解淀粉芽胞杆菌菌株HRH317对感染串珠镰孢菌玉米幼苗伏马毒素B1含量及相关防御酶活性的影响

为探讨解淀粉芽胞杆菌Bacillus amyloliquefaciens菌株HRH317对感染串珠镰孢菌Fusarium moniliforme玉米幼苗产生伏马毒素B_1(FB_1)的影响,采用牛津杯法测定菌株HRH317对串珠镰孢菌的抑制活性,并通过浸种处理进行盆栽试验,应用高效液相色谱技术对生长至3叶期后不同时间玉米幼苗叶片中FB_1含量进行测定,同时于室内测定玉米幼苗叶片防御酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)的活性。结果表明:解淀粉芽胞杆菌菌株HRH317能明显抑制串珠镰孢菌生长,抑菌圈直径平均可达33.31 mm;玉米幼苗生长至3叶期后1～6 d,菌株HRH317能有效抑制玉米植株体内FB_1含量,经串珠镰孢菌分生孢子悬浮液与菌株HRH317菌悬液1∶1混合液处理玉米种子后,对幼苗中FB_1的抑制率为59.20%～75.70%;而玉米种子先接种菌株HRH317菌悬液后接种串珠镰孢菌分生孢子悬浮液处理对幼苗中FB_1的抑制率为76.77%～88.10%。且这2种处理中幼苗叶片的SOD、CAT、PAL和POD活性均较对照有不同程度提高,其峰值是对照的1.24～5.45倍。表明解淀粉芽胞杆菌菌株HRH317可通过抑制FB_1产生来降低串珠镰孢菌对玉米幼苗的侵害,同时能诱导玉米植株体内防御酶活性的表达而增强其系统抗性,在防治玉米穗腐病方面具有潜在的应用价值。     

伏马菌素B1单克隆抗体的制备及鉴定

 【目的】真菌毒素可导致严重的食品安全问题。本试验旨在制备可用于检测伏马菌素B1的特异性单克隆抗体。【方法】制备伏马菌素B1人工抗原,杂交瘤细胞法筛选杂交瘤细胞株制备腹水型单克隆抗体,并对其特性进行鉴定。【结果】筛选得到杂交瘤细胞株F3,分泌的单克隆抗体亚类为IgG1,轻链类型为κ链;10%变性聚丙烯酰胺凝胶电泳显示单抗蛋白重链分子质量约为50 kD,轻链分子质量约为25 kD;间接酶联免疫吸附法测定杂交瘤细胞F3细胞培养上清和腹水效价分别为1:3 200和1:51 200;亲和力常数为1.60×10-8 mol•L-1;IC50值可达3.589 ng•mL-1;对其它真菌毒素不存在交叉反应;免疫转印法鉴定抗体具有高特异性。【结论】本试验制备FB1单克隆抗体具有较好亲和力和高特异性,具有良好的应用价值。     

Fumonisin B1 Metabolism by Bovine Liver Microsomes

Only limited and contrasting information is available about the metabolic fate in cattle of fumonisin B₁, a mycotoxin produced by moulds of Fusarium. This study was carried out to evaluate the hepatic metabolism of fumonisin B₁ by bovine liver microsomes. No biodegradation or metabolization of the mycotoxin by liver microsomes was detectable after incubating fumonisin B₁ with bovine microsomes in the presence of a regenerating system for 1 h. No aminopolyol 1, aminopolyol 2 or aminopentol, metabolites of fumonisin B₁, were detected in any of the incubated samples. The tolerance of ruminants to fumonisin B₁ is apparently not dependent on its detoxification in the rumen.     

The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides

The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi.     

镰刀菌及其毒素

镰刀菌易在温度为10℃,湿度40％-70％,pH值呈酸性和中性的马铃薯-葡萄糖琼脂培养基或者察氏培养基上生长。镰刀菌毒素主要为玉米赤霉烯酮、呕吐素、T-2毒素、伏马菌素、串珠镰刀菌素,对人类和养殖业具有不同程度的危害。     

玉米弯孢叶斑病菌Clg2基因克隆、序列特征和不同发育阶段表达分析

异源三聚体G信号通路在调控植物病原真菌的形态、侵染结构形成和致病力等方面具有重要作用。本研究根据玉米弯孢叶斑病菌新月弯孢全长cDNA文库中的EST序列,克隆获得一个含1 074bp开放读码框(ORF)Gα基因。该基因包含5个外显子和4个内含子,编码357个氨基酸,被命名为Clg2。通过序列比对和进化树分析,证明Clg2蛋白具有Gα蛋白结构特征和保守序列区,与小麦黄斑叶枯病菌(Pyrenophora tritici-repentis Pt-1C-BFP)和大斑刚毛座腔菌(Setosphaeria turcica)中的Gα蛋白相似性高达96%和97%,处于同一分支。采用实时荧光定量PCR技术分析了Clg2基因在病菌不同发育阶段的表达水平,显示在菌丝中表达水平最低,在分生孢子中表达量最高,暗示Clg2基因在病菌分生孢子形成期起重要作用。上述结果将为开展Clg2蛋白功能研究奠定基础。     

The food contaminant fumonisin B1 reduces the maturation of porcine CD11R1+ intestinal antigen presenting cells and antigen-specific immune responses, leading to a prolonged intestinal ETEC infection

Consumption of food or feed contaminated with fumonisin B₁ (FB₁), a mycotoxin produced by Fusarium verticillioides, can lead to disease in humans and animals. The present study was conducted to examine the effect of FB₁ intake on the intestinal immune system. Piglets were used as a target and as a model species for humans since their gastro-intestinal tract is very similar. The animals were orally exposed to a low dose of FB₁ (1 mg/kg body weight FB₁) for 10 days which did not result in clinical signs. However, when compared to non-exposed animals, FB₁-exposed animals showed a longer shedding of F4⁺ enterotoxigenic Escherichia coli (ETEC) following infection and a lower induction of the antigen-specific immune response following oral immunization. Further analyses to elucidate the mechanisms behind these observations revealed a reduced intestinal expression of IL-12p40, an impaired function of intestinal antigen presenting cells (APC), with decreased upregulation of Major Histocompatibility Complex Class II molecule (MHC-II) and reduced T cell stimulatory capacity upon stimulation. Taken together, these results indicate an FB₁-mediated reduction of in vivo APC maturation.     

Quantitative detection of Fusarium pathogens and their mycotoxins in South African maize

The distribution and co‐occurrence of four Fusarium species and their mycotoxins were investigated in maize samples from two susceptible cultivars collected at 14 localities in South Africa during 2008 and 2009. Real‐time PCR was used to quantify the respective Fusarium species in maize grain, and mycotoxins were quantified by multi‐toxin analysis using HPLC‐MS. In 2008, F. graminearum was the predominant species associated with maize ear rot in the eastern Free State, Mpumalanga and KwaZulu‐Natal provinces, while F. verticillioides was predominant in the Northwest, the western Free State and the Northern Cape provinces. In 2009, maize ear rot infection was higher and F. graminearum became the predominant species found in the Northwest province. Fusarium subglutinans was associated with maize ear rot in both years at most of the localities, while F. proliferatum was not detected from any of the localities. Type B trichothecenes, especially deoxynivalenol, and zearalenone were well correlated with the amount of F. graminearum, fumonisins with F. verticillioides, and moniliformin and beauvericin with F. subglutinans. This information is of great importance to aid understanding of the distribution and epidemiology of Fusarium species in South Africa, and for predicting mycotoxin contamination risks and implementing preventative disease management strategies.     

Impact of push–pull cropping system on pest management and occurrence of ear rots and mycotoxin contamination of maize in western Kenya

Push–pull involves intercropping of cereals with Desmodium as a “push” crop and planting Napier grass/Brachiaria as the “pull” crop at the border. The technology has been reported to effectively control stemborers, striga weed, and fall armyworm (FAW), and to improve soil nutrition, resulting in increased grain yield. This study evaluated the impact of stemborer and FAW management using this technology on incidence of maize ear rots and preharvest contamination of grains with aflatoxin and fumonisin in western Kenya. The study was conducted during three cropping seasons on maize grown under the push–pull system and as a monocrop. Incidence of stemborer and FAW damage was determined as percentage of damaged plants, while incidence of ear rots was determined as percentage of ears with symptoms. At harvest, fungi were isolated from kernels and aflatoxin and fumonisin were quantified using enzyme-linked immunosorbent assay. Stemborer and FAW damage was significantly (p = .001) reduced by over 50% under the push–pull system. There was also a significant (p < .001) reduction in the incidence of Fusarium verticillioides (60%) and Aspergillus flavus (86%), which was reflected in a reduced incidence of ear rots (50%) with the push–pull system (p = .001). Fumonisin in maize from push-pull farms was significantly (p = .048) reduced (39%) but the technology had no significant (p > .05) effect on aflatoxin. The study showed that push–pull is an effective strategy for managing maize ear rots and fumonisins, and therefore could play a role in improving food safety among smallholder maize farmers in the region.     

伏马毒素B1单克隆抗体的制备及间接竞争ELISA方法的建立

采用戊二醛法将半抗原伏马毒素B1分别与载体蛋白钥孔血蓝蛋白（KLH）和蛋白卵清白蛋白（OVA）偶联成为完全抗原KLH-FB1和OVA-FB1。OVA-FB1作为包被抗原建立ELISA方法;KLH-FB1作为免疫原免疫8周龄Balb/c小鼠,应用杂交瘤细胞融合技术制备单克隆抗体。经三次亚克隆后,筛选出两株（6H3,6H11）能特异、稳定分泌抗FB1单克隆抗体的杂交瘤细胞株。选择其中一株杂交瘤细胞通过体内诱生腹水的方法制备单克隆抗体,经检测表明单克隆抗体的亚型为IgG1,轻链为κ型。采用间接ELISA测得腹水纯化后效价达l∶16 000,抗体对伏马毒素的半数阻断浓度（IC50）为8.26 ng/mL,对玉米赤霉烯酮、黄曲霉素B1等结构类似物几乎不存在交叉反应,与其他结构的氯丙嗪、链霉素等无交叉反应。利用获得的抗FB1单克隆抗体,建立了间接竞争ELISA检测方法,检测灵敏度为0.44 ng/mL,检测线性范围为0.93~73.06 ng/mL,加标回收率在线性范围内达到回收率可达78.4%~102%。