Eutypa dieback in grapevines: differential production of acetylenic phenol metabolites by strains of Eutypa lata

The production of acetylenic phenol metabolites in vitro by three strains of the ascomycete Eutypa lata, the causative agent of dying-arm disease in grapevines, has been investigated. Metabolite composition and yields differed significantly between strains and with growth medium but usually reached a maximum after 24-30 days of fungal growth. A general method for the analysis and identification of metabolites by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives was developed and individual compounds were quantitated by analytical high-performance liquid chromatography (HPLC) and separated by preparative HPLC. The phenolic aldehyde, eutypine (1), reported to be the grape phytotoxin, occurred in only one of the strains examined whereas the primary metabolite was the corresponding alcohol, eutypinol (2), the presumptive detoxification product. A novel metabolite was isolated as a major constituent, together with a minor component, and their structures were established by spectroscopic methods as a methoxyquinol, named eulatinol (4), and a chromene analog (9) of 2, respectively. The evidence suggests that 1 is not solely responsible for phytotoxicity in grapevines but that dying-arm disease may result from a suite of compounds elaborated by the fungus, with the composition dependent on fungal strain and nutritional source.     

Transformation of Eutypa dieback and esca disease pathogen toxins by antagonistic fungal strains reveals a second detoxification pathway not present in Vitis vinifera

Eutypine, 4-hydroxybenzaldehyde, and 3-phenyllactic acid are some of the phytotoxins produced by the pathogens causing Eutypa dieback and esca disease, two trunk diseases of grapevine (Vitis vinifera). Known biocontrol agents such as Fusarium lateritium and Trichoderma sp. were screened for their ability to consume these toxins. Transformation time courses were performed, and an high-performance liquid chromatography-based method was developed to analyze toxin metabolism and to identify and quantify the converted products. The results show that the aldehyde function of eutypine was reduced to eutypinol, as by V. vinifera cv. Merlot, the cultivar tolerant to Eutypa dieback. We revealed a supplementary detoxification pathway, not known in Merlot, where the aldehyde function was oxidized to eutypinic acid. Moreover, some strains tested could further metabolize the transformation products. Every strain tested could transform 4-hydroxybenzaldehyde to the corresponding alcohol and acid, and these intermediates disappeared totally at the end of the time courses. When biological assays on cells of V. vinifera cv. Chasselas were carried out, the transformation products exhibited a lower toxicity than the toxins. The possibility of selecting new biocontrol agents against trunk diseases of grapevine based on microbial detoxification is discussed.     

葡萄器官酚类物质的含量研究

葡萄植株总酚含量的积累在其年生长期间呈双S型曲线.浆果膨大期开始积累,浆果着色期含量下降,浆果成熟期含量再次升高,随后急速下降.新梢中的总酚含量最高,卷须中的极低.不同品种叶片内总酚含量存在显著或极显著的差异.     

Hydroxycinnamic acids as affected by different fertilization of Rebula grapevines

The phenolic composition of white grapes is important since browning reactions may dramatically compromise the overall quality of musts and wines. Only few scientific contributions are available, which investigated how fertilization can influence this class of compounds. Thus, the aim of this work was to examine the effects of NPK soil fertilization coupled with soil or foliar applications of Mg, Fe, and Zn on the concentrations of K, Mg, Fe, and Zn of grape berries and leaf petioles of potted Rebula grapevines (Vitis vinifera L.), also revealing the change of hydroxycinnamic acids in grape juice. The results obtained over the three‐year study (2009–2011) showed that NPK fertilization positively affected K and Zn concentrations of petioles and grape berries, and negatively Mg. In addition, K (synergistic) and Mg (antagonistic) had an influence on Zn uptake. Our findings suggest that the supply of NPK was profitable for a reduction of trans‐caftaric acid in grape juice, while only few differences were observed with further application of nutrients.     

Fruit quality and nutrient composition of grapevines: a review

Grape quality and its nutrient composition vary depending on agronomical management practices (fertilization, irrigation, weed, and pest control), and agrochemicals treatments (such as kaolin, hormones, and sucrose), viticultural (grape cultivars and varieties, training, pruning, cluster thinning, and trunk girdling), and biotechnological techniques, as well as growth stage and environmental changes (soil, climate, and season). Understanding the mentioned agro-biotechnological techniques assists grape growers and geneticists in breeding grapevines to improve yield, tolerance, quality, and nutraceutical values based on their usage purposes. Thus, this review article focuses on the up-to-date approaches and incentivizes further studies on the unknown mechanisms related to engineering grape flavonoid/phenylpropanoid biosynthetic pathways to improve its health-promoting effects in both grape and human. The engineering/breeding strategies and viticultural practices have been proposed based on the grape usage purposes and environmental conditions.     

Cynodontin: a fungal metabolite with antifungal properties

A red pigment that accumulates in cultures of a Drechslera avenae pathotype with specificity for Avena sterilis was isolated and identified as the anthraquinone cynodontin (3-methyl-1,4,5,8-tetrahydroxyanthraquinone). Satisfactory yield of the compound was obtained with 20-60 day incubations at temperatures between 20 and 27 degrees C. Cynodontin was tested in vitro for fungitoxicity and was found to be a potent inhibitor of the growth of Sclerotinia minor, Sclerotinia sclerotiorum, and Botrytis cinerea and, to a lesser extent, of Verticillium dahliae. The ED(50) values obtained with these fungi were of the same order of magnitude as those of the commercial fungicides dicloran and carbendazim, which were used as reference chemicals. In contrast, the growth of a number of other fungi was not significantly inhibited by cynodontin. Anthraquinone and two other anthraquinone derivatives, emodin and chrysophanol, which were also included in the tests, did not affect the growth of the cynodontin-sensitive fungi. It thus appears that the type and position of the substitutions at the C-ring play a role in the expression of antifungal activity.     

双推理核山羊疾病诊断专家系统

专家系统在动植物疾病虫害诊断领域得到了广泛应用。为辅助兽医求解当地主要经济动物山羊疾病诊断治疗问题,建模山羊疾病诊断专家系统体系架构,提出双推理机核的推理机制,设计重权关联因子可信度预测推理和支持机器学习的贝叶斯（Bayesian）推理算法,构造原型系统,开展现场试验。试验结果表明：双推理机核推理诊断同传统单一推理机制相比,显著改善了准确度,提高知识库利用率,求解模型的综合准确率达到了90%,填补了对比度空白,获得满意的效果。     

Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Increase of secondary metabolite content in marigold by inoculation with plant growth-promoting rhizobacteria

The effects of single inoculation and co-inoculation of two plant growth-promoting rhizobacteria (PGPR) (Pseudomonas fluorescens, Azospirillum brasilense) on growth and essential oil (EO) composition and phenolic content were evaluated in marigold (Tagetes minuta). Plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) were measured. EO yield increased 70% in P. fluorescens-inoculated and co-inoculated plants in comparison with control (non-inoculated) plants, without altering EO composition. The biosynthesis of the major EO components was increased in the inoculated plants. Shoot fresh weight and EO yield were significantly higher in P. fluorescens-inoculated and in co-inoculated plants than in control plants. The total phenolic content was 2-fold higher in singly-inoculated or co-inoculated treatments than in controls. In view of the economic importance of monoterpenes and phenolic compounds for a variety of applications in the food and cosmetic industries, P. fluorescens and other PGPR have clear potential for improving the productivity of cultivated aromatic plants. Better understanding of the processes that affect the accumulation of secondary metabolites will lead to increased yields of these commercially valuable natural products.     

Targeted metabolite analysis and antioxidant potential of Rumex induratus

Targeted metabolite analysis of aqueous extract of Rumex induratus leaves, in terms of phenolic compounds and organic acids, and the study of its antioxidant activity against the DPPH(*) radical, a reactive oxygen species, hypochlorous acid, and a reactive nitrogen species, nitric oxide ((*)NO), were performed. The samples were collected in several locations, spontaneously occurring or from greenhouse culture, at different stages of development and seasons. The phenolic composition was achieved by high-performance liquid chromatography (HPLC)-diode array detection, and four hydroxycinnamic acid derivatives and 10 flavonoid glycosides (C- and O-heterosides) were determined. Organic acids composition was established by HPLC-UV, revealing five compounds. The total amount of phenolic compounds and organic acids were affected by growing conditions and developmental phase. The aqueous extract exhibited a dose-related activity against all tested reactive species.     

电子鼻对草莓采后贮藏早期霉菌感染的检测

为了实现电子鼻对草莓贮藏期常见霉菌感染的早期检测,对草莓果实分别接种灰霉、扩展青霉和根霉3种主要病原菌,以无菌水处理为对照组,每2d采用PEN3电子鼻获取草莓的气味,并用气质联用技术分析草莓气味。结果表明,草莓接种病原菌2d后,主成分分析能够正确区分正常果实(对照组)与病害果实,且可以较好区分草莓感染病原菌种类,多元方差分析结果也显示接种不同病原菌对草莓果实挥发性物质的影响差异显著(P<0.05),通过Fisher判别建立的回归函数对3种病原菌灰霉、扩展青霉和根霉及对照组的判别正确率分别为100%、93.3%、86.7%和100%。载荷分析及气质联用技术结果表明病原菌对草莓果实挥发性物质的影响主要体现在烃类及酯类的变化。研究结果可为实现草莓采后贮藏和流通过程中质量变化和病原微生物的感染进行无损快速检测和监测提供参考。     

Reduction of latent infection and enhancement of disease resistance in muskmelon by preharvest application of harpin

Latent infection was analyzed when harpin at 50 mg/L was sprayed on muskmelon 4 times at four different stages: florescence, young fruit, fruit enlarging, and netting periods. Results showed that the latent infection was significantly lower in sprayed muskmelons than in the control fruit. Meanwhile, the activities of peroxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, and chitinase increased significantly in the fruit treated with harpin. It also resulted in an increase of contents of total phenolic compounds, flavanoids, and lignin. In addition, the treatment of harpin increased the activity of superoxide dismutase, enhanced the content of hydrogen peroxide, and reduced catalase activity. Furthermore, harpin treatments contributed to the reinforcement of cell walls of pericarp in fruit, the reduction of postharvest decay, and the improvement of postharvest qualities. These results suggested that harpin effectively participated in inducing resistance and could be a new strategy for preventing latent infection in muskmelon.     

Gas chromatographic analysis of milk for deoxynivalenol and its metabolite DOM-1

A gas chromatographic method is described for the determination of deoxynivalenol (DON) and its metabolite DOM-1 in milk. Milk samples were extracted with ethyl acetate on a commercially available disposable extraction column, followed by hexane-acetonitrile partitioning. Final purification was accomplished on a reverse phase C-18 cartridge. The trimethylsilyl ether (TMS) derivatives of DON were prepared, chromatographed on an OV-17 column, and quantitated with an electron capture detector. Chromatography of the TMS derivatives of milk extracts was compared to that of the corresponding heptafluorobutyryl derivatives. The limit of detection using TMS derivatives was 1 ng/mL for both toxins with recoveries averaging 82% +/- 9% at 2.5 and 10 ng/mL milk for DON and 85% +/- 6% at 10 ng/mL for DOM-1.     

甘蔗黑穗病流行学参数和病情指数调查与分析系统开发

根据甘蔗黑穗病流行学参数和病情指数调查与分析中遇到的困难,提出了利用条形码自动识别系统来管理调查过程中的数据,并利用专门开发的计算机分析系统来分析这些数据。调查系统主要包括用户登录界面、条码扫描界面、数据输入界面等三大部分;分析系统具有最短潜伏期（IP）、发病持续期（SSD）、最终累计茎感染率（ISmax）、最终累计丛感染率（IPmax）和病害进展曲线下面积（AUDPC）的计算和抗性等级自动划分等功能。实践表明,此系统有效的提高了工作效率,并有助于减少人工记录和分析过程中的失误率。     

Changes in phenolic composition induced by Pseudomonas savastanoi pv. Savastanoi infection in olive tree: presence of large amounts of verbascoside in nodules of tuberculosis disease

A study was carried out to determine the changes in phenolic composition induced by tuberculosis infection in olive trees. Four ethanolic extracts were compared: olive leaf from shoots affected by Pseudomonas savastanoi pv. Savastanoi, nodules induced by this bacteria, leaf from healthy (asymptomatic) shoots, and shoots. Among the differences found, the presence of a phenolic compound in nodules was significant in much larger quantities than in leaf or shoots. Mass spectrometric analysis showed this compound to be verbascoside. The enhancement of its biosynthesis could be related to the defense mechanisms of the tree in the nodules induced by P. savastanoi and suggests the possibility of exploration of natural and biotechnological sources of this compound.     

Seasonal variation of defense-related gene expression in leaves from Bois noir affected and recovered grapevines

Although Bois noir is one of the main phytoplasma diseases of grapevine, the gene expression and enzyme activities that underlie physiological changes occurring in symptomatic and recovered (with spontaneous or induced symptom remission) plants are mostly unknown. Bois noir symptomatic leaves (September 2006, 2007) and symptomless leaves from infected symptomatic plants (September 2007) of Sangiovese (moderately susceptible) and Chardonnay (highly susceptible) cultivars were collected. Moreover, leaves from infected symptomless plants of both cultivars were harvested in June 2007. Leaves from recovered plants were also collected in the same periods. In recovered plants of both cultivars, class III chitinase and almost every time phenylalanine ammonia-lyase and chalcone synthase expression were increased for all collection periods. In symptomatic leaves of both cultivars, the expressions of the same genes were up-regulated and also those of β-1,3-glucanase and flavanone 3-hydroxylase. The activities of chitinase, phenylalanine ammonia-lyase, β-1,3-glucanase, and superoxide dismutase generally correlated with gene expression. For the moderately susceptible Sangiovese, the defense genes were generally up-regulated in both symptomatic and symptomless leaves (for all collection periods). This behavior was not observed in the highly susceptible Chardonnay, in which changes in gene expression were linked to evident symptom display. Therefore, the physiological response of the plants to this pathogen infection appear to be the reason for the resistance of the cultivar to the disease.     

Residue analysis of glyphosate and its principal metabolite in certain cereals, oilseeds, and pulses by liquid chromatography and postcolumn fluorescence detection.

A postcolumn liquid chromatographic method to determine the extractable residues of glyphosate (GLYPH) and its principal metabolite, (aminomethyl)phosphonic acid (AMPA), in various cereals and beans is described. The finely ground sample is extracted with a mixture of chloroform and water, and the resulting aqueous layer is passed through a cation exchange column. The eluate is adjusted to pH 7-10 and passed through an anion exchange column. The second column is eluted with 0.3M HCl solution and the resulting acidic eluate is analyzed with liquid chromatography coupled with postcolumn fluorescence detection. The mean recoveries for GLYPH in barley, canola, dry pea, flax, soybean, wheat, and white bean ranged from 90.0 to 98.1%, with coefficients of variation (CV) from 2.9 to 10.0% and limits of detection (LOD) from 0.07 to 0.14 ppm. Similarly, mean recoveries for AMPA in the same crops ranged from 87.4 to 98.9%, with CV from 4.6 to 7.7 and LOD from 0.05 to 0.12 ppm. Using this method, an analyst can routinely analyze 6 samples per 1.5 days. The advantages of this procedure are discussed.     

Determination of ciprofloxacin, the major metabolite of enrofloxacin, in milk by isopotential fluorimetry

A new method for the determination of ciprofloxacin, the major metabolite of enrofloxacin, for concentrations between 20 and 200 ng/mL by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as ciprofloxacin in whey, without the need of tedious preseparation. The determination was performed in an ethanol/water medium (20% v/v) at pH 4.8, provided by adding a sodium acetate/acetic acid buffer solution. Since enrofloxacin is widely used as an antibacterial agent in veterinary medicine, the method was successfully applied to the determination of its main metabolite in milk. An exhaustive statistical analysis has been developed to all calibration graphs. This treatment includes robust regression such as least median of squares, which also detects outliers and leverage points. The overall least-squares regression has been applied to find the more exact straight line that fits the experimental data. The error propagation has been considered to calculate the detection limit and the repeatability of the method.     

Sap analysis for diagnosis of nitrate accumulation in cereal forages

Abstract

Development of a quantitative, preharvest quiektest for NO₃ levels in cereal forages would improve crop management options to avoid NO₃ toxicity in livestock. Our objective was to determine if concentrations of NO₃ in sap expressed from oat (Avenasativa) and barley (Hordeum vulgare) are correlated with those in dry tissue of simultaneously harvested hay, and to test the reliability of the Cardy portable NO₃ meter for sap analysis in these species. In 1993, whole plant samples were gathered from plots fertilized with variable nitrogen (N) rates at four environments in Montana, and were analyzed for NO₃ concentration in lower‐internode sap and in whole plant dry matter. In 1994 and 1995, the study was repeated at two environments. The sampling technique included three subsamples from each plot for sap analysis, followed immediately by harvest of the entire plot for hay, and further subsampling for dry matter NO₃ analysis after drying. Linear correlations between dry matter and sap NO₃ concentrations were found across species at each environment in 1993 with r values of 0.64 to 0.81. No relationship was established for oat at one environment. Locations differed in the coefficient of correlation, indicating environmental influences on the relationship and/ or variability due to sampling technique. In 1994 and 1995, each species fit a separate linear correlation across site‐years with r values of 0.89 (oat) and 0.87 (barley). The consistency across site‐years (1994–1995) indicates that the variability in preliminary results was overcome with sampling technique. We propose a quantitative quiektest for NO₃ levels in cereal forages using conditional predictions of dry matter NO₃ based on observed values of sap NO₃. Since sap NO₃ readings with the Cardy portable nitrate meter were well correlated (r=0.93) with Accumet ISE readings across critical ranges, quiektest procedures are practical.