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。     

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...     

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。     

红枣及其制品链格孢毒素的QuEChERS-UPLC-MS/MS检测方法建立及其污染分析

为进一步掌握红枣中链格孢毒素的污染水平,本研究以市售红枣及其制品为研究对象,探索以QuChERS为核心的真菌毒素提取方法,建立超高效液相色谱-串联质谱的红枣5种链格孢毒素分析技术,并分析了红枣及其制品中链格孢毒素的污染水平。结果表明:采用QuChERS方法提取固体样品中链格孢毒素回收率在74.89%~98.04%;采用固相萃取法提取液体样品中链格孢毒素回收率在68.35%~99.63%,相对标准偏差均小于10%。对阿克苏、喀什、和田三个地区的骏枣原料中5种链格孢毒素进行测定,共检出细交链孢菌酮酸（TeA）、交链格孢酚（AOH）2种毒素,其中喀什地区红枣中TeA最高,达到5.12~10.76 μg/kg。在11种红枣制品中检出TeA、AOH、ALT 3种链格孢毒素,其中红枣酒中TeA达到127.08 μg/kg,仅紫晶枣中检出AOH,含量在34.76~98.61 μg/kg;在冻干枣片、香酥脆枣、紫晶枣中检出ALT,含量在2.04~399.64 μg/kg。本文建立的UPLC-MS/MS方法可满足不同形态红枣样品中链格孢毒素的测定要求,为红枣的食用安全提供理论依据。     

短波紫外线处理对3种链格孢毒素的降解作用研究

目的研究短波紫外线(UV-C)辐照对交链孢酚单甲醚(alternariol monomethyl ether, AME)和交链孢酚(alternariol,AOH)、腾毒素(tentoxin,TEN)3种链格孢毒素的降解效果。方法测定不同辐照时间、不同辐照距离、不同pH值条件下、不同照射强度下UV-C辐照对3种毒素的影响,采用高效液相色谱技术测定毒素含量。结果经过UV-C辐照过后, 3种毒素的浓度均随着辐照时间的延长而降低,随着辐照强度的增强而降低,但是辐照距离与毒素溶液的pH值大小与毒素的降解情况并无显著关联。在pH为5,毒素浓度为1.0μg/mL,UV-C灯功率为36 W、辐照距离为25 cm条件下照射120 min后, AME、AOH、TEN 3种毒素的降解率分别为16.10%、63.92%、89.99%。结论在偏酸性环境及长时间的UV-C辐照暴露下, UV-C对链格孢毒素有较为明显的降解作用。     

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。     

加压毛细管电色谱-固相萃取测定链格孢霉毒素

以霉变柑橘为材料,建立同时检测链格孢酚甲基乙醚(AME)、链格孢酚(AOH)和链格孢毒素(ALT)3种链格孢霉菌毒素的固相萃取-加压毛细管电色谱(SPE-p CEC)快速检测、通过乙腈提取,无水Mg SO4和Na Cl脱水盐析,C_(18)SPE萃取净化,以C_(18)毛细管色谱柱为分离柱,以含0.1%甲酸的乙腈为流动相进行梯度洗脱,利用荧光检测器进行检测。该方法在2μg/L~200μg/L范围内线性关系良好,相关系数r2≥0.991 5,检出限为0.1μg/kg~0.9μg/kg。结果表明,该方法快速、准确、灵敏,适用于食品、水果中常见的3中链格孢霉毒素的快速分析及检测。     

QuEChERS-UPLC-MS/MS测定焙烤食品中4种链格孢菌毒素

建立了超高效液相色谱-串联质谱法测定焙烤食品中4种链格孢菌毒素(腾毒素、链格孢菌酚、交链胞酚单甲醚、细交链胞菌酮酸)快速检测方法。样品经乙腈-0.1 mol/L盐酸溶液超声萃取,萃取液经QuEChERS净化处理,在RP18色谱柱(2.1 mm×100mm,1.7μm)上以乙腈和1 mmol/L碳酸氢铵为流动相梯度洗脱,采用电喷雾离子源、正负离子多反应监测模式检测,外标法定量。结果表明,4种待测物在相应的浓度范围内线性良好,相关系数均大于0.999;方法定量限(S/N≥10)为0.03～0.3μg/kg;进行3个水平的添加回收实验,平均回收率(n=6)为83.2%～105.3%;相对标准偏差(RSD,n=6)为1.2%～6.4%。应用本方法检测45个焙烤食品样品,4种真菌毒素均有检出。其中24份样品检出AME,含量为0.1～4.6μg/kg;6份样品检出AOH,含量为0.47～1.5μg/kg;27份样品检出Te A,含量为0.6～3.7μg/kg;9份样品检出TEN,含量为0.3～2.1μg/kg。     

鲜食葡萄表皮生物性危害因子及链格孢毒素分析

为确定链格孢属真菌发酵液和鲜食葡萄中链格孢酚(AOH)、链格孢酚甲基乙醚(AME)、链格孢霉素(ALT)和细交链孢菌酮酸(TeA)4种链格孢霉毒素的含量,对我国两个鲜食主栽品种‘巨峰’‘红地球’的50份样品表皮的生物性危害因子进行分离鉴定。结果表明,鲜食葡萄表皮的生物性危害因子主要为曲霉属、链格孢属和青霉属真菌,其中链格孢属真菌主要为Alternaria alternata,而A. alternata的产毒发酵液中能检测到AOH、AME和TeA,其中TeA的含量较高。基于链格孢毒素的毒性,要加强葡萄及其制品的风险监测。     

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。     

臭氧处理对交链孢菌生长及其毒素积累的抑制作用

交链孢菌（Alternaria spp.）易侵染农作物,引起农产品病害,而且能够代谢产生交链孢毒素,包括细交链孢酮酸（tenuazonic acid,TeA）、交链孢酚（alternariol,AOH）、交链孢酚单甲醚（alternariol monomethyl ether,AME）等,严重影响人体健康。因此,亟需高效、安全的方法用以防控交链孢菌及其毒素积累。本实验研究了臭氧处理对体外互隔交链孢（A. alternata）生长及其产毒能力的影响。结果表明,臭氧处理组的菌落直径显著低于对照组（P＜0.05）,且臭氧处理可显著抑制互隔交链孢产生TeA、AOH、AME这3 种交链孢毒素;利用扫描电子显微镜观察臭氧处理后互隔交链孢的微观形态,发现孢子和菌丝发生了凹陷、褶皱、断裂等异常现象;臭氧处理后的交链孢菌对番茄果实的致病力明显减弱,同时交链孢菌的产毒能力明显降低,20 mg/L臭氧处理条件下TeA、AOH、AME含量比对照组分别减少了36.1%、89.9%、93.2%。此外,臭氧对TeA、AOH、AME具有降解作用,降解率随着臭氧质量浓度的增加和处理时间的延长而显著提高,TeA经过20 mg/L臭氧处理30 min即可被完全降解,AOH及AME经20 mg/L臭氧处理120 min后降解率可达90%以上。综上,臭氧处理可以作为农产品及其制品中互隔交链孢及其毒素污染的防治手段。     

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.     

Mycotoxin production by Alternaria strains isolated from Argentinean wheat

The toxigenic potential of Alternaria strains isolated from Argentinean wheat was investigated. A total of 123 strains were assayed for the production of tenuazonic acid (TA), alternariol (AOH) and alternariol monomethyl ether (AME). All but one of the isolates were able to produce at least one of the three mycotoxins. TA was produced by 72% of the strains (1-14782 mg/kg), AOH by 87% (4-622 mg/kg) and AME by 91% (7-2625 mg/kg). The average level of TA detected for all strains (1757 mg/kg) was higher than the average level of both alternariols (162 mg/kg for AOH and 620 mg/kg for AME). TA was the toxin produced at the highest concentration but in lower frequency. Most of the strains were able to synthesize more than one toxin: 74 isolates (60%) were positive for all three toxins, 30 (24%) for both AOH and AME, 5 (4%) for both TA and AME, and 2 (2%) for TA and AOH. The widespread occurrence of Alternaria in wheat and its ability to produce mycotoxins suggests the possible occurrence of its toxins in wheat naturally infected with this fungus.     

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.     

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...     

Production of alternariol and alternariol methyl ether by Alternaria alternata grown on fruits at various temperatures

Two toxigenic strains of the fungus Alternaria alternata (ATCC 56836 and ATCC 66868) were grown on surface-disinfected, fresh, ripe fruits and tested for the production of alternariol (AOH) and alternariol methyl ether (AME). Examined fruits included strawberries; red and green seedless grapes; concord grapes; red delicious, golden delicious, and gala apples; and blueberries. After inoculation, fruits were incubated at 4, 10 degrees C, or room temperature (approximately 21 degrees C) for up to 3 weeks. At weekly intervals, duplicate samples were analyzed for AOH and AME by using liquid chromatography. Results indicated that A. alternata and its metabolites were not a major problem in strawberries due to the presence of fast-growing molds like Rhizopus and Botrytis that outgrew and possibly inhibited Alternaria. Both Alternaria strains showed limited growth on apples, although fast-growing molds were not present after surface disinfection; AOH and AME were produced only by the ATCC 56836 strain on the golden delicious and gala varieties, (ranging from <0.1 to 5 microg/g and <0.1 to 14 microg/g for AOH and AME, respectively). Restricted growth of both strains without toxin production occurred in blueberries, whereas moderate growth and AOH (<0.1 to 3,336 microg/g) and AME (<0.1 to 1,716 microg/g) production took place in grapes.     

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.     

Contamination of fresh and dried tomato by Alternaria toxins in southern Italy

In the present investigation, fresh and dried tomato samples from markets and packinghouses located in Apulia region (southern Italy) were analysed for Alternaria toxins. All samples proved to be contaminated by tenuazonic acid (TeA); in particular, dried tomatoes were contaminated in the range 425–81,592 µg/kg, while fresh tomatoes were in the range 11–4560 µg/kg. The second most abundant toxin was alternariol monomethyl ether (AME), followed by tentoxin (TEN) and alternariol (AOH). Overall dried tomatoes were more contaminated than fresh ones, although this seemed not directly related to the presence of sodium chloride, utilized in the drying process. Five representative Alternaria isolates within those collected from samples proved to be one Alternaria arborescens (A215) and four Alternaria alternata. Within the latter species, one strain belonged to morphotype tenuissima (A216), and three to alternata (A214, A217 and A218). They were confirmed to produce TeA, AOH, and AME in vitro. This study demonstrates the possible risk for consumers’ health related to the consumption of contaminated fresh and dried tomatoes, and thus the need for suitable control strategies.