菌株(NBRC 30800、NBRC 33126、NBRC 100633和NBRC 33253)购自日本生物资源库(NITE Biological Resource Center, Department of Biotechnology, National Institute of Technology and Evaluation, Japan)，Pyt. chondricola菌株(JS151205、PYTHT201801-1、RZ201902和LS201903)由本实验室分离自患赤腐病紫菜(邱丽萍, 2018; 何礼娟, 2020)。上述菌株保藏于中国水产科学研究院黄海水产研究所养殖生物病害控制与分子病理学研究室，在4℃传代保藏。培养紫菜腐霉的培养基为海水玉米培养基(SCM) (玉米粉2 g/L、酵母粉1 g/L、琼脂15 g/L、盐度为20的海水、pH调为7.0~7.5)，常规培养温度为24℃。培养细菌的培养基为2216E海水培养基(MA) (蛋白胨5 g/L、酵母膏1 g/L、FePO4 0.01 g/L、琼脂15 g/L、陈海水)，常规培养温度28℃，震荡条件为150 r/min。

1.2 细菌的分离和纯化

采集紫菜的苗期贝壳丝状体、海区栽培期的叶状体、海带(Saccharina japonica)苗及相应的栽培水体进行细菌的分离。采集的样品放入无菌采样袋(海博生物)及水样采集袋(海博生物)带回实验室处理。紫菜贝壳丝状体用灭菌海水清洗表面3次，用无菌刮刀刮取约1 cm2贝壳丝状体；紫菜叶状体及海带苗用无菌刷子清除表面附着的杂质，再用灭菌海水清洗3次。所得的藻类样品用无菌研磨棒分别匀浆，用灭菌海水分别将匀浆液进行梯度稀释，取稀释度为10–2、10–3、10–4的稀释液100 μL涂布于MA上。水样也分别稀释至10–1、10–2和10–3，取100 μL涂布于MA上。将所有平板置于28℃培养2 d，挑取形态不同的单菌落进行纯化，–80℃保存在30%甘油里。

1.3 拮抗菌初筛

用平板对峙法进行拮抗菌的初筛(Farhaoui et al, 2022)。用接种环挑取细菌菌株在MA上划线，在28℃培养活化2 d备用。供试紫菜腐霉菌株NBRC 33253接种在SCM上，在24℃培养7 d至长满平板后，用打孔器沿边缘菌丝将紫菜腐霉打成5 mm的圆形菌饼，将其接种在新的SCM平板中央，于24℃培养1 d后，以紫菜腐霉菌饼为中心画“十”字作为定位线，挑取细菌菌落在距离紫菜腐霉菌饼中心2 cm、垂直于定位线画1 cm短线，每平板可划4条短线，即接种4株菌株，对照组不接种细菌。将平板置于24℃培养，为高效筛得目标菌株，本实验选用腐霉最适生长温度24℃(何礼娟, 2020)，待对照组紫菜腐霉直径长至约为4 cm时，观察接种细菌的周围是否出现抑菌圈。选出对紫菜腐霉有拮抗效果的菌株重复上述实验，在同一平板上接种相同菌株，每组3个平行，用游标卡尺量取定位线上紫菜腐霉边缘与细菌之间的距离。对具有拮抗能力的菌株重复上述筛选2次。

1.4 拮抗菌复筛

用含毒介质法进行拮抗菌的复筛(何碧珀等, 2019)。将初筛得到的细菌单菌落接种到100 mL MA液体培养基中，在28℃、150 r/min条件下震荡培养72 h，取30 mL菌液于50 mL离心管中5000 r/min离心10 min，取上清液用0.22 μm膜过滤获得无菌滤液。将灭菌SCM冷却至40℃左右，按无菌滤液∶SCM= 1∶5的比例充分混匀后制备含无菌滤液平板。将紫菜腐霉NBRC 33253制成5 mm菌饼接种在含拮抗菌无菌滤液的平板中央，作为实验组；根据初筛结果选择1株无拮抗效果的菌株制备无菌滤液，用上述方法制备平板，接种紫菜腐霉菌饼作为阴性对照组；在不含无菌滤液的SCM平板上接种紫菜腐霉菌饼作为空白对照组。每组平板设3个平行。将平板置于24℃培养，至空白对照组紫菜腐霉长满平板(约7 d)，用菌落分析仪(Synbiosis Protocol 2, 英国)拍摄平板照片，然后用显微镜(OLYMPUS BX53, 日本)的自带软件AJ-VERT的“任意多边形”工具圈取腐霉生长面积，计算抑菌率。重复上述实验2次。

$ \text { 计算公式：抑菌率 }=\left(A_{\mathrm{b}}-A_{\mathrm{e}}\right) /\left(A_{\mathrm{b}}-A_{\mathrm{i}}\right) \times 100 \% $

式中，Ab为空白对照组的腐霉面积，Ae为实验组的腐霉面积，Ai为菌饼面积。

1.5 拮抗谱的测定

用平板对峙法测定3株拮抗菌(P3、P6和P19)对8株腐霉的拮抗谱及拮抗能力。具体步骤为：挑取单菌落接种到MA液体培养基培养12 h，按10%的接种量转接至新鲜的MA液体培养基继续培养1~2 h，将细菌培养液5000 r/min离心10 min去除上清液，用灭菌海水悬浮细菌，重复离心、悬浮步骤3次，最后制成108 CFU/mL的菌悬液备用。将5 mm紫菜腐霉菌饼接种在SCM中央，1 d后按1.3中的定位方法，在距离菌饼2 cm处，点种菌悬液2 μL作为实验组，点种2 μL灭菌海水作为空白对照组。每组设3个平行，将所有平板置于15℃。为筛得具有防治赤腐病潜力的菌株，本实验选用温度为实验室腐霉侵染紫菜温度15℃，待空白组腐霉长满平板(10~15 d)，计算抑制率，统计方法同1.4。重复上述实验2次。

用打孔器将抑菌圈边缘的腐霉菌丝打成5 mm直径的菌饼，用乳酸酚棉蓝染液(G1600, Solarbio)对腐霉菌饼进行染色，染色完成后用灭菌海水洗去菌饼表面残余的染液后制片，以空白对照组腐霉边缘菌丝为对照，用显微镜观察抑菌圈边缘菌丝的形态变化。

1.6 拮抗细菌的鉴定

对细菌进行菌落形态观察、革兰氏染色(G1060, Solarbio)。用通用引物27F/1492R扩增拮抗菌的16S rRNA基因进行细菌鉴定(杨慧超, 2019)。对假交替单胞菌属细菌(Pseudoalteromonas sp.)进一步扩增管家基因dnaA、dnaN、recA基因序列，进行多位点序列分析(multilocus sequence analysis, MLSA)。根据GenBank中假交替单胞菌的dnaA、dnaN、recA序列，利用BioEdit软件(Alzohairy, 2011)，识别序列中的高保守区域，设计引物。dnaA的引物序列为PdnaAF (5´-TTTAGCATGTGGGTAAGACC-3´)和PdnaAR (5´-TCTTGGTCTAGCAACT­GAAC-3´)，dnaN为PdnaNF (5´-TGCAAATAACG­­ATTC­CAAGAG-3´)和PdnaNR (T TTGCATCAGAAAGCGTAAA)，recA为recA-F/recA-R (Beurmann et al, 2017)。利用MEGA-X软件将16S rRNA (1499 bp)、dnaA (1146 bp)、dnaN (955 bp)和recA (811 bp)基因序列按顺序连接，并进行比对，构建N-J系统发育树。

1.7 数据分析

利用Excel和SPSS软件对实验数据进行统计分析，实验结果取平均值±标准差$ (\bar x \pm SD,\;n = 3) $，利用Duncan检验法进行多重比较，显著性为P < 0.05。

2 结果 2.1 细菌的分离及拮抗菌筛选

从紫菜的贝壳丝状体、叶状体、海带及栽培水体中分离到385株细菌用于筛选拮抗菌。用平板对峙法进行初筛时，得到9株对腐霉NBRC 33253具有拮抗作用的菌株(图 1)。2次重复实验显示，拮抗菌的抑菌带宽度在1.65~16.54 mm之间(表 1)。

图 1 对峙生长法初筛腐霉拮抗菌 Fig.1 Preliminary screening of Pyt. porphyrae antagonistic bacteria by dual culture bioassays A：空白对照；B~J：P3(B)、P4(C)、P5(D)、P6(E)、P7(F)、P8(G)、P12(H)、P19(I)、B1(J)与腐霉的对峙生长 A: Blank control; B~J: Antagonistic growth of P3(B), P4(C), P5(D), P6(E), P7(F), P8(G), P12(H), P19(I), and B1(J) against Pyt. porphyrae
表 1 拮抗菌的筛选 Tab.1 Screening of antagonistic bacteria

用含毒介质法对这9株细菌进行复筛时，有3株菌株(P3、P6和P19)的无菌滤液对腐霉的生长产生了显著的抑制(图 2)。2次重复实验显示，这3株细菌的抑菌率为20.04%~30.09% (表 1)。根据这些结果，选取P3、P6和P19进行下一步实验。

图 2 含毒介质法复筛拮抗菌 Fig.2 Secondary screening of antagonistic bacteria by toxic media method A：空白对照；B：阴性对照组；C~E：腐霉在含有P3(C)、P6(D)、P19(E)无菌滤液的培养基上生长 A: Blank control; B: Negative control; C~E: Pyt. porphyrae grew on the medium containing extracellular products from P3(C), P6(D) and P19(E)
2.2 拮抗谱

用平板对峙法检测了P3、P6和P19对8株腐霉的拮抗谱和拮抗能力。2次重复实验结果显示，15℃条件下，3株拮抗菌对8株腐霉均具有拮抗作用(图 3)，其中，P3的拮抗能力最强，抑菌率达到(52.09%~ 97.95%)，其次为P6 (26.81%~78.04%)和P19 (10.47%~ 41.91%) (表 2)。用乳酸棉酚蓝对对峙边缘的腐霉菌丝进行染色观察，结果显示，与对照组的腐霉菌丝相比，与拮抗菌P3和P19对峙的腐霉菌丝密度变稀疏、着色变浅，而与拮抗菌P6对峙的腐霉菌丝无明显变化(图 4)。

图 3 8株腐霉与3株拮抗菌的对峙生长图 Fig.3 Confrontation growth between Pythium and antagonistic bacteria 00、26、33、53、JS、HT、LS、RZ分别为腐霉NBRC 30800、NBRC 33126、NBRC 100633、NBRC 33253、JS151205、PYTHT201801-1、LS201903、RZ201902；00-P3、26-P3、33-P3、53-P3、JS-P3、HT-P3、LS-P3、RZ-P3分别为P3与腐霉NBRC 30800、NBRC 33126、NBRC 100633、NBRC 33253、JS151205、PYTHT201801-1、LS201903、RZ201902的对峙生长；00-P6、26-P6、33-P6、53-P6、JS-P6、HT-P6、LS-P6、RZ-P6分别为P6与腐霉NBRC 30800、NBRC 33126、NBRC 100633、NBRC 33253、JS151205、PYTHT201801-1、LS201903、RZ201902的对峙生长；00-P19、26-P19、33-P19、53-P19、JS-P19、HT-P19、LS-P19、RZ-P19分别为P19与腐霉NBRC 30800、NBRC 33126、NBRC 100633、NBRC 33253、JS151205、PYTHT201801-1、LS201903、RZ201902的对峙生长。 00, 26, 33, 53, JS, HT, LS, RZ represent Pythium NBRC 30800, NBRC 33126, NBRC 100633, NBRC 33253, JS151205, PYTHT201801-1, LS201903, RZ201902, respectively; 00-P3, 26-P3, 33-P3, 53-P3, JS-P3, HT-P3, LS-P3, RZ-P3 represent antagonistic growth of P3 against Pythium NBRC 30800, NBRC 33126, NBRC 100633, NBRC 33253, JS151205, PYTHT201801-1, LS201903, RZ201902, respectively; 00-P6, 26-P6, 33-P6, 53-P6, JS-P6, HT-P6, LS-P6, RZ-P6 represent antagonistic growth of P6 against Pythium NBRC 30800, NBRC 33126, NBRC 100633, NBRC 33253, JS151205, PYTHT201801-1, LS201903, RZ201902, respectively; 00-P19, 26-P19, 33-P19, 53-P19, JS-P19, HT-P19, LS-P19, RZ-P19 represent antagonistic growth of P19 against Pythium NBRC 30800, NBRC 33126, NBRC 100633, NBRC 33253, JS151205, PYTHT201801-1, LS201903, RZ201902, respectively.
表 2 拮抗菌对腐霉生长的抑菌率 Tab.2 Growth inhibition rate of antagonistic bacteria against Pythium strains
图 4 拮抗菌和腐霉对峙区域的腐霉菌丝显微观察(乳棉酚蓝染色) Fig.4 Microscopic observation of the Pythium mycelia growing in the confrontation area between Pythium and antagonistic bacteria (lactophenol cotton blue staining)
2.3 拮抗菌的鉴定

菌株P3、P6和P19革兰氏染色均为阴性，其中，P6产黄色色素，P19产棕色色素，P3无色素产生。16S rRNA基因序列分析结果显示，3株拮抗菌均与假交替单胞菌的相似性最高，P3和P6为99.93%，P19为99.86%。将P3、P6和P19的4个基因串联(16S rRNA-dnaA-dnaN-recA)构建多位点序列的系统进化树，分析结果显示，P3和P6与杀鱼假交替单胞菌(Pseudoalteromonas piscicida)聚为一支，P19与解肽假交替单胞菌(Pseudoalteromonas peptidolytica)聚为一支(图 5)。

图 5 基于拮抗菌P3、P6和P19的16S rRNA-dnaA-dnaN-recA构建的N-J系统发育树 Fig.5 N-J phylogenetic tree based on 16S rRNA-dnaA-dnaN-recA of P3, P6, and P19
3 讨论

大型海藻在生长发育过程中会产生多种代谢物质到藻体表面，为微生物定植提供了适宜的基质和营养(Steinberg et al, 2002; Staufenberger et al, 2008; Singh et al, 2013)。附着在海藻表面的微生物群落可产生多种活性物质，这些物质不仅影响海藻的正常形态及生长发育，还可保护宿主免受有害生物的侵害。因此，大型海藻的附着微生物成为生物和医药领域的优选目标(Singh et al, 2014、2015)。本研究从紫菜和海带及其栽培水体分离细菌，通过平板对峙法筛选出9株具有抑制腐霉生长活性的细菌，并通过含毒介质法检测出3株细菌(P3、P6和P19)的胞外产物能够抑制腐霉的生长，表明这3株细菌通过拮抗作用抑制了腐霉的生长。其他6株细菌的胞外产物未检测到抑制腐霉生长的情况，说明这6株细菌通过竞争作用抑制了腐霉的生长。

通过16S rRNA鉴定及多位点序列分析，将P3和P6鉴定为杀鱼假交替单胞菌，将P19鉴定为解肽假交替单胞菌。多个研究表明，从海洋环境分离的假交替单胞菌能分泌具有抗菌、防污、杀藻等作用的活性物质，包括抗生素、胞外酶类物质和胞外毒素等(Holmstrom et al, 1999; Egan et al, 2001; Bowman, 2007; 孙星等, 2018; 练小军等, 2020)。大多数的研究报道了抗细菌活性物质的分离和鉴定，而对抗真菌、抗卵菌的活性物质的报道不多。Moree等(2014)报道了从健康珊瑚分离的一株假交替单胞菌产生的一种酰胺聚酮具有抗真菌活性；Franks等(2005)报道了一株P. tunicata产生的黄色色素具有抗真菌活性，并鉴定为生物碱。分离自海洋环境的一些杀鱼假交替单胞菌菌株被证实能够有效拮抗多种病原菌，包括创伤弧菌(Vibrio vulnificus)、副溶血弧菌(V. parahaemolyticus)、美人鱼发光杆菌(Photobacterium damselae)、金黄色葡萄球菌(Staphylococcus aureus)、白色念珠菌(Candida albicans)及枯草芽孢杆菌(Bacillus subtilis)等(Richards et al, 2017; Eliseikina et al, 2021)；一些解肽假交替单胞菌具有淀粉酶、几丁质酶、木质纤维素酶活性(Venkateswaran et al, 2000; Johnson et al, 2021)。但这些分离株产生的抑菌物质尚不清楚。在本研究中，拮抗菌P3和P19与腐霉相互作用区域的腐霉菌丝出现乳酸棉酚蓝着色浅的情况，而与P6相互作用区域的菌丝无明显变化，说明3株细菌可能通过不同方式抑制腐霉生长。当阳性菌株溶解或死亡时，乳酸酚棉蓝染色常呈阴性，说明P3和P19分泌的活性物质有可能降解了腐霉菌丝或影响菌丝活性，导致着色变浅。

在水产养殖中，假交替单胞菌作为益生菌在防治养殖动物的病害中显示了良好的效果(Fjellheim et al, 2010; Goulden et al, 2012; Offret et al, 2019)。本研究筛选得到的拮抗菌P3、P6和P19，对引起紫菜赤腐病的致病性腐霉展示了显著的拮抗能力，显示了其作为益生菌用于防治紫菜赤腐病的生防潜力。在后续的研究中，我们将开展拮抗菌的安全性评价及其在预防和治疗紫菜赤腐病中的生防效果和应用，在此基础上探究拮抗菌的活性物质的鉴定及其生防机制。

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