柠檬醛抗黄曲霉作用的分子机理

以多组分山苍子[Litsea cubeba(Lour)Per]香精油作为复合中药模型。以该香精油中主要抗菌成分柠檬醛为中药靶部位,以能分泌致癌毒素的黄曲霉单细胞作为药物作用对象,吸收当今医学影像领域先进的科学技术,采用多学科交叉策略,将多维显微、瑞利光散射(Rayleigh scattering)、电镜与生化分析4项技术构筑平台, 从细胞、亚细胞和生物大分子三个水平,研究柠檬醛作用于黄曲霉的动静态过程,阐明模拟的中药方剂靶部位对细胞整体的作用规律.发现该醛不仅能改变黄曲霉细胞膜的形态结构、物理学特性及其生物学功能(如对物质吸收的选择通透性,细胞体积调节机制等),而且使细胞膜产生脂质过氧化损伤;进入细胞后,既作用于细胞器(如线粒体、细胞核等),使其产生损伤及区域性分布;又通过干扰细胞内大分子拥挤状态,导致细胞内生物大分子构象的改变、高含量类大分子缔合反应不可逆增强以及因生化反应区域效应丧失而产生的新陈代谢紊乱,揭示该醛能使黄曲霉孢子失去萌发力、菌丝体生长被抑制及产生孢子的能力,在于黄曲霉细胞膜、细胞器及大分子失去了正常结构、功能及相关的调节机制.在实现对柠檬醛抗黄曲霉机理阐明的过程中,在研究思路和方法上进行全新的探索.     

柠檬醛胁迫环境下黄曲霉线粒体的畸变

通过对黄曲霉细胞受柠檬醛损伤后线粒体形态畸变的透射电镜观察,发现柠檬醛所产生胁迫环境影响线粒体DNA复制系统,产生增生变异的巨型线粒体而与之应答。丙二醛法测定黄曲霉细胞内自由基,结果表明药物进入细胞后还通过诱发自由基使线粒体损伤,致使氧化还原系统及细胞能量代谢途径受到影响。     

柠檬醛致黄曲霉孢子丧失萌发力的机制

通过由倒置显微镜、衍射光栅和线阵光电偶合器件CCD(chargecoupleddevice)等构成的显微多道分光光度系统及由计算机DEPHI编程工具编制的单细胞凝胶电泳SCGE(single cellgelelectro phoresis)图像分析系统 ,摄取荧光显微镜所呈图像 ,再由图像捕捉卡将CCD产生的图像信号送入计算机 ,将柠檬醛对黄曲霉质膜和核DNA损伤的图像进行显示存储和分析处理 ,测定彗星长度、荧光强度、矩类及头尾DNA含量比等彗星参数指标 .结果发现Olive尾矩、尾长、尾分布矩等彗星尾参数指标与柠檬醛致黄曲霉损伤浓度呈正相关性 ,当致损浓度达到 1 5mg L以上时 ,DNA损伤为致死性损伤 ,不能被细胞内修复系统所修复 .揭示柠檬醛通过损伤质膜而进入细胞 ,对DNA产生不可逆损伤 ,使孢子失去萌发力的机制 .实现将DNA损伤的生化定性检测推进到数值化研究范围 ,为柠檬醛的开发应用提供了重要理论依据 .与国内外同类技术相比 ,本检测观察系统还具有高灵敏度、快速、无扰、多光谱显微测定之特点 .     

柠檬醛损伤黄曲霉线粒体生化机理的研究

应用生物化学方法并结合扫描电镜,研究柠檬醛掺入黄曲霉细胞,并通过损伤线粒体(Mt),导致抑制其生长的机理。结果表明,在药物致敏浓度时,菌丝体经该醛作用后,胞内Mt呈不规则增多,氧化还原反应系统受到破坏,与对照组相比,柠檬醛组的琥珀酸脱氢酶(Succinate Dehydrogenase,SDH)、苹果酸脱氢酶(Malate Dehydrogenase,MDH)活性分别呈不可逆下降271%和242%,随着药物浓度的升高,SDH、MDH的活性直至消失;以琥珀酸、α酮戊二酸和丙酮酸为底物时,线粒体呼吸速率分别下降24.1%、14.3%和36.1%,提示柠檬醛能使菌丝体DNA、RNA、脂类和蛋白质等生物合成受到抑制,促进细胞死亡。     

柠檬醛顺反异构体对黄曲霉超微结构及膜功能的影响

研究柠檬醛顺反异构体（香叶醛和橙花醛）抗菌性是阐明该醛抗菌机理核心所在.用酶转化法合成香叶醛和橙花醛后,用液态或气态的异构单体分别对黄曲霉孢子及菌丝体进行毒化.采用透射电镜、多维显微及激光拉曼散射技术对毒化的黄曲霉孢子及菌丝体进行显微结构观察和膜相关参数的测定.结果表明,无论是液体还是气体毒化方式,柠檬醛顺反异构体单独存在时均有抗黄曲霉作用;二者混合物的抗菌总活性与单体相比表现出一定程度协同性;二个异构单体的抗菌作用不仅表现为破坏黄曲霉超微结构,而且还反映在损伤其细胞膜体积调节功能及变形能力.     

植物精油对大菱鲆弧菌的体外和体内抗菌活性

【目的】研究天然植物精油对大菱鲆弧菌的体外和体内抗菌活性。【方法】采用纸片扩散法和微量肉汤稀释法对14种植物精油或其组分的体外抑菌活性进行检测;通过细菌形态透射电镜观察、胞内乳酸脱氢酶及核酸释放研究山苍子精油对大菱鲆弧菌的膜损伤作用;采用大菱鲆人工攻毒感染实验研究山苍子精油的体内抗菌作用。【结果】14种植物精油或其组分对大菱鲆弧菌具有不同程度的抑制效果,其中肉桂醛的抗菌活性最强,最低抑菌浓度为0.25μL/m L;百里香酚、丁香酚、柠檬醛和山苍子的抗菌活性次之,最低抑菌浓度为0.5μL/m L;山苍子精油可破坏大菱鲆弧菌的细胞膜,并导致胞内蛋白酶和核酸外泄;经200μL/L山苍子精油浸浴后,大菱鲆攻毒后死亡率由对照组50%降至0。【结论】富含芳香醛、芳香酚和柠檬醛的植物精油对大菱鲆弧菌具有良好抗菌活性,有望替代抗生素用于大菱鲆弧菌病的防治。     

彗星系统定量检测柠檬醛损伤黄曲霉DNA的研究

理化因素致细胞DNA损伤,彗星测试提供了一个直观的方法。采用新型SCGE图象分析系统(IMI10),将细胞显微分光光度分析与显微成像及图象分析结合,直接检测柠檬醛致黄曲霉核DNA损伤,与国际流行的SCGE图象分析系统相比,具有分析速度快、便于分析,同时具有中英文可切换界面和多格式输出打印特点。该系统使彗星试验的检测时间缩短2/3,并提高了准确性,可实现对活细胞多种结构参数、细胞内分子与膜的变化状况同时进行长时间连续的动态瞬间监测,具有广阔应用前景。     

小麦纹枯病菌侵染过程的组织学研究

本文报道了小麦纹枯病菌(Rhizoctonia cerealis)侵染小麦的过程。病菌在穿透寄主之前产生侵染垫、菌丝圈以及形态简单的单附着胞等侵染结构。由侵染垫基部菌丝或附着胞产生的侵染菌丝直接或通过气孔侵入寄主,也可见菌丝直接侵入寄主;菌丝侵入寄主表皮后,迅速在受侵细胞内呈网状扩展,并直接穿透毗邻细胞壁,向其它细胞纵横扩展。受病组织出现细胞变形、变空;接近菌丝的质膜发生质壁分离,质膜断裂：叶绿体变形、变小或接近消失,类囊体被破坏,叶绿体内嗜饿颗料减少或无;线粒体解体等系列组织病变。     

柠檬醛熏蒸对互隔交链孢生长及其产毒的抑制作用

互隔交链孢是一种重要的能产生交链孢酚（AOH）等真菌毒素的植物病原菌。精油是重要的抑制病原菌侵染的挥发性植物提取物,其活性组分包括柠檬醛等。本研究表明柠檬醛可高效地抑制互隔交链孢的生长和AOH毒素的产生。柠檬醛熏蒸能够引起互隔交链孢菌丝断裂影响其延伸,而对其分生孢子结构的影响不明显。柠檬醛能够引起互隔交链孢活性氧生成的紊乱,这可能是导致AOH显著下降的原因之一。由于柠檬醛能高效抑制互隔交链孢生长和产毒,因此其可作为传统熏蒸剂的潜在替代品,以防控互隔交链孢引起的病害以及毒素污染。柠檬醛抑制互隔交链孢生长产毒的研究为其开发与应用奠定了良好的基础。     

小麦纹枯病菌侵染过程的组织学研究

本文报道了小麦纹枯病菌侵染小麦的过程,病菌在穿透寄主之前产生侵染热 丝圈以及形态简单的单附着胞等侵染结构,由染垫基部丝或附产丰胞产生的侵染菌丝直接或通过气孔侵入寄主,也可见菌丝直接侵入寄主;菌丝侵入寄主表皮后,迅速在受侵细胞内呈网状产扩展,并直接穿透毗邻细胞壁,向其它细胞纵横扩展,受病组织出现细胞变形,变空;接近菌丝的质膜发生质壁分离,质膜断裂,叶绿体有,变小或接近消失,类本被破坏,叶绿体内嗜颗粒     

Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent

Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 μm membrane. Toxigenic and atoxigenic conidial mixtures (50∶50) placed on both sides of these filters restored inhibition. There was ～50% inhibition when a 12 μm pore size filter was used. Conidial and mycelial diameters were in the 3.5-7.0 μm range and could pass through the 12 μm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3-5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.     

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates,collected from peanut fields in China

Aims: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Methods and Results: Analysis of random‐amplified polymorphic DNA and microsatellite‐primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Conclusions: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Significance and Impact of the Study: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.     

Comparison of growth,nutritional utilisation patterns,and niche overlap indices of toxigenic and atoxigenic Aspergillus flavus strains

The effects of temperatures (20–30 °C) and water activity (0.90–0.99 a_w) on the lag phase duration, mycelial growth, and nutritional utilisation patterns of two toxigenic (AFL1⁺ & AFL2⁺) and three atoxigenic (AFL1⁻, AFL2⁻, & AFL3⁻) Aspergillus flavus strains were evaluated in vitro. Both temperature and a_w and their interactions had a significant influence on the growth and nutritional utilisation patterns (p < 0.05). There were no significant differences between toxigenic and atoxigenic strains in terms of lag phase prior to growth and mycelial growth rates. Based on carbon source (CS) utilisation patterns, toxigenic and atoxigenic strains' niche size was greater at higher temperatures and in wetter conditions. Additionally, based on niche overlap indices (NOIs), regardless of temperature, when water was freely available, atoxigenic and toxigenic strains co-existed. However, under moisture stress, the nutritional competitiveness was variable. Temporal carbon utilisation sequences (TCUS) of toxigenic and atoxigenic strains were compared. At 0.99 a_w most CS sources were utilised by the strains and the time to detection (TTD) of each strain was shortest on monosaccharides at the same level of a_w. Conversely, under moisture stress the least number of CS was utilised. The current study has demonstrated that carbon utilisation patterns are equally important as are other determinants of competitiveness and that growth rate alone is not a key attribute which determines competitiveness.     

Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.

Five strains of Aspergillus flavus lacking the ability to produce aflatoxins were examined in greenhouse tests for the ability to prevent a toxigenic strain from contaminating developing cottonseed with aflatoxins. All atoxigenic strains reduced contamination when inoculated into developing bolls 24 h prior to the toxigenic strain. However, only one strain, AF36, was highly effective when inoculated simultaneously with the toxigenic strain. All five strains were able to inhibit aflatoxin production by the toxigenic strain in liquid fermentation. Thus, in vitro activity did not predict the ability of an atoxigenic strain to prevent contamination of developing bolls. Therefore, strain selection for competitive exclusion to prevent aflatoxin contamination should include evaluation of efficacy in developing crops prior to field release. Atoxigenic strains were also characterized by the ability to convert several aflatoxin precursors into aflatoxin B1. Four atoxigenic strains failed to convert any of the aflatoxin biosynthetic precursors to aflatoxins. However, the strain (AF36) most effective in preventing aflatoxin contamination in developing bolls converted all tested precursors into aflatoxin B1, indicating that this strain made enzymes in the aflatoxin biosynthetic pathway.     

Biological control of sporidesmin-producing strains of Pithomyces chartarum by biocompetitive exclusion

The feasibility of using atoxigenic strains of Pithomyces chartarum for the biological control of toxigenic strains of P. chartarum was examined. Pasture, treated with atoxigenic strains of P. chartarum , contained up to 80% less sporidesmin than found in untreated pasture. Maximum sporidesmin levels of 26 ng g⁻¹ grass in treated pasture and 113 ng g⁻¹ grass in untreated pasture (means of 24 and four plots, respectively) were recorded 14 weeks after treatment, when spore numbers had reached a maximum of 80 000 spores g⁻¹ grass in the untreated plots and 50 000 spores g⁻¹ grass in the treated plots. This trial demonstrated that sporidesmin-producing spores of P. chartarum could be successfully reduced in pasture by the addition of atoxigenic strains, thereby reducing the risk of facial eczema in livestock.     

The relative effect of citral on mitotic microtubules in wheat roots and BY2 cells

The plant volatile monoterpene citral is a highly active compound with suggested allelopathic traits. Seed germination and seedling development are inhibited in the presence of citral, and it disrupts microtubules in both plant and animal cells in interphase. We addressed the following additional questions: can citral interfere with cell division; what is the relative effect of citral on mitotic microtubules compared to interphase cortical microtubules; what is its effect on newly formed cell plates; and how does it affect the association of microtubules with γ‐tubulin? In wheat seedlings, citral led to inhibition of root elongation, curvature of newly formed cell walls and deformation of microtubule arrays. Citral’s effect on microtubules was both dose‐ and time‐dependent, with mitotic microtubules appearing to be more sensitive to citral than cortical microtubules. Association of γ‐tubulin with microtubules was more sensitive to citral than were the microtubules themselves. To reveal the role of disrupted mitotic microtubules in dictating aberrations in cell plates in the presence of citral, we used tobacco BY2 cells expressing GFP‐Tua6. Citral disrupted mitotic microtubules, inhibited the cell cycle and increased the frequency of asymmetric cell plates in these cells. The time scale of citral’s effect in BY2 cells suggested a direct influence on cell plates during their formation. Taken together, we suggest that at lower concentrations, citral interferes with cell division by disrupting mitotic microtubules and cell plates, and at higher concentrations it inhibits cell elongation by disrupting cortical microtubules.     

Environmental distribution and genetic diversity of vegetative compatibility groups determine biocontrol strategies to mitigate aflatoxin contamination of maize by Aspergillus flavus

Maize infected by aflatoxin‐producing Aspergillus flavus may become contaminated with aflatoxins, and as a result, threaten human health, food security and farmers' income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the effectiveness of atoxigenic isolates in mitigating aflatoxin contamination. However, such information has not been used to facilitate selection and deployment of atoxigenic isolates. A total of 35 isolates of A. flavus isolated from maize samples collected from three agro‐ecological zones of Nigeria were used in this study. Ecophysiological characteristics, distribution and genetic diversity of the isolates were determined to identify vegetative compatibility groups (VCGs). The generated data were used to inform selection and deployment of native atoxigenic isolates to mitigate aflatoxin contamination in maize. In co‐inoculation with toxigenic isolates, atoxigenic isolates reduced aflatoxin contamination in grain by > 96%. A total of 25 VCGs were inferred from the collected isolates based on complementation tests involving nitrate non‐utilizing (nit^?) mutants. To determine genetic diversity and distribution of VCGs across agro‐ecological zones, 832 nit^? mutants from 52 locations in 11 administrative districts were paired with one self‐complementary nitrate auxotroph tester‐pair for each VCG. Atoxigenic VCGs accounted for 81.1% of the 153 positive complementations recorded. Genetic diversity of VCGs was highest in the derived savannah agro‐ecological zone (H = 2.61) compared with the southern Guinea savannah (H = 1.90) and northern Guinea savannah (H = 0.94) zones. Genetic richness (H = 2.60) and evenness (E₅ = 0.96) of VCGs were high across all agro‐ecological zones. Ten VCGs (40%) had members restricted to the original location of isolation, whereas 15 VCGs (60%) had members located between the original source of isolation and a distance > 400 km away. The present study identified widely distributed VCGs in Nigeria such as AV0222, AV3279, AV3304 and AV16127, whose atoxigenic members can be deployed for a region‐wide biocontrol of toxigenic isolates to reduce aflatoxin contamination in maize.     

Proteomic analysis reveals an aflatoxin-triggered immune response in cotyledons of Arachis hypogaea infected with Aspergillus flavus

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (Arachis hypogaea) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of Aspergillus flavus. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and A. flavus growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant-pathogen interaction.