光照对交链孢毒素合成的调控

目的 明确光调控交链孢（Alternaria）产真菌毒素的作用。方法 在光/暗培养条件下,比较分析光照对2种交链孢菌株（ATCC 66981和Pear-3）的菌丝生长、孢子形成以及毒素合成的影响。结果 持续白光照射对交链孢菌丝生长影响不显著,而显著抑制其孢子形成。光照刺激交链孢菌ATCC 66981中交链孢酚（alternariol, AOH）、交链孢酚单甲醚（alternariol monomethyl ether, AME）和腾毒素（tentoxin, TEN）3种毒素的产生。菌株持续光照培养30天,AOH、AME和TEN毒素浓度分别为120、182和173 g/L;较之黑暗培养,分别增加了1.1、5.9和9.6倍。光照抑制交链孢菌Pear-3中细交链孢菌酮酸（tenuazonic acid, TeA）毒素的合成。菌株光照培养6天,TeA毒素浓度即已达142 g/L;而黑暗培养甚至高达325 g/L,毒素产率反而增加了1.3倍。结论 光照不影响交链孢菌丝生长,而对孢子形成影响显著;持续白光照射能够促进交链孢中AOH、AME和TEN毒素的产生,而抑制TeA毒素的产生。     

胶束电动毛细管色谱法分析3种交链孢毒素主成分含量

目的 建立胶束电动毛细管色谱法(micellar electrokinetic chromatographic, MEKC)测定3种交链孢毒素[交链孢酚(alternariol, AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)和交链孢菌酮酸(tenuazonic acid, TeA)]主成分含量的新分析方法。方法 以未涂层熔融石英毛细管柱(50μm×30 cm, 20 cm, i.d.)为分离柱,分别以3种分离缓冲溶液[40 mmol/L Na_2B_4O_7-120 mmol/L十二烷基硫酸钠(sodium dodecyl sulfate, SDS)(含体积分数为5%的乙腈)、30mmol/LNa_2B_4O_7-40mmol/LSDS(含体积分数为30%的异丙醇)和60mmol/L Na_2B_4O_7-180 mmol/L脱氧胆酸钠]及2种样品缓冲溶液[8 mmol/L硼砂(含体积分数为57%的甲醇用于AOH和TeA)、8mmol/L硼砂(含体积分数为57%的异丙醇用于AME)]分析AOH、AME和TeA的主成分含量。结果 AOH、AME、TeA的方法精密度分别为0.05%、0.01%和0.21%(n=5),检出限分别为5、15和0.5 mg/L。主成分含量分别为99.94%、99.78%和97.28%,且与高效液相色谱法(high performance liquid chromatography, HPLC)的结果无显著差异(α=0.05)。结论 MEKC法可作为HPLC法测定3种交链孢毒素主成分含量方法的互补方法 ,能够满足3种交链孢菌毒素主成分含量分析的要求。     

农产品及其制品中交链孢酚和交链孢酚单甲醚研究进展

交链孢菌(Alternaria spp.)是一类重要的植物病原体,能产生多种毒素,其中交链孢酚(alternariol,AOH)和交链孢酚单甲醚(alternariol monomethyl ether,AME)是目前从食物中检出的比较普遍和主要的2种交链孢毒素,可广泛污染蔬菜、水果及谷物等农产品及其制品。本文对AOH和AME的结构及理化性质、毒性、产生影响因素、生物及化学合成和污染现状等进行了综述,并对交链孢毒素限量标准的制定、快速检测方法等进行了展望。     

番茄交链孢病原菌鉴定及产毒分析

目的 通过对番茄病果中链格孢病原菌的分离鉴定及产毒分析,为番茄采收后病害防控提供理论依据。方法 采集福建省5个县市番茄病果,通过切片和组织分离法从番茄病害部位分离纯化病原菌,结合形态学特征和分子生物学对病原菌进行鉴定。根据柯赫氏法则确定病原菌的致病性,并利用超高效液相串联质谱法对分离得到的交链格孢菌产毒能力进行检测分析。结果 在发病番茄病害部位共分离得到的 3株互隔链格孢菌株均具产毒能力,但毒素种类和产毒能力各有不同,其中NH06菌株产毒量最高,可产生细交链格孢酮酸(tenuazonic acid,TeA )、交链孢酚 (alternariol, AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)、交链孢烯(altenuene,ALT )、腾毒素 (tentoxin,TEN ) 5种毒素,而NP002和NH07仅检出产生4种毒素。结论 福建省番茄种植产区内存在交链格孢菌的污染,且产毒种类多,因此,在番茄种植产区内病果的处理应引起关注和重视。     

固相萃取-同位素稀释液相色谱-串联质谱法测定橄榄油中4种交链孢霉毒素含量及其分布状况

目的：建立固相萃取-同位素稀释液相色谱-串联质谱法测定橄榄油中4种交链孢霉毒素含量的方法并描述其在橄榄油中的分布状况。方法：将橄榄油混匀后取5 g(精确至0.001 g),经乙腈-磷酸二氢钠溶液混合溶液提取，固相萃取柱净化，收集净化液经水浴氮吹浓缩后，采用超高效液相色谱质谱仪测定，内标法定量。结果：交链孢菌酮酸(tenuazonic acid, TeA)在0～50 ng/mL、交链孢酚(alternariol, AOH)在0～20 ng/mL、腾毒素(tentoxin, TEN)在0～10 ng/mL、交链孢酚单甲醚(alternariol methyl ether, AME)在0～2 ng/mL范围内线性良好(R>0.99)。TeA、AOH、TEN、AME的检出限在0.20～2.00μg/kg范围之间。4种生物毒素加标回收率为92.25%～124.66%,精密度范围3.79%～10.58%,表明本方法样本检测量小、灵敏度高、准确度高、精密度高，重现性好，是检测橄榄油中交链孢霉毒素的理想方法。结论：橄榄油普遍受到交链孢霉毒素污染，检出率93.33%(28/30),AME毒素是橄榄...     

番茄交链孢菌病斑及其外延组织中交链孢毒素的分布

目的 检测不同大小的交链孢菌病斑及其外延组织中交链孢毒素残留量, 明确交链孢毒素在番茄中的迁移规律。方法 样品经80%乙腈溶液提取后, 通过自制固相萃取柱排除杂质干扰, 流出液经氮吹至近干后, 采用超高效液相色谱-串联质谱仪(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)测定交链孢毒素残留量。结果 不同大小病斑组织中均检测到高浓度的4种交链孢毒素残留, 包括交链孢酚(alternariol, AOH), 交链孢酚单甲醚(alternariol monomethyl ether, AME), 细交链格孢酮酸(tenuazonic acid, TeA)和交链孢烯(altenuene, ALT); 外延组织中仅在病斑周围1 cm处检测到TeA, 含量为病斑组织的1/10左右。结论 番茄交链孢毒素能向病斑外延组织扩散, 但扩散情况与病斑大小无直接联系, 病斑周围2 cm处虽然用超高效液相色谱-串联质谱仪检测不到交链孢毒素, 但仍存在安全风险, 因此, 建议食用时或生产中将病斑及其外延2 cm范围内组织均剔除。     

白光光照对交链孢毒素合成的调控研究

目的研究白光光照对交链孢(Alternaria)毒素合成的影响。方法在光/暗培养条件下,比较分析白光光照对2种交链孢菌株(ATCC 66981和Pear-3)的菌丝生长、孢子形成以及毒素合成的影响。结果持续白光照射对交链孢菌丝生长影响不显著,而显著抑制其孢子形成。光照刺激交链孢菌ATCC66981中交链孢酚(alternariol, AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)和腾毒素(tentoxin, TEN)3种毒素的产生。菌株持续光照培养30d,AOH、AME和TEN毒素浓度分别为120、182和173?g/L;较之黑暗培养,分别增加了1.1、5.9和9.6倍。光照抑制交链孢菌Pear-3中细交链孢菌酮酸(tenuazonic acid, TeA)毒素的合成。菌株光照培养6 d, TeA毒素浓度达到142?g/L;而黑暗培养后其浓度高达325?g/L,毒素产率增加了1.3倍。结论光照不影响交链孢菌丝生长,而对孢子形成影响显著;持续白光照射能够促进交链孢中AOH、AME和TEN毒素的产生,而抑制TeA毒素的产生。     

食品中4种交链孢毒素对肝脏细胞的联合毒性

交链孢毒素是一类广泛存在的食品污染物,交链孢酚(AOH)、交链孢酚单甲醚(AME)、细交链孢菌酮酸(TeA)、腾毒素(TEN)经常同时出现,然而目前对其联合毒性的评估十分有限。目的:以BRL-3A细胞为研究模型,评估常见4种交链孢毒素对肝脏细胞的联合毒性。方法:应用CCK-8试剂盒检测10~200 μmol/L交链孢毒素单独或联合处理BRL-3A细胞24,48,72 h细胞活性的变化。基于组合指数法,分析它们混合暴露后的联合毒性效应。结果:AOH、AME、TeA和TEN单独处理BRL-3A细胞后,能够降低细胞存活率,并呈现剂量和时间依赖性,其IC50分别为38.11,41.25,49.05,358.58 μmol/L(48 h)。AOH-AME、AME-TEN和TeA-TEN组合的毒性在IC10~IC90水平上都表现出累加或协同效应。AOH-AME-TeA、AME-TeA-TEN和AOH-AME-TeA-TEN组合的毒性在低剂量时具有拮抗效应,在高剂量时具有协同效应,而其它组合与之相反。结论:食品中常见的4种交链孢毒素均能不同程度地抑制BRL-3A细胞的增殖,具有肝脏细胞毒性,其毒性强度顺序为AOH > AME > TeA >> TEN。当它们同时出现时,其毒性作用可能为协同效应,提示今后应重点关注毒素的联合毒性风险,以更精准地评估其安全风险。     

小麦产毒病原菌及其毒素防控的研究进展

小麦是全球第二大粮食作物,每年因病害造成小麦严重减产,品质下降。一些病原菌还能够产生真菌毒素,进一步危害小麦及其制品的质量安全,对人畜健康造成巨大危害。由镰刀菌引起的小麦赤霉病是我国最主要的小麦病害之一,由交链孢引起的小麦黑胚病也备受关注。这2种真菌既能引起小麦病害,又能产生真菌毒素,故称之为产毒病害。镰刀菌产生的脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)、玉米赤霉烯酮(zearalenone,ZEN)、伏马菌素等和交链孢产生的交链孢酚(alternariol,AOH)、交链孢酚单甲醚(alternariol monomethyl ether,AME)和细交链格孢酮酸(tenuazonic acid,Te A)等是2类病原菌产生的主要真菌毒素。本文综述了能引起小麦产毒病害的镰刀菌和交链孢的特点、真菌毒素以及病害和毒素的防控技术,尤其是2类病原菌引起的小麦病害和真菌毒素的防控。这将为后期防治小麦产毒病害及控制毒素产生的研究提供有利参考。     

链格孢霉菌侵染番茄产毒机制

为研究链格孢霉侵染番茄后的产毒机制,本实验将链格孢霉孢子悬液接种于蕃茄上,并分别置于4 ℃和25 ℃下培养,15 d内进行3 次取样。样品经高分辨质谱检测后,使用MZmine、Xcalibur软件对检测结果进行分析,原质谱数据集经Mzmine软件处理后导入全球天然产物社会分子网络平台,采用基于特征的分子网络（feature-based molecular networking,FBMN）建立真菌毒素及代谢物分子网络,实现对链格孢毒素代谢通路的可视化分析。结果表明,接种后番茄在4 ℃下未产生任何毒素,25 ℃下链格孢酚（alternariol,AOH）、链格孢甲基醚（alternariol monomethyl ether,AME）、细链格孢菌酮酸（tenuazonic acid,TeA）、腾毒素（tentoxin,TEN）在5、10、15 d时均被检出,链格孢霉烯（altenuene,ALT）直至15 d时才被检出,3 次取样中TeA含量均最高。利用MZmine软件的碎片诊断过滤功能和Xcalibur软件精确分子质量技术验证了AOH和AME硫酸盐结合的隐蔽型真菌毒素的存在。FBMN分析结果表明3 种毒素（AOH、AME、TeA）有明显的代谢通路,代谢产物的种类较多,而未在番茄中检测到TEN和ALT代谢产物。本研究阐明了链格孢霉毒素在番茄中的产生及代谢机制,可为番茄中链格孢毒素的有效控制及番茄的贮藏保鲜提供理论支持。     

继代培养对链格孢菌生长及产毒的影响

目的 探究连续继代培养对链格孢菌(Alternaria alternata)菌株的生长以及产毒的影响。方法 将10株链格孢菌在马铃薯葡萄糖琼脂培养基(potato dextrose agar, PDA)上连续继代培养,分析菌株的生长、产孢情况;利用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)检测并分析主要链格孢霉毒素[包括交链孢酚(alternariol,AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)、交链孢烯(altenuene, ALT)、细交链格孢菌酮酸(tenuazonic acid, TeA)]的含量变化。结果 在PDA培养基中,连续继代培养链格孢菌极易出现角变、菌丝变白、菌丝生长速率加快等菌株退化现象。随着继代培养次数的增加,菌株生长速度加快,菌落直径变大,角变率增大;10株链格孢菌中有7株(MY-4、MY-12、MY-15、MY-73、MY-80、MY-103、MY-117)产孢量随着继代次...     

Toxin production by Alternaria alternata in tomatoes and apples stored under various conditions and quantitation of the toxins by high-performance liquid chromatography

Alternaria alternata strain 8442-3 was inoculated into tomatoes (Lycopersicon esculentum Mill.) and Red Delicious cultivar apples (Malus domestica Borkh.). Half of the lots of each fruit were shrink-wrapped in high-density polyethylene film. Wrapped and unwrapped fruits were incubated under darkness at 4, 15 and 25 degrees C for up to 5 weeks. A high-performance liquid chromatography method was developed to quantitate tenuazonic acid (TeA), alternariol (AOH), alternariol methyl ether (AME), and altenuene (ALT). Shrink-wrapping retarded, but did not completely inhibit growth in tomatoes for 3-7 days. Concentrations of up to 120.6 mg of AOH and 63.7 mg of AME per 100 g of tissue were produced in unwrapped tomatoes stored at 15 degrees C for 4 weeks; 19.0 mg of ALT per 100 g of tomato tissue was produced after 3 weeks at 25 degrees C. AOH, AME and ALT were also produced in unwrapped tomatoes stored at 4 degrees C; however, no TeA was detected in decayed tomatoes, regardless of type of wrapping or storage temperature. Shrink-wrapping resulted in decreased production of AOH, AME, and ALT. Alternaria toxins were not detected in apples stored at 4 and 15 degrees C. The highest concentration of AOH produced (48.8 mg per 100 g of tissue) was in unwrapped apples stored at 25 degrees C for 2 weeks; 12.3 mg per 100 g of tissue of shrink-wrapped apples was detected after 5 weeks of storage at 25 degrees C, while ALT reached 5.7 mg per 100 g after 4 weeks. TeA was not detected in apples infected with A. alternata.     

PRODUCTION OF ALTERNARIOL AND ALTERNARIOL METHYL ETHER BY ALTERNARIA SPP.

Five isolates of Alternaria alternata, five isolates of A. tenuis, one isolate of A. hemicota and three isolates of unidentified Alternaria spp. were examined for their production of alternariol methyl ether (AME) and alternariol (AOH) in complex liquid media. The data were compared with the production of these two toxins on solid substrate rice cultures. A new high-performance liquid chromatography method with minimum detection levels for AME and AOH of 20 ng and 40 ng, respectively, was used to quantitate the toxins. All isolates produced AME and AOH, but the amount varied considerably with the isolates and media tested. Of the three different liquid media tested, the highest quantity of AME and AOH was produced in a modified Czapek-Dox broth. The highest yield of toxins was obtained in the solid rice cultures. As much as 125 mg of AME and 16 mg of AOH were produced by one A. tenuis isolate on 300 g of rice after 14 days of incubation at 25°C in darkness.     

Production of alternaria mycotoxins by Alternaria alternata isolated from weather-damaged wheat

The production of Alternaria mycotoxins by Alternaria alternata isolated from Chinese weathered wheat kernels were first investigated on polished rice and durum wheat grains. These mycotoxins included alternariol (AOH) and its monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), and tenuazonic acid (TA). Of 25 isolates tested, all were AOH and AME producers, 21 (84%) coproduced ALT and ATX-I, and 8 (32%) produced TA in rice culture. TA was the most abundant toxin produced at a level ranging from 1,369 to 3,563 mg/kg. Much smaller amounts of AOH, AME, ALT, and ATX-I were present with average concentrations of 54, 40, 44, and 8 mg/kg, respectively. There were linear correlations between the level of AOH and AME (r = 0.846), alternariols (AOH plus AME) and ALT (r = 0.785), and ATX-I and TA (r = 0.553). Polished rice medium seems to support a bit more production of Alternaria metabolites than wheat but with an insignificant difference in concentrations (P > 0.05). A study of the time-course of toxin production by A. alternata isolates indicated that AOH production began faster than any other toxins monitored, and ALT production exhibited a progressive increase throughout the experiment. TA producers might reveal their considerably higher ability to produce toxin in the field despite their low frequency.     

人工接种温州蜜柑后链格孢霉毒素的产生及分布规律

采用超高效液相色谱-串联质谱法结合Qu ECh ERS(quick,easy,cheap,effective,rugged,safe)前处理方法,研究温州蜜柑人工接种链格孢菌(Alternaria alternate)后,病斑部位全果、非病斑部位果皮和果肉中腾毒素(tentoxin,Ten)、链格孢酚(alternariol,AOH)、交链格孢酚单甲醚(alternariol monomethyl ether,AME)、交链孢烯(altenuene,ALT)和细交链格孢菌酮酸(tenuazonic acid,Te A)5种链格孢霉毒素的产生和分布规律。结果表明,病斑部位全果、非病斑部位果皮和果肉中的链格孢霉毒素含量随着病斑直径的扩大,不同链格孢霉毒素的变化趋势不同。Te A、Ten、AME和AOH在所有部位中均有检出,其中Te A检出含量最高,在病斑部位的含量范围为3.05×10~3~55.88×10~3μg/kg,在非病斑部位的含量范围为65.35~40.68×10~3μg/kg;Ten、AME和AOH在病斑部位的含量范围分别为69.16~373.94、22.63~1 395.82μg/kg和8.18~689.19μg/kg;非病斑部位的含量范围分别为0~67.56、0~195.96μg/kg和0~301.91μg/kg;ALT在非病斑部位果肉中未检出,但在非病斑部位的果皮和病斑部位的全果中均有检出,含量最高达16.61μg/kg。研究表明,由于柑橘感染链格孢菌后产生的毒素会从发病部位扩散到健康部位累积,因此,在鲜食加工和风险评估中应引起关注和重视。     

Natural occurrence of Alternaria mycotoxins in olives--their production and possible transfer into the oil

A limited survey of the natural occurrence of the major Alternaria mycotoxins, i.e. alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), and tenuazonic acid (TA) has been carried out on olives and related processing products (oil and husks). The toxigenicity of Alternaria strains isolated from olives and the possible mycotoxin transfer into the oil have also been investigated. Four out of 13 olive samples were contaminated by 2 to 4 Alternaria mycotoxins. The highest contamination was found in a badly damaged sample containing 2.9, 2.3, 1.4 and 0.3 mg/kg of AME, AOH, ALT and TA, respectively. No mycotoxins were detected in olive-oil destined for human consumption (6 samples) or olive-husks (3 samples) collected from oil-mills after the first pressing of olives. An oil sample produced in our laboratory by processing the most contaminated olive sample contained AME (0.79 mg/kg) and AOH (0.29 mg/kg). The estimated mycotoxin amount transferred into the oil was 4% for AME, 1.8% for AOH, and zero for ALT and TA (considering 15% the oil yield). Although Alternaria species, mostly A. alternata (Fr.) Keissler, were present at various extent on all the examined olive samples, mycotoxins were only detected in samples of physically damaged olives. The production of mycotoxins by A. alternata isolated from olives was much higher (up to 3 order of magnitude for TA) on rice cultures than on olive cultures.     

肉桂醛对链格孢菌生长及非寄主选择性毒素合成的影响

目的 初步探究肉桂醛对葡萄采后链格孢菌菌丝体生长及非寄主选择性毒素合成的影响。方法 采用扫描电镜(scanning electron microscopy, SEM)观察肉桂醛处理后链格孢菌菌丝体的形态结构; 测定处理后菌丝体脂质和麦角固醇含量, 胞外电导率、OD260值和菌丝体荧光强度; 高效液相色谱(high performance liquid chromatography, HPLC)测定处理后链格孢酚(alternariol, AOH)、交链格孢酚单甲醚(alternariol monomethyl ether, AME)含量的变化。结果 扫描电镜结果显示肉桂醛处理后链格孢菌菌丝体形态结构明显被破坏; 脂质和麦角固醇含量显著下降; 胞外电导率和OD260值以及菌丝体荧光强度显著增加; AOH、AME的含量显著下降。 结论 肉桂醛通过破坏细胞膜通透性和完整性来抑制链格孢菌菌丝体生长, 同时体内主要非寄主选择性毒素合成受到抑制。