Flavonoid biosynthesis and degradation play a role in early defence responses of bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis>) against biotic stress

Bilberry (Vaccinium myrtillus) represents one of the richest flavonoid sources among plants. Flavonoids play variable, species-dependent roles in plant defences. In bilberry, flavonoid metabolism is activated in response to solar radiation but not against mechanical injury. In this paper, the defence reaction and biosynthesis of phenolic compounds of bilberry was studied after infection by a fungal endophyte (Paraphaeosphaeria sp.) and a pathogen (Botrytis cinerea). The defence response of bilberry was faster against the endophyte than the pathogen. All flavonoid biosynthesis genes tested were activated by each infection. Biosynthesis and accumulation of phenolic acids, flavan-3-ols and oligomeric proanthocyanidins were clearly elevated in both infected samples. Infection by the pathogen promoted specifically accumulation of epigallocatechin, quercetin-3-glucoside, quercetin-3-O-α-rhamnoside, quercetin-3-O-(4”-HMG)-R-rhamnoside, chlorogenic acid and coumaroyl quinic acid. The endophyte-infected plants had a higher content of quercetin-3-glucuronide and coumaroyl iridoid. Therefore, accumulation of individual phenolic compounds could be specific for each infection. Quantity of insoluble proanthocyanidins was the highest in control plants, suggesting that they might act as storage compounds and become activated by degradation upon infection.     

Accumulation of phytoalexins in leaves of plane tree (Platanus spp.) expressing susceptibility or resistance toCeratocystis fimbriata f. sp.platani

Inoculation of leaves of resistantPlatanus occidentalis and susceptiblePlatanus acerifolia leaves withCeratocystis fimbriata f. sp.platani, the canker stain disease agent, induced foliar necrosis and biosynthesis of two phytoalexins, scopoletin and umbelliferone. Foliar symptoms keep localized and accumulation of coumarin phytoalexins was rapid for incompatible interactions. Necrosis spread widely and accumulation of these phenolic compounds was much later and lower for compatible interactions. The differential response could be used in a genetic improvement program for resistance against canker stain.     

Concentration-dependent effect of salicylic acid application on wheat seedling resistance to take-all fungus, <Emphasis Type="Italic">Gaeumannomyces graminis</Emphasis> var. <Emphasis Type="Italic">tritici</Emphasis>

Application of 0.1 and 0.2 mM salicylic acid (SA) significantly reduced take-all disease caused by Gaeumannomyces graminis var. tritici (Ggt) and increased the root and shoot lengths and biomass, whereas 0.5 and 1 mM SA had no significant effect. The effective SA concentrations also increased the activities of soluble peroxidase (SPOX) and cell-wall-bound peroxidase (CWPOX) and the concentration of total phenolic compounds. SPOX activity was highest at days 4 and 3 in healthy roots and those inoculated with Ggt, respectively, and that of CWPOX at day 6 in both healthy and inoculated roots. The concentration of phenolic compound was also highest at day 3 in both healthy roots and those inoculated with Ggt. The results indicate that the protective effect of SA depends on certain concentrations which increase peroxidase activity and phenolic compounds accumulation in the wheat roots; higher SA concentrations did not differ from the controls.     

Wheat leaf resistance to Pyrenophora tritici‐repentis induced by silicon activation of phenylpropanoid metabolism

Tan spot caused by Pyrenophora tritici‐repentis is a disease present in all wheat‐producing countries and silicon (Si) treatment of wheat plants has been shown to increase plant resistance to tan spot. In this study, the effect of phenylpropanoid metabolism on resistance to tan spot was evaluated and some phenolic compounds that accumulated in response to P. tritici‐repentis attack were identified. Furthermore, the effect of Si on phenylalanine ammonia‐lyase (PAL) activity and phenolic compound accumulation were determined in situ. Antifungal activity of differentially accumulated phenolic compounds was also evaluated in in vitro tests. Results showed that the increase in concentration of phenolic compounds was greatest at the onset of infection, and that some compounds showed fungitoxic effects including fungal tip swelling, granulation of germ tube and hyphae, and hyphal hyperbranching. Silicon‐induced reduction in both lesion size and tan spot disease progression were associated with activation of phenylpropanoid metabolism. PAL activity and accumulation of antifungal phenolic compounds were greater in pathogen‐inoculated plants supplied with Si. In these plants, fluorescence indicative of accumulation of phenolic compounds occurred early in epidermal cells and its intensity increased during the evaluation period, showing higher numbers of fluorescent cells around infected cells. Thus, the combined responses of cell fluorescence at sites of infection, increased PAL activity and accumulation of phenols indicate that Si strengthened wheat defence responses to infection by P. tritici‐repentis, reducing the severity of tan spot.     

Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten

The phenolic composition of olive roots and stems was studied by high performance liquid chromatography–mass spectrometry. The in vivo levels of the principal phenolic compounds found in olive plants infected by Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten differed from the levels observed in non-infected plants. When the antifungal activity of these compounds against both fungi was studied in vitro, the most active were quercetin and luteolin aglycons, followed by rutin, oleuropein, p-coumaric acid, luteolin-7-glucoside, tyrosol and catechin. Microscopic study showed that these phenolic compounds affected the growth, morphology and ultrastructure of the fungi. Taken together, these findings suggest that the phenolic compounds present in olive plants play an active role in the protection against pathogen attack.     

氯虫苯甲酰胺和氟虫腈对黏虫细胞色素P450基因表达的影响

为筛选出黏虫Mythimna separata参与杀虫剂解毒代谢的主效细胞色素P450基因,采用叶片浸渍法测定了用于处理黏虫3龄幼虫的亚致死浓度,通过构建转录组测序文库并结合数字基因表达(digital gene expression,DGE)对不同处理的黏虫进行测序,并运用实时荧光定量PCR(realtime quantitative polymerase chain reaction,RT-qPCR)技术验证12个P450基因的表达情况。结果表明,用于处理黏虫的氯虫苯甲酰胺和氟虫腈亚致死浓度LC_(10)分别为0.15、13.66 mg/L;对照样品、氟虫腈处理样品和氯虫苯甲酰胺处理样品分别获得59 521 504、64 838 148和41 722 990个原始序列数据,分别获得57 441 216、62 368 912和40 285 164个过滤后的序列数据;过滤后的序列长度分别为8.62、9.36和6.04 G;碱基错误率均为0.02%;Phred数值大于20、30的碱基占总碱基的百分比均高于90.59%;鸟嘌呤+胞嘧啶(guanine cytosine,GC)含量分别为47.16%、48.94%和47.55%,表明转录组测序质量较高;黏虫受氯虫苯甲酰胺胁迫后,29个P450基因表达量上调,27个P450基因表达量下调;黏虫受氟虫腈胁迫后,23个P450基因表达量上调,26个P450基因表达量下调;12个P450基因表达量的RT-qPCR技术检测结果与DGE测序文库显示的结果基本一致。     

Phenolic compounds as defence response of pepper fruits to Colletotrichum coccodes

Qualitative and quantitative changes of individual and total phenolics induced by Colletotrichum coccodes fungal infection have been studied in two susceptible sweet pepper cultivars ‘Soroksari’ and ‘Bagoly’, and the role of soluble phenolic compounds in plant's defence mechanism has been evaluated. Three distinct parts were analysed on pepper fruit: healthy tissue, anthracnose lesion, and bordering tissue, and individual phenolic compounds have been identified with the use of HPLC-MS system. In pepper fruit pericarp 21 phenolic compounds have been determined; the prevalent apigenin, quercetin and luteolin glycosides, chlorogenic acid and one chrysoeriol glucoside. C. coccodes infection increased the accumulation of chlorogenic acid, chrysoeriol glucoside, quercetin and luteolin glycosides in infected bordering tissue of both analysed pepper cultivars compared to healthy pepper tissue or symptomatic spot. Total apigenin derivatives did not show a significant increase in bordering tissue compared to the healthy pepper fruit in contrast to other groups of phenolics. This suggests a lesser role of apigenin glycosides in pepper plant defence against the Colletotrichum fungus. Intense phenolic synthesis was characteristic for the bordering zone between the healthy and infected plant tissue resulting in higher total phenolic content which might hinder the fungus to spread from the infected cells into the healthy tissue.     

Changes in phenolic content induced by infection with Didymella applanata and Leptosphaeria coniothyrium,the causal agents of raspberry spur and cane blight

The phenolic profile of healthy and infected raspberry canes was investigated in three raspberry cultivars: Autumn Bliss, Himbo Top and Polka. The content of total phenols and tannins was determined using spectrophotometric methods, whilst individual phenolic acids, flavan‐3‐ols, ellagic acid derivatives and glycosides of quercetin were analysed using HPLC/MS analysis. The content of secondary metabolites varied considerably among the analysed raspberry cultivars. Moreover, Didymella applanata and Leptosphaeria coniothyrium infection significantly altered the metabolism of phenolic compounds. Flavanols represented the greatest share of all identified phenolics in raspberry canes (90%), followed by glycosides of quercetin (6%), derivatives of ellagic acid (3%) and traces of hydroxycinnamic acid derivatives. Spur and cane blight diseases caused an increase of flavan‐3‐ols and tannins but the levels of hydroxycinnamic acid derivatives, conjugates of ellagic acid and quercetin glycosides were significantly reduced. Cultivars Himbo Top and Polka contained higher levels of hydroxycinnamic acid and ellagic acid derivatives in healthy and infected canes compared to cv. Autumn Bliss. Cultivar Polka also contained the highest level of flavanols and tannins. However, despite high levels of flavanols and total phenols measured in cv. Polka, the canes were highly diseased following infection with D. applanata and L. coniothyrium. The results of the study provide evidence that the level of phenolic compounds in the canes could be causally linked to the differences in disease susceptibility.     

四种P450基因调控小菜蛾对氯虫苯甲酰胺的抗性

为研究CYP6家族P450基因在小菜蛾Plutella xylostella代谢氯虫苯甲酰胺中的作用,利用浸叶法测定不同小菜蛾种群3龄幼虫对氯虫苯甲酰的抗性水平,采用实时荧光定量PCR(quantitative realtime PCR,q RT-PCR)方法分析CYP1v3、CYP1v4、CYP6B6和CYP6f这4种P450基因在小菜蛾不同抗性种群体内的表达差异及杀虫剂的短期诱导效应,并通过RNA干扰技术沉默4种P450基因后分析小菜蛾对氯虫苯甲酰胺的敏感性。结果显示,4种P450基因在中等抗性水平小菜蛾种群体内高表达;氯虫苯甲酰胺处理可诱导4种基因显著上调表达。分别沉默4种P450基因后,处理组小菜蛾的P450酶活力显著下降37.60%～54.82%,且处理组小菜蛾的死亡率显著高于对照组;同时沉默4种基因处理组的小菜蛾P450酶活力显著低于其他各处理组。表明4种P450基因可能同时参于小菜蛾对氯虫苯甲酰胺的代谢。     

Functional Analysis of ABC Transporter Genes From Botrytis cinerea Identifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA–BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.     

Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster

Two protein bands, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, account for most of the cytochrome P-450 in Drosophila melanogaster. P-450-A is ubiquitous among strains tested; whereas P-450-B is unique to certain strains. Dimethylnitrosamine demethylase activity is associated with P-450-B. Biochemical and genetic analyses showed that genes located on chromosome II were required for P-450-B expression. These genes were mapped within an interval that includes a major insecticide-resistance locus. Regulatory loci on chromosome III were required for maximum expression of P-450-B. One of these regulatory loci was mapped at another major resistance locus on chromosome III. There was a good correlation between P-450-B expression and resistance to phenylurea among the different strains tested. These results indicate that Drosophila can be a useful model to study the molecular mechanisms of insecticide resistance.     

Effect of leaf scald (<Emphasis Type="Italic">Xanthomonas albilineans</Emphasis>) on polyamine and phenolic acid metabolism of two sugarcane cultivars

Polyamine and phenolic acid levels as well as activities of some enzymes of their biosynthetic metabolism were examined in two sugarcane (Saccharum officinarum) cultivars differing in susceptibility to leaf scald, a disease caused by the bacterium, Xanthomonas albilineans. Juice obtained from both infected cultivars showed significantly increased levels of free putrescine and ornithine decarboxylase activity. However, the pathogen induced different changes in the two cultivars in subsequent metabolic steps. Whereas acid insoluble conjugated spermidine completely disappeared from the highly susceptible cv. C 439-52, an increase in acid insoluble conjugated polyamines was observed in the moderately susceptible cv. L 55-5. Phenolic acid metabolism also differed in the two cultivars. Since total phenolic acid content and phenylalanine ammonium lyase activity was greater in both cultivars after infection, distribution of phenolic acids between free or conjugated forms diverted into different pathways. The level of susceptibility of the two cultivars is discussed in terms of changes in these compounds.     

Epidemiological and histological components of crown rust resistance in oat genotypes

Crown rust, caused by Puccinia coronata f. sp. avenae, can cause significant damage in all regions where oats (Avena sativa L.) are cultivated. The primary means of controlling crown rust has been through genetic resistance, although in most cases resistance has been quickly overcome by the pathogen. More durable partial or non-specific resistance may possess different mechanisms from those underlying genes with specific effects. We studied the epidemiological and histological components of crown rust resistance with potential use in plant protection. Among the components evaluated, pustule density showed the clearest effect on resistance, while the latent period was not an important component. Cell death associated with the accumulation of autofluorescent and phenolic compounds was common in the resistant genotypes, but temporally distinct for the genotypes studied. Genotype Pc68/5*Starter, which has race-specific resistance, showed rapid cell death that prevented the development of pathogen colonies. Conversely, with cultivar URS 21 and genotypes 04B7113-1 and 04B7119-2, cell death and associated accumulation of autofluorescent and phenolic compounds was delayed until pathogen colonies were already established. Pathogen colonies developed normally in susceptible plants genotypes, and had usually produced sporogenic tissue by 5 days after inoculation. The data suggest that the resistance mechanisms, especially hypersensitivity and phenolic compound production, active in resistant plants are similar but may be differently expressed over time. The temporal variation in the expression of hypersensitivity and phenolic compound production reflects the level of field resistance in these genotypes.     

Natural mechanisms for cereal resistance to the accumulation of <Emphasis Type="Italic">Fusarium</Emphasis> trichothecenes

This review describes the naturally occurring mechanisms in cereals that lead to a reduction of Fusarium trichothecene mycotoxin accumulation in grains. A reduction in mycotoxin contamination in grains could also limit fungal infection, as trichothecenes have been reported to act as virulence factors. The mechanisms explaining the low toxin accumulation trait, generally referred to as type V resistance to Fusarium, can be subdivided into two classes. Class 1 includes mechanisms by which the plants chemically transform the trichothecenes, leading to their degradation or detoxification. Among the detoxification strategies, glycosylation of trichothecenes is a natural process already reported in wheat. According to the structure and the toxicity of trichothecenes, two other detoxification processes, acetylation and de-epoxidation, can be expressed, at least in transgenic plants. Class 2 comprises mechanisms that lead to reduced mycotoxin accumulation by inhibition of their biosynthesis through the action of plant endogenous compounds. These include both grain constitutive compounds and compounds induced in response to pathogen infection. There are already many compounds with antioxidant properties, like phenolic compounds, peptides or carotenoids, and with prooxidant properties, like hydrogen peroxide or linoleic acid-derived hydroperoxides, that have been described as ‘modulators’ of mycotoxin biosynthesis. This review addresses for the first time different studies reporting specific in vitro effects of such compounds on the biosynthesis of Fusarium mycotoxins. A better understanding of the natural processes limiting accumulation of trichothecenes in the plant will open the way to the development of novel breeding varieties with reduced ‘mycotoxin risk’.     

Dynamic root exudation of phenolic allelochemicals from Johnson grass (Sorghum halepense)

The phenolic allelochemicals from the root exudates of Johnson grass (Sorghum halepense) were collected at different developmental stages and analyzed using high performance liquid chromatography‐ultraviolet/photo‐diode array. The results revealed that three simple phenolic components, p‐hydroxybenzoic acid, p‐hydroxybenzaldehyde and ethyl p‐hydroxybenzoate, along with two flavonoids, diosmetin (3′,5,7‐trihydroxy‐4′‐methoxyflavone) and tricin (5,7,4′‐trihydroxy‐3′,5′‐dimethoxyflavone), were found in the root exudates of Johnson grass. However, the major phenolic compounds varied and the quality of each compound was dynamic at different developmental stages: the only phenolic compound that was detected in the seedling stage was p‐hydroxybenzoic acid; three simple phenolic allelochemicals, p‐hydroxybenzoic acid, p‐hydroxybenzaldehyde and ethyl p‐hydroxybenzoate, were found in the jointing stage; and five compounds, including the two flavonoids, tricin and diosmetin, appeared in the reproductive stage along with the other three simple phenolic allelochemicals. The highest diversity in phenolic constituents was in the reproductive stage, with five allelochemicals. Only p‐hydroxybenzoic acid was found in the whole life cycle of Johnson grass, with a range of 2.9 nmol to 6.8 nmol per plant per day. p‐Hydroxybenzaldehyde had the highest quality among all the compounds, with the highest releasing rate of 26.8 nmol per plant per day. The lowest‐content compounds were the two flavonoids, with the highest quality of 1.8 nmol per plant per day. The results suggest that the temporal dynamic of phenolic allelochemicals in the root exudates of Johnson grass corresponds with the dynamic of development.     

Molecular cloning and characterization of the cinnamate 4‐hydroxylase gene from Eupatorium adenophorum

Cinnamate‐4‐hydroxylase (CA4H), a cytochrome P450‐dependent monooxygenase, plays crucial roles in phenylpropanoid metabolism and plant defense. Previously, the authors showed that the expression of CA4H was induced in response to an allelopathic substance in Eupatorium adenophorum. Here, the full‐length cDNA of EaCA4H was cloned by using rapid amplification of cDNA ends. The 1518 bp open reading frame of EaCA4H was deduced to encode a protein of 505 amino acid residues. Like other CA4H proteins, the predicted EaCA4H polypeptides contained conserved domains of cytochrome P450. A Southern blot analysis indicated that at least five copies of EaCA4H exists in the genome of E. adenophorum. Subcellular localization revealed nuclear‐localized EaCA4H–green fluorescent protein fusion protein in onion epidermal cells. Heterologous silencing of endogenous CA4H in tobacco by a conserved EaCA4H fragment resulted in reduced expressions of key enzymatic genes and the production of downstream flavonoids in the phenylpropanoid pathway. Intriguingly, similar effects were observed in transgenic tobacco plants overexpressing EaCA4H. Altogether, the results indicate that the disturbed expression of CA4H in plants leads to relatively low expression levels of key enzymatic genes and the accumulation of the flavonoids that are involved in phenylpropanoid metabolism.