作物病害作为制约农业生产的重要因素，严重威胁了全球农业和食品安全。传统的化学防治方法虽可在一定程度上控制病害的发生，但是也存在环境污染、农药残留和破坏生态等问题，故探索可持续、环保的作物病害防治技术[1]。生物防治是指利用有益生物及其代谢产物进行植物病害防治，与化学防治相比，其绿色安全，不易产生抗性，选择性强。因此，利用生防微生物的防治方式逐渐引起人们的关注，国内外已有大量的相关研究，发展前景广阔[2]。防御假单胞菌（Pseudomonas protegens）是植物根际促生微生物的主要类群之一，其营养需求简单、增殖速度快、根际定殖力强，不但能产生多种抑菌活性物质，同时还能分泌促进作物生长的代谢物质。除此之外，与其他假单胞菌相比，防御假单胞菌可分泌一些杀虫毒素，其生物防治效果更加突出[3-4]。
硒作为“生命元素”，是生物体生长所必需的，在提高机体免疫力、调节代谢、抗氧化、解毒、促进生殖等方面均有重要的生理功能[5⇓-7]。硒在地球上的分布并不均匀，中国约72%地区处于低硒、缺硒带，日常饮食过程中硒摄入量的不足严重影响着几亿人口的身体健康[8]。目前，随着人们保健和养生意识的增强，各种富硒产品相继出现，一些农户为增加卖点，通过在种植过程中施用无机硒（亚硒酸钠、硒酸钠）来提高产品中硒含量。但是无机硒的毒性大，被《常用危险化学品的分类及标志：GB 13690-2009》将其列为第六类剧毒物质，无机硒的使用严重影响人类和环境健康[9]，而纳米硒毒性低且活性高，有望成为最佳补硒形态。微生物转化合成纳米硒条件温和，分散性好，结构稳定，应用前景良好[10]。
高效安全的微生物制剂一直是作物病害防治研究的重点，但目前少有多重防效且使作物富硒的研究成果，更缺乏推广应用。本试验将从生物技术方面进行探索，筛选出使作物富硒且防效优良的微生物制剂。
1 材料与方法
1.1 试验材料
供试生防菌株为防御假单胞菌KBD-3，该菌种从云南保山高发病烟区采集的健康烟草植株根际土壤中分离得到，现保存于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.26145，保藏日期为2022年11月14日。供试材料为烟草（K326和枯斑三生烟）、绿豆、青枯雷尔氏菌（Ralstonia solanacearum）和烟草花叶病毒（TMV）毒源，均由中国农业科学院烟草研究所植物保护研究中心保存提供。供试培养基为LB液体培养基、LB固体培养基、NB液体培养基和NA固体培养基。供试试剂为亚硒酸钠（上海泰锐尔生物科技有限公司提供）和农用硫酸链霉素72%可溶性粉剂（石家庄曙光制药厂提供）。
1.2 试验方法
1.2.1 对TMV抑制效果的测定
挑选生长良好的单一KBD-3菌落接种于LB液体培养基中，在37 ℃、150转/min条件下振荡培养20 h，得到KBD-3发酵菌液，将其用无菌水稀释成1×108 cfu/mL菌液备用。
试验采用半叶法。取KBD-3菌液（浓度108 cfu/mL）、LB液体培养基分别与等体积TMV汁液（将带TMV的烟草叶片冷冻研磨成粉末，按1:40加去离子水，纱布过滤）混合15 min后摩擦接种，接种前接种叶片喷洒少量石英砂。左半片叶接种KBD-3菌液与TMV汁液的混合液，右半片叶接种LB液体培养基与TMV汁液的混合液（空白对照，CK），每半片叶接种量为200 μL。接种后对叶面进行冲洗，接种3株枯斑三生烟，每株接种上部2片叶，重复3次，3~5 d调查枯斑数并计算抑制率。
抑制率（%）=[1-（处理平均枯斑数/对照平均枯斑数）]×100。
1.2.2 对青枯雷尔氏菌抑制效果的测定
在NA培养基正中心滴加20 μL防御假单胞菌KBD-3菌液（浓度108 cfu/mL），待菌液被培养基充分吸收后，倒置于28 ℃培养箱中培养48 h，然后用喷壶喷洒青枯雷尔氏菌重悬液（OD600=0.3），倒置于28 ℃培养箱中进行培养。重复3次，设置空白对照（NA培养基中仅喷洒同等浓度的青枯雷尔氏菌重悬液），48 h后观察结果，并测量抑菌圈直径。
1.2.3 对烟草青枯病防治效果的测定
在育苗盘中播撒K326种子，待烟苗伸展4~5片叶时移栽到装有无菌土的花盆中，缓苗后用KBD-3菌液（浓度为108 cfu/mL）对烟苗进行第1次灌根处理，5 d后进行再次灌根，每次灌根10 mL/株，第2次灌根2 d后，将青枯雷尔氏菌菌液（OD600=0.3）接种于烟苗根际土壤中，用量10 mL/株。同时设置对照组，以施用农用硫酸链霉素72%可溶性粉剂（6470倍液）作为阳性对照（CK1），2次灌根间隔5 d，每次灌根10 mL/株，其他处理均与试验组相同；以只接种10 mL/株青枯雷尔氏杆菌菌液作为发病对照（CK2）；以只浇清水作为空白对照（CK3）。每组处理5株，3次重复。接种青枯雷尔氏杆菌7 d后，调查各烟苗的发病率（%）、病情指数及相对防治效果（%）。病情分级标准[11]如下：
0级：全株无病；1级：茎部部分出现褪绿斑，或发病一侧1/2以下叶片凋萎；3级：茎部出现黑色条斑，但不超过茎高1/2，或发病一侧1/2至2/3叶片凋萎；5级：茎部黑色条斑超过茎高1/2，但未达到茎顶部，或发病一侧2/3以上叶片凋萎；7级：茎部黑色条斑到达茎顶部，或病株叶片全部凋萎；9级：病株基本枯死。
发病率（%）=（发病株数/调查总株数）×100；病情指数=（∑各级病株数×该病级值）/（调查总株数×最高级值）×100；控制效果（%）=[（对照区病情指数-处理区病情指数）/对照区病情指数]×100。
1.2.4 对无机硒还原作用的测定
将活性菌株KBD-3接种到LB固体培养基上进行纯化，挑取生长良好的单一菌落接种于LB液体培养基中，37 ℃、150转/min振荡培养20 h，后以1%（v/v）接种量加入到含1 mmol/L亚硒酸钠的LB液体培养基中，30 ℃、150转/min振荡培养，2~5 d后观察菌液颜色，待出现红色后用扫描电镜观察[12]。
1.2.5 纳米硒的制备
按1%接种量将防御假单胞菌KBD-3菌液接种至含1 mmol/L亚硒酸钠的LB液体培养基中，在37 ℃、150转/min条件下振荡培养2~5 d，获得纳米硒活性菌液。将纳米硒活性菌液在13 000 g条件下离心10 min，沉淀物为纳米硒和菌体的混合物。向沉淀物中加入溶菌酶，使其终浓度为20 mg/mL，37 ℃孵育20 min，孵育期间间隔5 min取出倒置几次混匀。然后置于冰上超声30 min（超声30 s，停40 s）。将超声后的溶液在6000转/min条件下离心10 min，去沉淀收集上清液。将上清液在13 000 g条件下离心10 min，弃上清液，沉淀用超纯水溶解洗涤2次后获得纳米硒[12]。
1.2.6 富硒豆芽的培育
挑选饱满、无蛀虫霉烂的绿豆作为供试种豆，于50 ℃热水中浸泡30 min后沥干水分转移到盛有30 mL 15 μg/mL纳米硒溶液的烧杯中，室温下浸泡过夜。后取出沥干放置于铺有4层纱布的育苗托盘中，上部覆盖4层湿纱布，每12 h喷淋1次10 mL 15 μg/mL纳米硒溶液，同时设空白对照试验（CK）[13]。4 d后，分别取各处理培养的豆芽样品，去掉种皮，用去离子水反复漂洗后置于60 ℃烘箱中烘干（约6 h），粉碎过60目筛，后采用氢化物原子荧光分光光度计测定样品中的硒含量。
2 结果与分析
2.1 KBD-3对TMV的抑制效果
图1
图1
KBD-3菌液对TMV的钝化作用
左半叶：KBD-3发酵菌液；右半叶：空白对照。
Fig.1
Passivation of KBD-3 bacterial solution on TMV
Left half: KBD-3 bacterial solution; Right half: CK.
表1 KBD-3菌液对TMV的抑制作用
Table 1
| 处理 Treatment | 枯斑数 Number of local necrotic lesions | 抑制率 Inhibition rate (%) |
|---|---|---|
| KBD-3 | 3.33±0.04b | 95.77±1.05 |
| CK | 78.67±0.72a | － |
不同小写字母表示差异显著（P < 0.05）。下同。
Different lowercase letters indicate significant differences (P < 0.05). The same below.
2.2 KBD-3对青枯雷尔氏菌的抑制效果
待喷洒青枯雷尔氏菌重悬液后倒置于28 ℃培养箱中进行培养，48 h后进行观察测量，结果如图2所示，菌液对青枯雷尔氏菌的抑制效果较好，抑菌圈直径达6.23 cm。
图2
图2
KBD-3菌液对烟草青枯病菌的拮抗活性
左：KBD-3菌液；右：空白对照。
Fig.2
Antagonistic activity of KBD-3 bacterial solution against tobacco bacterial wilt
Left: KBD-3 bacterial solution; Right: CK.
2.3 KBD-3对烟草青枯病的防治效果
结果（表2）显示，KBD-3菌液处理的烟草青枯病发病率为40.24%，病情指数相对较低，相对防效达到70.89%，较CK1的防效高，而只接种青枯病菌的对照处理（CK2）的发病率高达89.31%。
表2 KBD-3菌液对烟草青枯病的防治效果
Table 2
| 处理 Treatment | 发病率 Incidence rate (%) | 病情指数 Disease index | 相对防效 Relative control effect (%) |
|---|---|---|---|
| KBD-3 | 40.24±0.07c | 17.02±0.05c | 70.89±0.12a |
| CK1 | 53.86±0.11b | 30.21±0.03b | 48.32±0.08b |
| CK2 | 89.31±0.14a | 58.46±0.08a | － |
| CK3 | 0 | 0 | － |
2.4 KBD-3对无机硒的还原作用
图3
图3
KBD-3菌液及其纳米硒生物合成活性菌液
左：KBD-3菌液；右：KBD-3纳米硒生物合成活性菌液。
Fig.3
KBD-3 bacterial solution and its nano-selenium biosynthesis active bacterial solution
Left: KBD-3 bacterial solution; Right: KBD-3 nano-selenium biosynthesis active bacterial solution.
图4
图4
菌株KBD-3生物合成纳米硒的SEM/EDS图谱
Fig.4
SEM/EDS spectrum of biosynthesis of nano-selenium from strain KBD-3
2.5 KBD-3对绿豆芽培育的富硒效果
如表3所示，经纳米硒溶液处理的绿豆芽中硒含量为45.35 μg/g，与对照相比，硒含量有了大幅提高。这对富硒食品的研究与开发具有一定的指导意义。
表3 不同处理对绿豆芽中硒含量的影响
Table 3
| 处理 Treatment | 样品质量 Sample mass (g) | 硒含量 Selenium content (μg/g) |
|---|---|---|
| 纳米硒溶液 Nano-selenium solution | 0.15 | 45.35 |
| CK | 0.15 | 0.00 |
3 讨论
目前，越来越多的生防菌种类被应用到作物病害防治中，生防菌的作用机理包括抗病物质产生、诱导抗病性、种群竞争等诸多方面[14]。这些生防菌包括细菌、真菌和其他微生物，可有效抑制病原菌的生长繁殖，从而达到防治作物病害的效果[1]。防御假单胞菌是近年来重新归类的一个新物种，有研究[15⇓⇓⇓⇓-20]表明，防御假单胞菌可产生一些抑菌次生代谢产物，如硝吡咯菌素、藤黄绿脓菌素、2,4-DAPG等，这些物质可有效抑制作物病害的发生，对番茄灰霉病、小麦全蚀病以及葡萄灰霉病等多种果蔬的侵染病害具有防控作用。本研究提供的防御假单胞菌KBD-3，同时对烟草花叶病和青枯病病原菌具有拮抗活性，对烟草花叶病和青枯病的防效分别为95.77%和70.89%，防效较好。
4 结论
本研究中，防御假单胞菌KBD-3同时具备拮抗烟草花叶病毒和烟草青枯病病原菌的活性，且具有硒元素还原能力，可将亚硒酸钠还原为纳米硒，因此，其不仅为作物病害防治提供新的微生物资源，还能较好地用于作物的富硒培育。
参考文献
Biological control of soil-borne pathogens by fluorescent pseudomonads
DOI:10.1038/nrmicro1129
PMID:15759041
[本文引用: 1]
Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.
Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens
Selenium as an anticancer nutrient: Roles in cell proliferation and tumor cell invasion
Selenium deficiency and viral infection
Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2, 4-diacetylphloroglucinol and pyoluteorin
DOI:10.1016/j.syapm.2010.10.005
PMID:21392918
[本文引用: 1]
Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl(+) Plt(+) strains isolated from tobacco, cotton or wheat on different continents. Phl(+) Plt(+) strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the 'P. syringae', 'P. fluorescens' and 'P. chlororaphis' species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl(+) Plt(+) strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl(+) strains, the Phl(+) Plt(+) strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl(+) Plt(+) fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0(T) (=CFBP 6595(T), =DSM 19095(T)) as the type strain.Copyright © 2011 Elsevier GmbH. All rights reserved.
Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens
DOI:S0944-5013(18)30270-2
PMID:30642463
[本文引用: 1]
Pseudomonas sp. MP12 was isolated from a soil sample collected in a typical warm-temperate deciduous forest near Brescia, Northern Italy. Phylogenetic analysis identified the species as Pseudomonas protegens. We evidenced in this strain the presence of the genes phlD, pltB and prnC responsible for the synthesis of the antifungal compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin and pyrrolnitrin, respectively. P. protegens MP12 was also shown to produce siderophores and ammonia, yielded positive results with the indole-3-acetic acid test, and was capable of phosphate solubilization. Moreover, P. protegens MP12 exhibited inhibitory effects on in vitro mycelial growth of prominent grapevine (Vitis vinifera) phytopathogens such as Botrytis cinerea, Alternaria alternata, Aspergillus niger, Penicillium expansum and Neofusicoccum parvum. The strain showed activity even against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum, which cause the devastating tracheomycosis/esca disease of grapevine trunks for which no efficacious control methods have been demonstrated so far. Furthermore, the MP12 strain manifested in vivo antifungal activity against B. cinerea on grapevine leaves. Culture-dependent and culture-independent analysis revealed the ability of P. protegens MP12 to efficiently and permanently colonize inner grapevine tissues. These results suggest that P. protegens MP12 could be worth of exploitation as an antifungal biocontrol agent for applications in viticulture.Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
Biological effects of a nano red elemental selenium
DOI:10.1002/biof.5520150103
PMID:11673642
[本文引用: 1]
A novel selenium form, nano red elemental selenium (Nano-Se) was prepared by adding bovine serum albumin to the redox system of selenite and glutathione. Nano-Se has a 7-fold lower acute toxicity than sodium selenite in mice (LD(50) 113 and 15 mg Se/kg body weight respectively). In Se-deficient rat, both Nano-Se and selenite can increase tissue selenium and GPx activity. The biological activities of Nano-Se and selenite were compared in terms of cell proliferation, enzyme induction and protection against free racial-mediated damage in human hepatoma HepG2 cells. Nano-Se and selenite are similarly cell growth inhibited and stimulated synthesis of glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and thioredoxin reductase (TR). When HepG2 cells were co-treated with selenium and glutathione, Nano-Se showed less pro-oxidative effects than selenite, as measured by cell growth. These results demonstrate that Nano-Se has a similar bioavailability in the rat and antioxidant effects on cells.
