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。     

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。     

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。     

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。     

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。     

继代培养对链格孢菌生长及产毒的影响

摘 要：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(tenuazonicacid，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)产孢量随着继代次数的增加先增加后降低，3株(MY-65、MY-67和MY-108)产孢量随着继代次数的增加显著降低；10株链格孢菌中有4株可以产生TeA毒素，且TeA毒素的含量随着继代次数的增加先上升后下降，第五代菌株(F5)产毒能力最强。继代培养对ALT毒素含量影响较大，除MY-103以外，随着继代次数的增加，ALT毒素的含量显著降低(达91.13%~99.85%)。AOH和AME毒素含量随着继代次数的增加呈下降或者呈先上升后下降的趋势。结论 连续继代培养5代对链格孢菌的生长、产孢、产毒等均会产生较大影响。在菌株的实际应用过程中，应该避免菌株过多次的继代培养，尽量使用5代以内的链格孢菌。     

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。     

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。     

超高效液相色谱-串联质谱法测定麦芯粉中四种交链孢毒素

探讨固相萃取方法,超高效液相色谱串联质谱仪(UPLC-MS/MS)测定麦芯粉中4种交链孢毒素。样品经0.05 mol/L Na H2PO4(pH 3.0)-乙腈-甲醇超声提取(450+450+100,V/V),上清液过HLB固相萃取柱净化,以1.0 mmol/L氨水溶液(pH 8.3)-甲醇为流动相,经Waters CORTECS C18(4.6×100 mm,2.7μm)柱分离梯度洗脱,采用电喷雾负离子(ESI-)、多反应监测(MRM)模式检测;以基质加标工作曲线定量。细交链孢菌酮酸(TeA)、交链孢酚(AOH)在2.0μg/L~100μg/L浓度范围内,腾毒素(TEN)、交链孢酚单甲醚(AME)在0.2μg/L~10.0μg/L浓度范围内线性关系良好,相关系数(R2)均大于0.998。回收率为76.3%~107.5%,TeA、AOH检出限和定量限分别为1.0μg/kg和3.0μg/kg、TEN、AME检出限和定量限分别为0.1μg/kg和0.3μg/kg。该方法灵敏度高,简便,准确。适于测定麦芯粉中的交链孢毒素。     

Optimization of the Method of Detection of Metabolites Produced by the Alternaria Genus: Alternariol, Alternariol Monomethyl Ether, Altenuene, Altertoxin I and Tentoxin

ABSTRACT: A simple method has been developed in this work for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), tenuazonic acid (TA), and tentoxin by means of thin-layer chromatography from cultures of several strains of Alternaria. Strains were incubated in 2% malt extract broth for 7 d at 25 °C. For extraction of metabolites, chloroform was used. The solvent system benzene/methanol/acetic acid (92:6:2) was chosen. TLC plates were observed under ultraviolet light (254/366 nm). Results show that A. alternata IMI 354942 produced AOH, AME, ALT, ATX-I, tentoxin, and TA; A. alternata IMI 354943, AOH, AME, and TA; A. triticina IMI 289962 tentoxin; and A. triticina IMI 289680 AME, ATX-I, and TA.     

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer’s storage conditions. Sample extraction was performed using a liquid–liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g^–1. AME levels ranged between 11 and 137 ng g^–1, which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.     

Alternaria toxins in wheat from the Autonomous Province of Vojvodina,Serbia: a preliminary survey

Although Fusarium species remain a main source of mycotoxin contamination of wheat, in recent years, due to the evident climatic changes, other mycotoxigenic fungi have been recognised as important wheat contaminants. Alternaria species, especially A. alternata, have been found as contaminants of wheat as well as wheat-based products. Under favourable conditions A. alternata very often produce alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and others Alternaria toxins. The aim of the present study was to examine the presence of three Alternaria toxins (AOH, AME and TeA) in wheat samples harvested during three years (2011–13). To this end, 92 samples were collected during wheat harvesting from different growing regions of the Autonomous Province of Vojvodina, which represents the most important wheat-growing area in Serbia. The presence of Alternaria toxins was analysed by HPLC with electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-MS/MS). Among all the analysed wheat samples, 63 (68.5%) were contaminated with TeA, 11 (12.0%) with AOH and 6 (6.5%) with AME. Furthermore, the maximum and mean toxin concentrations were 2676 and 92.4 µg kg^?1, 48.9 and 18.6 µg kg^?1, and 70.2 and 39.0 µg kg^?1 for TeA, AOH and AME, respectively. Co-occurrence of three Alternaria toxins in wheat samples was detected in six samples; a combination of two toxins was found in two samples; and 64 samples contained one toxin. The results showed that among 92 analysed wheat samples, only 20 (21.7%) samples were without Alternaria toxins. The presence of Alternaria toxins was also investigated in terms of weather conditions recorded during the period of investigation, as well as with the sampling region. This study represents the first preliminary report of the natural occurrence of Alternaria toxins in wheat (Triticum aestivum) from Serbia.     

Alternaria toxins in Argentinean wheat,bran, and flour

Alternaria species have been reported to infect a wide variety of vegetables, fruits, and cereal crops. Wheat is one of the most consumed cereal worldwide. A sensitive HPLC-DAD methodology was applied to quantify alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TeA) in 65 samples of whole wheat, bran, and flour. The extraction methodology allowed extracting the three toxins simultaneously. Limits of detection in wheat were 3.4, 4.5, and 0.5 µg kg^?1 for AOH, AME and TeA, respectively. For bran, these data were 3.1, 4.5, and 12 µg kg^?1 and for flour 50, 70, and 14 µg kg^?1, respectively. The studied recoveries were higher than 70% and RSD was below 10%. Wheat and bran samples showed low AOH and AME contamination compared to TeA. The averages levels found for TeA in wheat, bran and flour were 19,190, 16,760, and 7360 µg kg^?1, respectively.     

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection

ABSTRACT

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λ_exc = 258, λ_em = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD < 8%, n = 6) for AOH and 67–91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg^–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.