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.     

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.     

Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether

The Alternaria toxins alternariol (AOH; 3,7,9-trihydroxy-1-methyl-6H-benzo[c]chromen-6-one) and alternariol methyl ether (AME, 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one) are common contaminants of food and feed, but their oxidative metabolism in mammals is as yet unknown. We have therefore incubated AME and AOH with microsomes from rat, human, and porcine liver and analyzed the microsomal metabolites with HPLC and GC-MS/MS. Seven oxidative metabolites of AME and five of AOH were detected. Their chemical structures were derived from their mass spectra using deuterated trimethylsilyl (TMS) derivatives, and from the information obtained from enzymatic methylation. Several of the metabolites were identified by comparison with synthetic reference compounds. AME as well as AOH were monohydroxylated at each of the four possible aromatic carbon atoms and also at the methyl group. In addition, AME was demethylated to AOH and dihydroxylated to a small extent. As the four metabolites arising through aromatic hydroxylation of AME and AOH are either catechols or hydroquinones, the oxidative metabolism of these mycotoxins may be of toxicological significance.     

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.     

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.     

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds.     

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.     

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。     

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。     

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...     

Natural occurrence of Alternaria mycotoxins in sorghum and ragi from North Bihar, India

The natural occurrence of major Alternaria mycotoxins i.e. alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin-1 (ATX-1) and tenuazonic acid (TA) has been investigated in sorghum (Sorghum vulgare Pers.) and ragi (Eleusine coracana Gaertn.) collected from North Bihar. Nine out of 20 sorghum samples, and three out of eight ragi samples, were found to be contaminated with one to three Alternaria mycotoxins. The toxin elaborating potential of Alternaria alternata (Fr.) Keissler strains isolated from sorghum and ragi have also been investigated. Out of 16 isolates of A. alternata (Fr.) Keissler, mycotoxins were detected in only 10.     

Survey of Brazilian tomato products for alternariol, alternariol monomethyl ether, tenuazonic acid and cyclopiazonic acid.

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TEA) are secondary metabolites of Alternaria species. Cyclopiazonic acid (CPA) is produced by fungi belonging to the ubiquitous genera of Penicillium and Aspergillus and has been found in a wide range of foods. These toxins were searched for by liquid chromatography with diode-array detection in tomato products processed and sold in Brazil. Eighty samples of tomato products were examined (juice 11, pulp 22, purée 22, paste 24 and whole stewed tomato 1). Eleven brands sold nationwide were covered. TEA was found in seven samples of tomato pulp (39-111 ng/g) and four samples of tomato puree (29-76 ng/g). CPA was found in six samples of pulp (64-178 ng/g) and two samples of purée (36-117 ng/g). Co-occurrence of TEA and CPA was found in two samples of purée and one of pulp. This is the first time that the presence of CPA has been reported in tomato products. Neither AME nor AOH were detected in the samples.     

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.     

Toxigenic Alternaria species from Argentinean blueberries

Blueberries are traditionally consumed in North America, some European countries and Japan. In Argentina, the blueberry crop is profitable because production starts in November, when the northern hemisphere lacks fresh fruit. Fungal contaminants can grow and produce mycotoxins in fresh fruit. The aims of this work were to identify the main genera of the mycobiota of blueberries grown in Argentina and to determine the toxicogenic potential, pathogenicity and host specificity of the species isolated. The genus Alternaria was the main component of the blueberry mycobiota (95%); minor proportions of Phoma spp. (4%) and Penicillium spp. (1%) were also isolated. According to their sporulation patterns, 127 Alternaria isolates belonged to the Alternaria tenuissima species-group, 5 to the Alternaria alternata species-group and 2 to the Alternaria arborescens species-group. The last mentioned species-group was not isolated at 5°C. Of the 134 isolates, 61% were toxicogenic in autoclaved rice; 97% of these produced alternariol (AOH) in a range from 0.14 to 119.18 mg/kg, 95% produced alternariol methylether (AME) in a range from 1.23 to 901.74 mg/kg and 65% produced tenuazonic acid (TA) in a range from 0.13 to 2778 mg/kg. Fifty two isolates co-produced the three mycotoxins. According to the size of the lesion that they caused on blueberries, the isolates were classified as slightly pathogenic, moderately pathogenic and very pathogenic. No significant differences in pathogenicity were found on different blueberry varieties. In this work, high incidence and toxicogenic potential of the Alternaria isolates from blueberries were demonstrated. Thus, more studies should be done to evaluate the health risk posed by the presence of the Alternaria toxins in blueberries and in the manufactured by-products.     

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。     

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。     

AFLP variability, toxin production, and pathogenicity of Alternaria species from Argentinean tomato fruits and puree

Large amounts of tomato fruits and derived products are produced in Argentina and may be contaminated by Alternaria toxins. Limited information is available on the genetic variability, toxigenicity, and pathogenicity of Alternaria strains occurring on tomato. We analyzed 65 Alternaria strains isolated in Argentina from tomato fruits affected by black mould and from tomato puree, using amplified fragment length polymorphisms (AFLPs) technique. AFLP analysis resolved the set of strains in 3 main clusters (DICE similarity values of 58 and 60%) corresponding to A. alternata/tenuissima (44 strains), A. arborescens (15 strains) and to an unknown group (6 strains). Most of the representative strains, belonging to each AFLP cluster, when cultured on rice, produced tenuazonic acid (up to 46,760 mg/kg), alternariol monomethyl ether (AME, up to 1860 mg/kg), and alternariol (up to 70 mg/kg). The toxin profile related to the strains was not related to any AFLP cluster, except for AME which was produced at lower level by A. arborescens. Most of strains were pathogenic on two types of commonly cultivated tomato fruits. These findings provide new information on the variability within the Alternaria species complex associated with tomato disease.     

Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices in vitro

Scope: Monohydroxylation of alternariol (AOH) and alternariol‐9‐methyl ether (AME) has previously been reported as a prominent metabolic route under cell‐free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance. Methods and results: To clarify whether hydroxylation of AOH and AME occurs under in vivo‐like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision‐cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O‐methylation products, as catalyzed by catechol‐O‐methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct‐cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O‐methyl ethers. Conclusion: These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.     

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。