,1,3, 王靖1, 谢中玉1, 施焕1, 曹哲1, 江龙1, 马小舟1,3, 汪代斌4, 张帅5, 黄进,2,3, 孙现超,1,3Preparation of A Novel Silver Nanoparticle and Its Antifungal Mechanism Against Alternaria alternata
XIANG ShunYu,1,3, WANG Jing1, XIE ZhongYu1, SHI Huan1, CAO Zhe1, JIANG Long1, MA XiaoZhou1,3, WANG DaiBin4, ZHANG Shuai5, HUANG Jin,2,3, SUN XianChao,1,3通讯作者:
责任编辑: 岳梅
收稿日期:2020-01-2接受日期:2020-02-15网络出版日期:2020-07-16
| 基金资助: |
Received:2020-01-2Accepted:2020-02-15Online:2020-07-16
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向顺雨, 王靖, 谢中玉, 施焕, 曹哲, 江龙, 马小舟, 汪代斌, 张帅, 黄进, 孙现超. 一种新型银纳米颗粒的制备及其抑制烟草赤星病菌的机制[J]. 中国农业科学, 2020, 53(14): 2885-2896 doi:10.3864/j.issn.0578-1752.2020.14.012
XIANG ShunYu, WANG Jing, XIE ZhongYu, SHI Huan, CAO Zhe, JIANG Long, MA XiaoZhou, WANG DaiBin, ZHANG Shuai, HUANG Jin, SUN XianChao.
0 引言
【研究意义】我国是农业大国,经济作物的种植是我国大部分农民的主要经济来源。赤星病是植物生产中重要的叶部病害,除烟草以外,其还可对番茄、曼陀罗等作物的生产造成一定影响[1,2,3]。交链孢菌（Alternaria alternata）为其病原,属半知菌亚门真菌[4],具有潜育期短、单斑产孢量大,暴发迅速等特点[5]。赤星病菌主要危害植物叶片,使叶部产生大小不一的病斑,后期病斑破碎脱落,极大程度地影响植物叶片生长而降低作物品质[6,7]。因此,提高烟草赤星病田间防治效率对增加农业生产产值具有重大意义。【前人研究进展】目前,生产上主要通过使用无毒种苗、选育抗病品种、加强田间管理、喷施化学药剂等方式对赤星病进行综合防治[8,9,10]。其中,化学防治是我国农业生产中防治赤星病的主要措施之一[5],菌核净是我国各农业产区主要使用的烟草赤星病防治药剂。但是大量使用菌核净以及不合理的管理方式会导致产生耐药菌株,这为烟草赤星病的防治增加了难度[11]。银纳米颗粒由于其优越的抗菌活性,不易产生耐药性而被作为一种理想的抗菌材料被各行业广泛研究。到目前为止,其应用范围已经覆盖到医学器械消毒、医用抗菌剂、食品包装、水体消毒等多领域[12,13,14,15]。但研究发现,银纳米颗粒的合成方式一直是影响其抗菌效果以及生物相容性的直接因素。在诸多合成方法中,银纳米颗粒的绿色合成由于具有合成条件温和,不产生有毒副产物等优势而备受关注。ELBESHEHY等曾用短小芽孢杆菌（Bacillus pumilus）、波斯芽孢杆菌（B. persicus）、地衣芽孢杆菌（B. licheniformis）3种土壤细菌还原银离子而成功制备出对于人体致病真菌、细菌以及大豆花叶病毒（Bean yellow mosaic virus）具有良好抑制作用的银纳米颗粒[16]。但现有的绿色合成银纳米颗粒的方法难以实现控制银纳米颗粒稳定性与粒径统一进而影响其抗菌活性[17,18]。【本研究切入点】羧基与银纳米颗粒具有强烈的配对作用,且海藻酸钠表面活性羟基具有一定的还原性。本研究基于此,利用海藻酸钠表面羟基还原银离子生成银颗粒,而后其表面羧基迅速与银纳米颗粒产生配对作用使海藻酸钠包裹银纳米颗粒以控制银纳米颗粒粒径,增加银纳米颗粒稳定性,防止银纳米颗粒聚沉,从而长时间保持较高的抗菌活性。【拟解决的关键问题】明确海藻酸钠-银纳米颗粒（S-AgNPs）抗菌活性与抑制烟草赤星病菌的机理,为银纳米颗粒的制备及银纳米颗粒在植物叶部病害防控中的作用提供理论依据。1 材料与方法
试验于2019年在西南大学植物保护学院完成。1.1 材料
烟草K326由中国农业科学院烟草研究所赠予,氨水、盐酸（浓度为1.18 g·mL-1）、NaCl、葡萄糖均购自重庆川东化工有限公司,乙二醇购自重庆北碚精细化工厂,高碘酸钠购自成都市科龙化工试剂厂,海藻酸钠（SA）（黏度200±20 mPa·s）购自上海阿拉丁生化科技股份有限公司,琼脂购自北京鼎国昌盛生物技术责任有限公司,考马斯亮蓝G250购自上海康朗生物科技有限公司。紫外分光光度计PGENERAL（北京普析通用仪器有限责任公司）,布鲁克纳米表面仪（BRUKER,布鲁克（北京）科技有限公司）,离心机（Sartorius stedim,德国赛多利斯集团）,透射电镜（JEM-1200EX Japan JEOL,捷欧路（北京）科贸有限公司）,扫描电镜（su8020,日本日立公司）,Zeta粒径分析仪（ZS90,英国马尔文公司）,冷冻干燥机（FD-1A-50,上海豫明仪器有限公司）,电导仪（DDS-11C,青岛明博环保科技有限公司）,水浴锅（Keelrein,上海齐欣科学仪器有限公司）。
1.2 海藻酸钠合成银纳米颗粒
称取0.02 g海藻酸钠,用5 mL蒸馏水完全溶解备用。0.04 g硝酸银溶于2 mL水中然后逐滴加入2%氨水溶液直到看到黄色絮状物消失停止滴加,将海藻酸钠溶液加至银氨溶液并在65℃水浴中反应6 min,得到粒径为10 nm左右的银纳米颗粒,并在12 000 r/min,4℃条件下离心5 min,离心3次后将沉淀溶解在超纯水中备用。1.3 表征方法
采用透射电镜（TEM）、原子力显微镜（AFM）、扫描电镜（SEM）观察S-AgNPs的形貌特征、粒径分布;用紫外分光光度计获得S-AgNPs的紫外-可见吸收光谱;Zeta粒径分析仪测试S-AgNPs的粒径与电位;SEM观察S-AgNPs处理后的赤星病菌菌丝。1.4 S-AgNPs抑菌作用测定
制作PDA培养基[19],并将已知原始浓度的S-AgNPs添加到PDA中,使PDA培养基中S-AgNPs的浓度分别为0.0625、0.125、0.25、0.5、1.0 μg·mL-1,移入直径为0.8 cm的赤星病菌菌饼（病菌生长5 d后移接）,清水为空白对照。当对照板中菌丝体的生长到达培养皿的边缘时,计算抑制率,每个处理设置3次重复。抑制率（%）=100×（清水对照组菌圈直径-处理组菌圈直径）/清水对照组菌圈直径。1.5 S-AgNPs菌丝抑制作用测定
制作PDB培养基（不带琼脂的PDA培养基）,即将液体灭菌后,分装在锥形瓶中,每瓶100 mL培养基,并在每个锥形瓶中移入10个0.8 cm的培养5 d的赤星病菌菌饼,然后28℃,180 r/min的摇床中黑暗培养2 d后加入S-AgNPs,使S-AgNPs在PDB培养基中的浓度分别为0、1.0 μg·mL-1,继续在28℃,180 r/min摇床中黑暗培养4 d后用三层纱布收集菌丝,并用灭菌水洗涤2—3次后用滤纸吸干表面水分后称重（鲜重）。将收集好的菌丝冷冻干燥后称重（干重）,统计各处理组菌丝质量,每个处理设置3次重复。1.6 细胞通透率测定
培养基制作及菌饼放置方法同1.5,在28℃,180 r/min的摇床中黑暗培养6 d后收集菌丝并冷冻干燥。称取100 mg冷冻干燥的菌丝于试管中,加入S-AgNPs并定容至10 mL,使试管中S-AgNPs溶液浓度分别为0、1.0 μg·mL-1。并分别在0、5、10、15、20 min后用电导仪测试各处理组电导率。等待测试期间在25℃,160 r/min摇床中培养。若细胞膜通透性被破坏,细胞质将泄漏到细胞膜外,可通过测试细胞膜外溶液DNA或RNA浓度来判断细胞膜通透性受影响程度。因此,待测完电导率后,将每处理组的菌丝去除掉,收集培养液,并用紫外分光光度计在260 nm测定每处理组溶液中DNA的含量,每个处理设置3次重复。1.7 可溶性蛋白含量测定
菌丝收集方法同1.5,将收集好的菌丝冷冻干燥后备用。取0.25 g菌丝,液氮冷却灭菌好的研钵体后加入0.025 mol·L-1的Tris-HCl 2 mL研磨菌丝至糊状。12 000 r/min,4℃离心15 min,收集上清液备用。然后取上述蛋白质粗提液0.2 mL,加入考马斯亮蓝G-250染色液5 mL,25℃环境下静止3 min后在595 nm处测其吸光值。考马斯亮蓝G250染液配制方法：称取考马斯亮蓝G-250 100 mg,用50 mL 95%乙醇溶解后加入100 mL 85% m/v磷酸溶液,去离子水定容至1 L,每个处理设置3次重复。1.8 离体叶片病害控制效果测定
选用烟草品种为K326（40 d烟龄）。分别用不同浓度的S-AgNPs（0.125、0.25、0.5、1.0 μg·mL-1）喷施叶面,每株喷洒50 mL,每个浓度5株重复。喷施24 h后选取第4、5位叶片并用注射孢子悬浮液法（106个/mL）接种赤星病菌。在28℃恒温培养箱保湿培养3 d后观察叶片发病情况。病害分级标准0级：全叶无病,1级：病斑面积占叶面积1%以下,3级：病斑面积占叶面面积2%—5%,5级：病斑面积占叶面面积6%—10%,7级：病斑面积占叶面面积11%—20%,9级：病斑面积占叶面面积21%以上。依据赤星病叶面病害分级标准计算其病情指数：病情指数=100×Σ（各级病斑数×相应级数值）/（病斑总数×最大级数）,每个处理设置3次重复。1.9 S-AgNPs的水体安全性
选择若干条长度为3—4 cm的鲫鱼,配置好体积为1 L浓度为1.0 μg·mL-1的S-AgNPs并在其中放入10条生长良好的鲫鱼,纯自来水作为空白对照,每组重复3次。分别在24、48、72、96 h观察并记录鲫鱼的死亡数量。1.10 数据处理
采用Excel 2010与OriginPro 8处理数据及作图,采用Photoshop对图片进行调色处理,采用SPSS Statistics 20对数据进行单因素方差分析（Duncan法）。2 结果
2.1 S-AgNPs紫外吸收与X射线光电子能谱分析
S-AgNPs在421 nm左右有紫外特征吸收峰（图1-A）,且XPS能谱显示S-AgNPs表面结合能为3.01 keV,明确了银主要以零价态存在,说明S-AgNPs中的Ag元素主要是单质银（图1-B）,表明在整个反应中所合成的物质为金属银单质,且具有银纳米颗粒的紫外吸收特质,能够充分肯定该反应可成功合成纳米级银单质。图1
新窗口打开|下载原图ZIP|生成PPT图1S-AgNPs紫外可见吸收光谱（A）与X射线光电子能谱（B）
Fig. 1Uv-vis (A) and XPS (B) spectrum of S-AgNPs
2.2 S-AgNPs形貌与稳定性表征
经TEM观测,S-AgNPs为球形颗粒,形貌规则,具有较好的分散性,能均匀分散在溶剂中,团聚现象较少（图2-A、2-B）。此外,SEM与AFM观测到S-AgNPs粒径相对均一,颗粒形貌大小无明显差异（图2-E—2-H）。对TEM与AFM图像进行粒径统计（图2-I、2-J）得到该S-AgNPs粒径大约8.14 nm（TEM）与11.23 nm（AFM）。为明确S-AgNPs的稳定性,将S-AgNPs水溶液直接进行冷冻干燥制成S-AgNPs粉末,而后将其复溶在水中发现水接触S-AgNPs粉末后无需搅拌S-AgNPs立刻均匀分散在水中,肉眼观察复溶溶液颜色、状态与未冻干前S-AgNPs溶液极度相似。进一步将其进行TEM观察发现,S-AgNPs冻干后粒径与稳定性与冻干前无明显差异,只观察到少许颗粒发生局部团聚现象,但整体分散性与粒径无明显变化（图2-C、2-D）。图2
新窗口打开|下载原图ZIP|生成PPT图2S-AgNPs形貌与稳定性表征
A、B：S-AgNPs溶液TEM图;C、D：S-AgNPs冷冻干燥后复溶于水中的TEM图;E、F：S-AgNPs扫描电镜图;G、H：S-AgNPs原子力显微镜图;I：S-AgNPs的TEM粒径统计;J：S-AgNPs的AFM粒径统计图
Fig. 2Morphology and stability characterization of S-AgNPs
A、B：TEM image of S-AgNPs solution;C、D：TEM image of S-AgNPs solution re-dissolved in water after freeze-drying;E、F：SEM images of S-AgNPs;G、H：AFM images of S-AgNPs;I：TEM particle size statistics of S-AgNPs;J：AFM particle size statistics of S-AgNPs
2.3 S-AgNPs的Zeta电位与粒径统计分析
对S-AgNPs进行进一步表征以明确其粒径分布,Zeta粒径仪分析试验结果发现S-AgNPs粒径分布在5—12 nm,60%的S-AgNPs粒径集中在8—9 nm,统计所得平均粒径为9.83 nm（图3-A）。选用上海函郎新材料有限公司生产的无表面活性剂的银纳米颗粒（AgNPs）作为对照组分与S-AgNPs一起进行Zeta电位测定（图3-B）,结果显示AgNPs电位为+4.6 mV,这主要是AgNPs无表面活性剂干扰银颗粒的电位,而S-AgNPs电位为-32.29 mV,这是银纳米颗粒表面包裹的海藻酸钠中和了银颗粒表面电位而使其显负电,该结果证实了海藻酸钠还原银离子后又作为表面活性剂包覆在银纳米颗粒表面。图3
新窗口打开|下载原图ZIP|生成PPT图3S-AgNPs Zeta粒径统计图（A）与Zeta电位图（B）
Fig. 3Zeta particle size statistical graph (A) and Zeta potential graph (B) of S-AgNPs
2.4 S-AgNPs对烟草赤星病菌的抑制作用
生测结果发现S-AgNPs在0.0625 μg·mL-1时就对烟草赤星病菌具有一定抑制作用（抑制率10%）,且在浓度为1.0 μg·mL-1时抑制率可达83.9%。初步确定S-AgNPs具有较强的抗菌活性,在低浓度下能够明显抑制烟草赤星病菌菌丝生长（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4S-AgNPs对烟草赤星病菌的抑治效果（A）与抑制率（B）
柱上标有不同小写字母表示处理间差异显著（P<0.05）,竖条表示标准差。下同
Fig. 4Antifungal effect of S-AgNPs on A. alternata (A) and inhibition rate (B)
Different lowercases on the bars indicate significantly different among different treatments according to the Duncan’s multiple range test (p<0.05). Vertical bars indicate standard deviations. The same as below
2.5 S-AgNPs对烟草赤星病菌菌丝生长的影响
如图5所示,清水对照组（CK）菌丝生长迅速,培养基中充满了白色菌丝且饱满（图5-A）,而S-AgNPs（1.0 μg·mL-1）处理后的菌饼未观察到明显的菌丝生长,在菌饼周围仅看到少了菌丝附着,培养基较清澈且无菌丝漂浮在培养基中（图5-B）。将两组菌丝取出测定其菌丝鲜重与干重后发现,对照组的菌丝鲜重与干重均显著高于S-AgNPs组（图5-C、5-D）。图5
新窗口打开|下载原图ZIP|生成PPT图5S-AgNPs对菌丝生长的影响
A、B：健康菌丝与S-AgNPs处理后的菌丝;C：菌丝干重;D：菌丝鲜重
Fig. 5Effect of S-AgNPS on mycelia growth
A、B：Healthy mycelium and S-AgNPs treated mycelium;C：Dry weight of mycelium;D：Fresh weight of mycelium
2.6 S-AgNPs处理后菌丝表面观察
将S-AgNPs处理后菌丝采用扫描电镜观察表面是否受损。扫描电镜图片显示清水对照组菌丝生长良好,菌丝结构完整,饱满无损伤。S-AgNPs处理后的菌丝显示不同程度的损伤,菌丝表面凹陷,局部膨大,菌丝整体干瘪褶皱明显,表明S-AgNPs对烟草赤星病菌菌丝有较强的破坏作用（图6）。图6
新窗口打开|下载原图ZIP|生成PPT图6健康菌丝与S-AgNPs处理后的菌丝扫描电镜图
Fig. 6SEM images of mycelia of the healthy group and S-AgNPs treatment group
2.7 细胞膜通透性测定
抗菌机制试验结果显示,S-AgNPs处理后的菌丝在0—5 min时就出现了大量的细胞质渗漏,细胞外液电导率迅速提升,5—20 min电导率也呈缓慢上升。CK对照组电导率在0—20 min无明显变化（图7-A）。与此同时,S-AgNPs处理过后的菌丝在20 min内DNA累积渗漏量要显著高于CK对照组（图7-B）[20]。图7
新窗口打开|下载原图ZIP|生成PPT图7菌丝电导率变化（A）与DNA泄露（B）
Fig. 7Mycelium conductivity change (A) and DNA leakage (B)
2.8 可溶性蛋白含量测定
S-AgNPs组分的菌丝可溶性蛋白含量要远低于CK对照组（图8）,表明S-AgNPs抑制菌丝生长或破坏菌丝细胞膜可能与抑制其可溶性总蛋白有关[21]。图8
新窗口打开|下载原图ZIP|生成PPT图8菌丝可溶性蛋白含量
Fig. 8Soluble protein content of mycelium
2.9 离体叶片病害控制效果
如图9所示,清水对照组在接种3 d后发病较严重,能明显观察到深褐色病斑。而S-AgNPs处理组仅浓度为0.125 μg·mL-1时观察到发病,但发病程度与病斑大小要明显低于对照组,且其病情指数也显著低于对照组（表1）。说明S-AgNPs在较低浓度时能够抑制赤星病菌侵染植株,在较高浓度时能够完全保护植株不受侵染。图9
新窗口打开|下载原图ZIP|生成PPT图9不同处理下烟草赤星病发病情况
Fig. 9Incidence of tobacco brown spot under different treatments
Table 1
表1
表1不同处理下烟草赤星病病情指数
Table 1
| 处理Treatment | 病情指数Disease index |
|---|---|
| CK | 100a |
| 0.125 μg·mL-1 | 48.1b |
| 0.25 μg·mL-1 | 11.1c |
| 0.5 μg·mL-1 | 0d |
| 1.0 μg·mL-1 | 0d |
新窗口打开|下载CSV
2.10 S-AgNPs的安全性测定
将S-AgNPs加入水中后观察鲫鱼96 h内的生命活动,结果发现清水对照组与S-AgNPs处理组鲫鱼在96 h内生命活动均正常,无不安、上下翻滚的情况,初步判断S-AgNPs在抑菌范围浓度内对水生生物无明显毒害作用（表2）。Table 2
表2
表2S-AgNPs对鲫鱼的毒性测定
Table 2
| 处理 Treatment | 死亡数量 Number of deaths | |||
|---|---|---|---|---|
| 24 h | 48 h | 72 h | 96 h | |
| 水 Water | 0 | 0 | 0 | 1 |
| S-AgNPs (1.0 μg·mL-1) | 0 | 0 | 1 | 1 |
新窗口打开|下载CSV
3 讨论
赤星病属暴发性病害,在高温高湿等适于发病的田间环境中可短时间大面积暴发,且其田间防治主推药剂菌核净也面临着高抗药性威胁,因此研究高效的赤星病防治策略迫在眉睫[22]。在诸多研究中,化学新药剂的研发与生物防治是目前针对赤星病研究最多且最受关注的两大途经[23,24]。但新药剂的研发周期较长,无法短时间投入于赤星病防治工作中[25]。生物防治也存在见效慢,农民可接受度低等问题,还需要更进一步完善以提高其应用前景[26]。因此,亟需开发并评价新的烟草赤星病防控药剂。银纳米颗粒是公认的具有良好抗菌活性的金属纳米材料,具有低耐药性,抗菌机制多样等特点,其已被证实具有优越的广谱抗植物病原真菌/细菌活性[27,28]。因此,银纳米颗粒是可应用于烟草赤星病防治的理想备选材料。而绿色合成银纳米颗粒的方法也可改善银纳米抗菌活性以及提高生物安全性,研究中利用一些生物提取成分或者有效物质对银离子进行还原合成银纳米颗粒可减少有毒副产物的产生,同时生物活性成分的加入也可协同银纳米抗菌以增加抗菌活性[29,30,31]。其中,最常见的绿色合成银纳米颗粒的方式就是利用微生物还原银离子[16]。有报道评价了购自Quantum Sphere公司直径为20 nm左右的银纳米颗粒对植物病原真菌抑制活性,其浓度在高达50 μg·mL-1时才能抑制小麦根腐病菌（Bipolaris sorokiniana）侵染小麦[21]。但MISHRA等利用土壤细菌BHU-S4的上清液绿色合成了粒径为20 nm的银纳米颗粒（bsAgNPs）并同样用于小麦根腐病的防治,发现bsAgNPs在浓度为4.0 μg·mL-1时就能完全抑制病菌侵染植株[32],表明BHU-S4绿色合成银纳米颗粒确实很大程度上提高了银颗粒对小麦根腐病的防治效果,在达到相同的防治效果时bsAgNPs相比于所购买的普通银颗粒用量更少。微生物在合成银纳米颗粒过程中可能存在难以有效控制银纳米颗粒粒径,所合成的纳米颗粒易出现粒径分散不均匀甚至团聚等现象[33,34,35]。而银纳米颗粒在抗菌时具有很强的尺度依赖性,其粒径越小抗菌活性越强[36]。同时,银纳米颗粒的稳定性也会直接影响其药效时长[36]。因此,提高银纳米颗粒绿色合成的颗粒稳定性与粒径均一性是增强该方法抗菌应用前景的重要一步。在本研究中,笔者通过前期调查发现羧基与银纳米颗粒的配对作用最强,工业中常用带有羧基的表面活性剂控制银纳米颗粒粒径并增强其稳定性[37]。海藻酸钠表面具有丰富的羧基,在本研究中羧基与银颗粒强烈配对作用使海藻酸钠作为表面活性剂包裹在银颗粒表面以控制银颗粒粒径,提高银颗粒稳定性,最终合成平均粒径为9.83 nm的海藻酸钠-银纳米颗粒（S-AgNPs）。因此,S-AgNPs的高效抗菌效果可能与其超强稳定性、粒径统一以及小粒径特性有关。
此外,利用生物多糖合成银纳米颗粒也逐渐受到各界关注,特别是关于生物多糖协同银纳米颗粒抗菌相关工作的研究。WEI等利用壳聚糖为还原剂制备了壳聚糖基银纳米颗粒,试验证明壳聚糖合成的银纳米颗粒相比于纯壳聚糖与硝酸银溶液抗菌作用更迅速,抗菌时间更持久且抗菌作用更强,在5 μg·mL-1时就能对大肠杆菌具有较强的抑制作用[38]。该策略最终实现了壳聚糖抗菌与银纳米抗菌的双重抗菌作用,实现了快速、高效的协同抗菌行为。海藻酸钠是从褐藻类中提取而来的一种生物多糖,其降解产物海藻酸钠寡糖（AOS）具有促进植物生长、缓解非生物胁迫和诱导抗病的作用[39]。利用海藻酸钠所合成的银纳米颗粒（S-AgNPs）在1.0 μg·mL-1时就能高效抑制赤星病菌生长,一方面可能是病菌对药剂的敏感性存在差异,另一方面也可能是包覆在银纳米颗粒表面的海藻酸钠在使用过程中降解产生了海藻酸钠寡糖,增强了抗菌效果。
综上所述,绿色合成银纳米颗粒可成为增强银纳米颗粒抗菌活性、降低银纳米颗粒生物毒性的重要途经之一[32,38,40],同时此类方法也增强了银纳米颗粒在赤星病防治工作中的应用前景,有望成为烟草赤星病防治研究的一个新方向。
4 结论
利用海藻酸钠成功合成了粒径统一、稳定性高、分散性强的海藻酸钠-银纳米颗粒,所合成的银纳米颗粒在低浓度下便具有高效抑制赤星病菌的活性,其主要通过破坏菌丝细胞膜通透性,抑制可溶性蛋白合成等途经抑制菌丝生长,最终实现抑制病菌侵染植株,具有广阔的农业应用前景。参考文献 原文顺序
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OBJECTIVE: To compare rates of healing of donor sites in pigs between those dressed with silver-coated dressings and those dressed with petrolatum-impregnated absorbent gauze. DESIGN: Open study with each animal acting as its own control. SETTING: University research facility, Canada. ANIMALS: 6 young specific-pathogen-free domestic pigs. INTERVENTIONS: A total of 72 wounds about 1 cm x 2 cm x 0.4 mm were made in rows of eight on each pig with a dermatome. They were divided into three groups of 24, and dressed with petrolatum gauze, or silver-coated dressings moistened with sterile water either once only or daily for 10 days. All dressings were secured in place with an elastic bandage. MAIN OUTCOME MEASURES: Erythema, infection, epidermal migration, and healing. RESULTS: Wounds dressed with moistened silver-coated dressings re-epithelialised significantly more quickly. This resulted in complete re-epithelialisation within 70% of the time taken by those wounds dressed with petrolatum gauze. CONCLUSION: Silver-coated dressings provide a moist environment for the healing wound combined with an effective antimicrobial agent, and this significantly accelerates healing compared with wounds dressed with traditional petrolatum gauze dressings.
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Silver has been in use since time immemorial in the form of metallic silver, silver nitrate, silver sulfadiazine for the treatment of burns, wounds and several bacterial infections. But due to the emergence of several antibiotics the use of these silver compounds has been declined remarkably. Nanotechnology is gaining tremendous impetus in the present century due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical and optical properties of metals. Metallic silver in the form of silver nanoparticles has made a remarkable comeback as a potential antimicrobial agent. The use of silver nanoparticles is also important, as several pathogenic bacteria have developed resistance against various antibiotics. Hence, silver nanoparticles have emerged up with diverse medical applications ranging from silver based dressings, silver coated medicinal devices, such as nanogels, nanolotions, etc.
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Arbuscular Mycorrhizal Fungi (AMF) constitute a group of root obligate biotrophs that exchange mutual benefits with about 80% of plants. They are considered natural biofertilizers, since they provide the host with water, nutrients, and pathogen protection, in exchange for photosynthetic products. Thus, AMF are primary biotic soil components which, when missing or impoverished, can lead to a less efficient ecosystem functioning. The process of re-establishing the natural level of AMF richness can represent a valid alternative to conventional fertilization practices, with a view to sustainable agriculture. The main strategy that can be adopted to achieve this goal is the direct re-introduction of AMF propagules (inoculum) into a target soil. Originally, AMF were described to generally lack host- and niche-specificity, and therefore suggested as agriculturally suitable for a wide range of plants and environmental conditions. Unfortunately, the assumptions that have been made and the results that have been obtained so far are often worlds apart. The problem is that success is unpredictable since different plant species vary their response to the same AMF species mix. Many factors can affect the success of inoculation and AMF persistence in soil, including species compatibility with the target environment, the degree of spatial competition with other soil organisms in the target niche and the timing of inoculation. Thus, it is preferable to take these factors into account when
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Silver in ionic or nanoparticle forms has a high antimicrobial activity and is therefore widely used for various sterilization purposes including materials of medical devices and water sanitization. There have been relatively few studies on the applicability of silver to control plant diseases. Various forms of silver ions and nanoparticles were tested in the current study to examine the antifungal activity on two plant-pathogenic fungi, Bipolaris sorokiniana and Magnaporthe grisea. In vitro petri dish assays indicated that silver ions and nanoparticles had a significant effect on the colony formation of these two pathogens. Effective concentrations of the silver compounds inhibiting colony formation by 50% (EC50) were higher for B. sorokiniana than for M. grisea. The inhibitory effect on colony formation significantly diminished after silver cations were neutralized with chloride ions. Growth chamber inoculation assays further confirmed that both ionic and nanoparticle silver significantly reduced these two fungal diseases on perennial ryegrass (Lolium perenne). Particularly, silver ions and nanoparticles effectively reduced disease severity with an application at 3 h before spore inoculation, but their efficacy significantly diminished when applied at 24 h after inoculation. The in vitro and in planta evaluations of silver indicated that both silver ions and nanoparticles influence colony formation of spores and disease progress of plant-pathogenic fungi. In planta efficacy of silver ions and nanoparticles is much greater with preventative application, which may promote the direct contact of silver with spores and germ tubes, and inhibit their viability.
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Silver nanoparticles are nanoparticles of silver which are in the range of 1 and 100 nm in size. Silver nanoparticles have unique properties which help in molecular diagnostics, in therapies, as well as in devices that are used in several medical procedures. The major methods used for silver nanoparticle synthesis are the physical and chemical methods. The problem with the chemical and physical methods is that the synthesis is expensive and can also have toxic substances absorbed onto them. To overcome this, the biological method provides a feasible alternative. The major biological systems involved in this are bacteria, fungi, and plant extracts. The major applications of silver nanoparticles in the medical field include diagnostic applications and therapeutic applications. In most of the therapeutic applications, it is the antimicrobial property that is being majorly explored, though the anti-inflammatory property has its fair share of applications. Though silver nanoparticles are rampantly used in many medical procedures and devices as well as in various biological fields, they have their drawbacks due to nanotoxicity. This review provides a comprehensive view on the mechanism of action, production, applications in the medical field, and the health and environmental concerns that are allegedly caused due to these nanoparticles. The focus is on effective and efficient synthesis of silver nanoparticles while exploring their various prospective applications besides trying to understand the current scenario in the debates on the toxicity concerns these nanoparticles pose.
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The present study is focused on the extracellular synthesis of silver nanoparticles (AgNPs) using culture supernatant of an agriculturally important bacterium, Serratia sp. BHU-S4 and demonstrates its effective application for the management of spot blotch disease in wheat. The biosynthesis of AgNPs by Serratia sp. BHU-S4 (denoted as bsAgNPs) was monitored by UV-visible spectrum that showed the surface plasmon resonance (SPR) peak at 410 nm, an important characteristic of AgNPs. Furthermore, the structural, morphological, elemental, functional and thermal characterization of bsAgNPs was carried out using the X-ray diffraction (XRD), electron and atomic microscopies, energy dispersive X-ray (EDAX) spectrometer, FTIR spectroscopy and thermogravimetric analyzer (TGA), respectively. The bsAgNPs were spherical in shape with size range of approximately 10 to 20 nm. The XRD and EDAX analysis confirmed successful biosynthesis and crystalline nature of AgNPs. The bsAgNPs exhibited strong antifungal activity against Bipolaris sorokiniana, the spot blotch pathogen of wheat. Interestingly, 2, 4 and 10 microg/ml concentrations of bsAgNPs accounted for complete inhibition of conidial germination, whereas in the absence of bsAgNPs, conidial germination was 100%. A detached leaf bioassay revealed prominent conidial germination on wheat leaves infected with B. sorokiniana conidial suspension alone, while the germination of conidia was totally inhibited when the leaves were treated with bsAgNPs. The results were further authenticated under green house conditions, where application of bsAgNPs significantly reduced B. sorokiniana infection in wheat plants. Histochemical staining revealed a significant role of bsAgNPs treatment in inducing lignin deposition in vascular bundles. In summary, our findings represent the efficient application of bsAgNPs in plant disease management, indicating the exciting possibilities of nanofungicide employing agriculturally important bacteria.
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Mosquitoes transmit dreadful diseases, causing millions of deaths every year. Therefore, screening for larvicidal and pupicidal activity of microbial extracts attributes could lead to development of new and improved mosquito control methods that are economical and safe for nontarget organisms and are ecofriendly. Synthetic chemical insecticides occupy predominant position in control strategies. These hazardous chemicals exert unwarranted toxicity and lethal effects on nontarget organisms, develop physiological resistance in target, and cause adverse environmental effect. For vector control, fungal-mediated natural products have been a priority in this area at present. In the current study, effective larvicidal and pupicidal effect of mycosynthesized silver nanoparticles (Ag NPs) using an entomopathogenic fungi Trichoderma harzianum against developmental stages of the dengue vector Aedes aegypti was investigated. An attractive possibility of green nanotechnology is to use microorganisms in the synthesis of nanosilver especially Ag NPs. The mycosynthesized Ag NPs were characterized to find their unique properties through UV-visible spectrophotometer, X-ray diffraction analysis, Fourier transform infrared, and surface characteristics by scanning electron microscopy. To analyze the bioefficacy, different test concentrations for extracellular filtrate (0.2, 0.4, 0.6, 0.8, and 1.0 %) and Ag NPs (0.05, 0.10, 0.15, 0.20, and 0.25 %) were prepared to a final volume of 200 mL using deionized water; 20 larvae of each instars (I-IV) and pupa were exposed to each test concentration separately which included a set of control (distilled water) group with five replicates. Characterization of the synthesized Ag NPs were about 10-20 nm without aggregation. Susceptibility of larval instars to synthesized Ag NPs was higher than the extracellular filtrate of T. harzianum alone after 24-h exposure, where the highest mortality was recorded as 92 and 96 % for first and second instars and 100 % for third, fourth instars, and pupa. Lethal concentration 50 values of 0.079, 0.084, 0.087, 0.068, and 0.026 % were recorded for I-IV instars and pupa, respectively, when exposed to Ag NPs at 0.25 % concentration. Toxicity was exhibited against first (1.076 %), second (0.912 %), third (0.770 %), fourth (0.914 %) instars larvae, and pupa (0.387 %) with extracellular filtrate at a concentration of 1 % that was three- to fourfold higher compared to Ag NPs; no mortality was observed in the control. The present study is the first report on effective larvicidal and pupicidal activity of Ag NPs synthesized from an entomopathogenic fungi T. harzianum extracellular filtrate and could be an ideal ecofriendly, single-step, and inexpensive approach for the control of A. aegypti.
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The physical and chemical properties of silver nanoparticles that are responsible for their antimicrobial activities have been studied with spherical silver nanoparticles (average diameter approximately 9nm) synthesized by the borohydride reduction of Ag+ ions, in relation to their sensitivity to oxidation, activities towards silver-resistant bacteria, size-dependent activities, and dispersal in electrolytic solutions. Partially (surface) oxidized silver nanoparticles have antibacterial activities, but zero-valent nanoparticles do not. The levels of chemisorbed Ag+ that form on the particle’s surface, as revealed by changes in the surface plasmon resonance absorption during oxidation and reduction, correlate well with the observed antibacterial activities. Silver nanoparticles, like Ag+ in the form of AgNO3 solution, are tolerated by the bacteria strains resistant to Ag+. The antibacterial activities of silver nanoparticles are related to their size, with the smaller particles having higher activities on the basis of equivalent silver mass content. The silver nanoparticles aggregate in media with a high electrolyte content, resulting in a loss of antibacterial activities. However, complexation with albumin can stabilize the silver nanoparticles against aggregation, leading to a retention of the antibacterial activities. Taken together, the results show that the antibacterial activities of silver nanoparticles are dependent on chemisorbed Ag+, which is readily formed owing to extreme sensitivity to oxygen. The antibacterial activities of silver nanoparticles are dependent on optimally displayed oxidized surfaces, which are present in well-dispersed suspensions.
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Chitosan-based silver nanoparticles were synthesized by reducing silver nitrate salts with nontoxic and biodegradable chitosan. The silver nanoparticles thus obtained showed highly potent antibacterial activity toward both Gram-positive and Gram-negative bacteria, comparable with the highly active precursor silver salts. Silver-impregnated chitosan films were formed from the starting materials composed of silver nitrate and chitosan via thermal treatment. Compared with pure chitosan films, chitosan films with silver showed both fast and long-lasting antibacterial effectiveness against Escherichia coli. The silver antibacterial materials prepared in our present system are promising candidates for a wide range of biomedical and general applications.
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