Evaluation of wheat (Triticum aestivum L.) phenolic acids during grain development and their contribution to Fusarium resistance

The phenolic acid profiles of six cultivars of wheat with known tolerance to Fusarium head blight were evaluated during plant development from anthesis through maturity. Analysis by HPLC of grain at anthesis revealed that p-coumaric and ferulic acid were the two principal phenolic compounds present. The effect of these two phenolic acids on Fusarium species was evaluated in vitro. Both phenolic acids demonstrated significant reductions (p < 0.05) of Fusarium species growth at all concentrations tested. Ferulic acid is the primary phenolic acid in grain at all stages of development, and its concentration increased steadily during grain development prior to a 50% decrease during grain ripening. The accumulation of ferulic acid synthesis from anthesis until approximately 20 days after anthesis appears related with cultivar resistance to Fusarium. Concentrations of ferulic acid in the grain were similar at maturity, implying that the end-use quality would be similar for both resistant and susceptible cultivars.     

Fusarium mycotoxins: overlooked aquatic micropollutants?

Deoxynivalenol and zearalenone are among the most prevalent toxins produced by Fusarium spp. They have been investigated in food and feed products for decades but rarely in the environment. We therefore established solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) methods to quantify these mycotoxins at trace concentrations in aqueous natural samples. In a model emission study, we inoculated a winter wheat field with Fusarium graminearum and subsequently monitored deoxynivalenol and zearalenone in its drainage water. Before during and after harvest in June and July 2007, these toxins were emitted in concentrations from 23 ng/L to 4.9 microg/L for deoxynivalenol and from not detected to 35 ng/L for zearalenone. Simultaneously, in July and August 2007, deoxynivalenol was also detected in a number of Swiss rivers in concentrations up to 22 ng/L and zearalenone was present in several river samples below the method quantification limit. Other mycotoxins might be emitted from Fusarium-infected fields as well, because some of them are produced in similar amounts as deoxynivalenol and zearalenone and exhibit similar or even higher water solubility than deoxynivalenol. The ecotoxicological consequences of the presence of mycotoxins in surface waters remain to be elucidated.     

Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol

Deoxynivalenol belongs to a group of highly toxic fungal metabolites produced by Fusarium species that may contaminate food and animal feed, mostly grains. Three different monoclonal mouse anti-deoxynivalenol antibodies were compared for the development of a surface plasmon resonance (SPR)-based immunoassay for the selective and quantitative determination of deoxynivalenol in naturally contaminated matrices. A conjugate of deoxynivalenol with the protein casein was prepared and immobilized on the sensor chip surface. An excess of antibody was added to each test solution before the measurement. The assay was based on the competition for antibody binding between the immobilized deoxynivalenol conjugate on the sensor and the free deoxynivalenol molecules in the test solution. The deoxynivalenol-casein sensor could be reused more than 500 times without significant loss of activity using 6 M guanidine chloride solution for regeneration. The cross-reactivity of the three antibodies in the SPR assay was tested with other trichothecene mycotoxins (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, HT2-toxin, and T2-toxin). The only sample preparation was extraction with max 80 vol % acetonitrile and 10-fold dilution with the running buffer. The assay had an optimal range between 2.5 and 30 ng/mL deoxynivalenol in the test solution. Most results of the SPR-based assay were in agreement with liquid chromatography/tandem mass spectrometry measurements of naturally contaminated wheat samples.     

Reduction in fusarium toxin levels in corn silage with low dry matter and storage time

Under unfavorable climatic conditions, Fusarium spp. can contaminate corn plants in the field and produce toxins that are present at the time of ensiling. The stability of deoxynivalenol, fumonisins B1 and B2, and zearalenone in corn silage was tested over two consecutive years. Variables studied were corn dry matter (DM) and storage length and temperature. The concentration of all Fusarium toxins decreased upon ensiling ( P < 0.001). Increasing the length of storage and ensiling with low DM resulted in a higher rate of toxin disappearance, particularly for the water soluble toxins deoxynivalenol and fumonisin B1. Toxin disappearance ranged from 50% for zearalenone to 100% for deoxynivalenol. In contrast, temperature did not have any effect on stability ( P > 0.05). These results indicate that low DM at ensiling as well as a prolonged storage could be a practical way to reduce or eliminate some Fusarium toxins in contaminated silages.     

An improved method for the purification of the trichothecene deoxynivalenol (vomitoxin) from Fusarium graminearum culture

It was hypothesized that a simplified and efficient strategy could be developed for large-scale production and purification of the mycotoxin deoxynivalenol from Fusarium graminearum rice cultures for toxicological studies. F. graminearum R6576 was cultured on rice and extracted with methanol, and the extract was concentrated and subjected to silica gel low-pressure liquid chromatography (LPLC) under a hexane-acetone gradient system. Deoxynivalenol isolation was monitored by thin-layer chromatography, and fractions containing deoxynivalenol were pooled, concentrated, and applied to a second LPLC column under the same conditions. An enriched deoxynivalenol fraction was obtained, which yielded a crystalline material. Repeated crystallization yielded spectroscopically pure deoxynivalenol. The identity of this compound was confirmed by HPLC comparison to an authentic deoxynivalenol standard, FABMS analysis, and comparison of the (1)H and (13)C NMR spectra with published data. This simplified purification scheme eliminated many laborious steps and equipment previously required to obtain gram quantities of crystalline deoxynivalenol for biological testing in animal models.     

不同抗性蚕豆品种根系分泌物对枯萎病菌的化感作用及根系分泌物组分分析

蚕豆枯萎病是土传病害,其发生与蚕豆根系分泌物有密切关系。本文以3个枯萎病不同抗性蚕豆品种——‘89-147’(高抗)、‘8363’(中抗)和‘云豆324’(感病)为材料,通过水培试验收集根系分泌物,测定根系分泌物对镰刀菌孢子萌发和菌丝生长的影响,分析对枯萎病表现出不同抗性的蚕豆品种根系分泌物中糖、氨基酸和有机酸的含量,分离鉴定了根系分泌物中氨基酸和有机酸的组分。结果表明,抗病品种的根系分泌物抑制了尖孢镰刀菌的孢子萌发和菌丝生长,在加入5 mL中抗品种根系分泌物时,显著促进尖孢镰刀菌孢子萌发,但对菌丝生长无显著影响;而在加入1 mL感病品种根系分泌物时,就能显著促进尖孢镰刀菌孢子萌发和菌丝生长。不同抗性蚕豆品种根系分泌物中氨基酸总量和总糖含量随抗性的降低而升高,有机酸分泌总量则随蚕豆品种对枯萎病的抗性增加而升高。感病品种和中抗品种中检出15种氨基酸,而高抗品种中检出14种,组氨酸只存在于中抗品种中,脯氨酸仅在感病品种中检出,3个蚕豆品种根系分泌物中均未检出精氨酸。蚕豆根系分泌物中天门冬氨酸、谷氨酸、苯丙氨酸、酪氨酸和亮氨酸含量高,可能会促进枯萎病的发生,而蛋氨酸、赖氨酸和丝氨酸含量高可能抑制枯萎病发生。酒石酸仅在抗病品种中存在,根系分泌物中有机酸种类丰富,有助于提高蚕豆对枯萎病的抗性。蚕豆对枯萎病的抗性不同,根分泌物对镰刀菌孢子萌发和菌丝生长的影响也不同,而这种抗病性差异与蚕豆根系分泌物中糖、氨基酸、有机酸的含量和组分密切相关。     

Comparison of the Distribution and Occurrence of Fusarium graminearum and Deoxynivalenol in Hard Red Winter Wheat for 1993-1996

Hard red winter wheat samples collected from different locations in Kansas from the 1993, 1994, 1995, and 1996 harvests were plated to determine Fusarium graminearum infection and analyzed for deoxynivalenol by thin-layer and gas chromatography. Rainfall, temperature, and cultivar were important factors affecting the severity of F. graminearum infection as well as deoxynivalenol production. The 1993 and 1995 growing seasons had high percentages of samples infected with F. graminearum and contaminated with deoxynivalenol. Averaged over the four years, cultivars 2163 and Karl had significantly higher levels of infection than did TAM 107. These widely grown cultivars were used in comparison. Northeastern Kansas had the highest levels of F. graminearum infection and deoxynivalenol contamination but also had the lowest acreage planted to hard red winter wheat.     

氯化钾对玉米根系糖和酚酸分泌的影响及其与茎腐病菌生长的关系

通过溶液培养试验,研究了氯化钾（KCl）对不同抗性品种玉米根系糖和酚酸分泌量的影响,以及在不同浓度糖和酚酸的培养基上禾谷镰刀菌（Fusarium graminearum Schwabe）的生长状况,探讨KCl抑制玉米茎腐病发生过程中,根系分泌物中糖和酚酸所发挥的作用。结果表明,感病品种吉单327的总糖、还原糖和其蔗糖分泌量均高于抗病品种吉单180;正常供钾条件下,总糖、还原糖和蔗糖的分泌量均表现出不同程度的下降,且还原糖下降幅度最大。阿魏酸和绿原酸是玉米根系分泌物中主要的两种酚酸组分。抗病品种吉单180分泌的阿魏酸量明显高于感病品种吉单327,而绿原酸分泌量低于吉单327。正常供钾时,阿魏酸分泌量明显增加,但绿原酸分泌量则有所减少。一定浓度的蔗糖和葡萄糖均能显著促进F. graminearum的生长,且还原糖（葡萄糖）的促进效果较蔗糖更明显;阿魏酸和绿原酸均能抑制F. graminearum的生长,但阿魏酸的抑制效果远远高于绿原酸。KCl 可明显减少不同抗性品种玉米根系的还原糖分泌量,增加阿魏酸的分泌,从而一定程度上抑制了病原菌F. graminearum在根际的快速生长。     

The effect of fungicidal treatment on selected quality parameters of barley and malt

Protection of barley grain against contamination by fungi such as Fusarium spp., particularly by those producing mycotoxins, secondary metabolites with adverse health effects, is of principal importance. Fungicides applied immediately after full heading of spring barley is one method of direct protection. In this work, extensive two-year field experiments combined with a detailed chemical laboratory analysis (barley and malt) were performed with the aim to study the effect of previous crops, different fungicides, and other conditions on the selected barley and malt quality parameters (content of beta-glucans, pentosans, oxalic acid, deoxynivalenol, and gushing), while the main task was to follow the effect of the fungicide (used as a treatment to protect against pathogens, mostly Fusarium) on changes of the chemical composition in barley and malt, and gushing. It was found that the relationship between the studied factors and the parameters usually applied to the evaluation of barley and malt quality is quite complex and not straightforward. The responses show typical features of a multifactorial influence with both positive and negative correlations resulting in a decrease or increase in grain quality (concentrations of beta-glucans, pentosans, deoxynivalenol, and other studied parameters). The role of previous crops was also found to be important. The fungicides should be applied at the time of heading but not at the very beginning of this period.     

Doehlert matrix design for optimization of the determination of bound deoxynivalenol in barley grain with trifluoroacetic acid (TFA)

Fusarium head blight (FHB) is an impediment to barley production in many regions of the world. Tricothecene toxins, associated with FHB-infected grain, particularly, deoxynivalenol (DON), pose a serious threat to human and animal health. Recent research has suggested that a portion of the DON present on grain is bound and escapes detection through conventional determination. The objective of this study was to optimize a method for determination of nonextractable DON in barley grain using trifluoroacetic acid (TFA). A Doehlert matrix design was performed to determine the optimal conditions for time, temperature, and TFA concentration. These conditions were treated with 1.25 N TFA in 86:14 acetontrile/water for 54 min at 133 degrees C. Cleanup, derivatization, and determination of DON by a gas chromatography electron capture detector (GC-ECD) was as normal. Treatment of the test sample resulted in the release of an additional 58% DON under the optimized conditions and an increase of 9-88% in a set of verification samples.     

Impact of physicochemical parameters on the decomposition of deoxynivalenol during extrusion cooking of wheat grits

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus and is known to cause a spectrum of diseases in animals such as vomiting and gastroenteritis. It is found in cereals and cereal products as most processing techniques lead only to a partial reduction of deoxynivalenol levels. One technique with a reported relatively high impact on deoxynivaleol decomposition is extrusion cooking. In the current work, systematic studies of a range of physicochemical parameters, such as temperature, moisture, compression, residence time in the extruder, pH value, and protein content, on their impact on deoxynivalenol decomposition during extrusion cooking were performed. The analysis of deoxynivalenol was made by high-performance liquid chromatography--tandem mass spectrometry using a quick, easy, cheap, effective, rugged, and safe-based cleanup with 15-d(1)-deoxynivalenol as an internal standard. It could be shown that the reduction of deoxynivalenol levels is dependent on a set of parameters partially interacting with each other. Especially the moisture content and compression are key factors for the reduction of deoxynivalenol levels. A correlation between residence time of the mycotoxin in the extruder and deoxynivalenol degradation was also observed when screws without a compression factor were used. Generally, the reduction of deoxynivalenol levels was increased by the use of screws with a high compression factor. As known from cooking, deoxynivalenol could also be easily degraded by extrusion under alkaline conditions. Furthermore, an increase of the protein content of the starting material resulted in higher reduction rates of deoxynivalenol.     

玉米品种抗顶腐病遗传多样性分析及其应用

经鉴定和调查, 从25个玉米品种中选择抗病性较稳定的14个品种为材料, 其中6个品种包括病、健株, 共20个样品, 进行抗病遗传多样性的RAPD分析.结果表明, 不同品种的抗性遗传背景丰富, 20个样品经PCR扩增聚类后, 以遗传距离15划分, 可分为7个类群, 同一类群内的品种在抗病性方面有较强的相似性;同一品种的健株与病株间能产生特异性条带, 且存在一定的遗传距离, 说明植株感病后, 其遗传特性会发生改变.田间试验表明, 遗传特性差异大的感病品种与抗病品种轮作对顶腐病有较高的防效, 可一定程度缓解生产中倒茬难而造成病害流行的难题.     

Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.     

甘薯抗薯瘟病的苯丙烷类代谢研究

甘薯瘟病是甘薯的重要病害, 严重影响甘薯的品质和产量.本试验在甘薯苗期, 通过剪叶法接菌研究甘薯抗感病品种苯丙烷类代谢的变化规律.结果表明: 甘薯抗感病品种接菌后PAL、POD活性均有所提高, 且提高速度与品种抗性呈正相关; 抗病品种接菌后木质素和绿原酸含量显著增加, 感病品种接菌后绿原酸、总酚和类黄酮含量波动较大, 木质素含量降低.相关性分析表明, 接菌后木质素和绿原酸的积累与甘薯品种抗病性密切相关.接菌后PAL、POD活性迅速提高并积累木质素和绿原酸等抗病物质是甘薯抗薯瘟病的苯丙烷类代谢基础, 在抗病品种鉴定方面有一定的参考价值.     

抗病性不同大豆品种根面及根际微生物区系的变化 Ⅱ.连作大豆(重茬)根面及根际微生物区系的变化

采用平板计数法测定了两个抗根腐病连作大豆品种生育期间根面和根际微生物区系动态变化。结果表明,两个品种大豆根面细菌随生育期增加呈递减趋势,品种间无差异。合丰25(H25)的根际细菌数量随生育期呈递减趋势,绥农10号(S10)根际细菌数量从三叶期到鼓粒初期不断增加,到成熟期又急剧减少。感病品种H25根瘤重明显低于抗病品种S10。H25根面真菌和镰孢霉总数在三叶期和成熟期均高于S10,苗期是根腐病的主要发病期。总之,在连作条件下,无论抗病品种还是感病品种大豆成熟期其根面和根际细菌减少,真菌和主要病原菌(镰孢霉Fusarium)都会大量富集,表明根系分泌物对根面和根际的微生物种群有选择性地促进或抑制作用。     

植物防御酶与桉树对焦枯病抗性的关系

为筛选鉴定桉树对焦枯病(CylindrocladiumqulnqueSeptatumMorgan)抗性的生化指标,对福建省11个桉树主栽种系接种焦枯病菌前后关键防御酶系中的过氧化物酶(POD)和超氧岐化酶(SOD)活性及其同工酶变化进行了研究.结果表明:无论是桉树健叶还是接种后的病叶,体内POD、SOD活性均表现为抗病种系>中抗种系>中感种系>感病种系,呈规律性变化;接种前后POD同工酶谱带数与桉树对焦枯病的抗性间不呈规律性变化,而SOD谱带数与抗病性成正比.从抗病育种角度出发可将接种前后桉树POD、SOD活性和接种后SOD同工酶谱带作为桉树对焦枯病抗性早期鉴定的生化指标.     

Deoxynivalenol Content of Cereal Grain from Naturally Infected and Artificially Inoculated Plants in Field Trials in Norway

The Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3-acDON) were determined in grain samples from naturally infected and Fusarium culmorum inoculated plants in field experiments in Norway during 1992–1996. The mean DON content in trials with inoculated plants was 11.8 μg/g in spring oats, 11.3μg/g in winter wheat, 28.9 μg/g in spring wheat and 31.4 μg/g in spring barley. In the natural infection trials the mean DON content was 0.32 μg/g in spring oats, 0.22μg/g in winter wheat, 1.48μg/g in spring wheat and 0.54 μg/g in spring barley. Only small differences in DON content were observed among cultivars, and significant differences were found only in winter wheat in the inoculation trials, and in spring wheat in the natural infection trials. A significant correlation was observed between the 3-acDON and DON contents in the inoculated trials in all grain species, the mean ratio of 3-acDON to DON ranging from 0.011 in wheat to 0.071 in oats.     

Deoxynivalenol removal from barley intended as swine feed through the use of an abrasive pearling procedure

Samples of naturally contaminated hulled barley, with varying deoxynivalenol concentrations, were subjected to an abrasive type dehulling procedure. The remaining grain fractions were analyzed for weight remaining (%), deoxynivalenol (ppm), crude protein (%CP), neutral detergent fiber (%NDF), ash (%ASH), gross energy (GE; kcal/kg), and calculated digestible energy values (DE; kcal/kg). Following the initial 15 s of pearling, 85% of the grain mass remained. Additional pearling resulted in a linear decline of grain mass. Following 15 s of pearling, the grain contained 34% of the initial deoxynivalenol content, irrespective of the initial level of contamination. Further pearling resulted in continued significant (p < 0.05) reductions in the percent of deoxynivalenol remaining to a level of 7.9% after 120 s but with significant losses in grain mass. Pearling can serve as an effective means of reducing the deoxynivalenol content of barley, with improvements in nutrient levels. However, the need to reduce the deoxynivalenol content of contaminated barley to less than 1 ppm for swine will necessitate the removal of a significant amount of the grain mass for heavily contaminated samples.     

Populations of Pseudomonas solanacearum biovar 3 in naturally infested soil

Populations of Pseudomonas solanacearum biovar 3 were monitored in a clay loam soil sampled from the root zone of infected tomato plants during 1978, 1979 and 1980. Soil numbers increased during symptom development and declined with the death of infected plants. The decline in population size in the soil was continuous where no cover crop was planted between the autumn and spring crops. This decline in population size was interrupted, however, following the planting of an oats cover crop numbers decreased with the ploughing under of the oats. Rainfall was associated with high soil numbers but soil temperature did not appear to directly affect population size. Soil populations in the root zone of susceptible tomato plants cultivar Floradel reached a maximum 1000-fold greater than in soil from the root zone of a resistant line. P. solanacearum survived in bare fallow soil for 21 months. Tomatoes planted 2 months later wilted rapidly.     

Changes in the Phenolic Acid Content and Antioxidant Activity During Kernel Development of Corn (Zea mays L.) and Relationship with Mycotoxin Contamination

Corn grain production could be affected by several fungal pathogens responsible for the production of mycotoxins. The aims of this study were to determine the evolution of phenolic acids and total antioxidant activity (TAA) during kernel development and to evaluate their potential protective role in minimizing mycotoxin contamination in six corn genotypes (four open‐pollinated varieties and two hybrids) characterized by a wide array of kernel traits. TAA and free and cell wall‐bound phenolics showed significant differences among corn genotypes at different stages of development, with the highest values found at the beginning of kernel development. Ferulic, p‐coumaric, and caffeic acids were the main cell wall‐bound phenolic acids during kernel development, whereas chlorogenic acid was the main free phenolic acid. A significant negative correlation was observed between deoxynivalenol contamination at harvest maturity and free phenolic acids and TAA at the beginning of kernel development, whereas no significant correlation was observed with fumonisin contamination. In conclusion, free phenolic acids are evidently involved in the resistance mechanism toward deoxynivalenol contamination, whereas their role toward fumonisin contamination was not elucidated under field conditions, implying that components other than phenolic acids may be responsible for this latter type of resistance.