根肿菌侵染油菜抗感病品种早期防御酶活性及转录组分析

为探究抗病和感病2种油菜品种抗病机理及基因表达差异,通过水培法观测油菜抗病品种6M80和感病品种中双11号接种根肿菌后的根毛侵染,利用紫外分光光度法和RNA-Seq技术分别测定根部防御酶的活性及所有的转录序列。结果表明,接菌后3~15 d油菜品种6M80的根毛侵染率显著低于油菜品种中双11号,其根内过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)活性在接菌后第12天达到峰值,且显著高于油菜品种中双11号,分别为382.50、2 044.44和3 342.22 U·g~(-1)·min~(-1)。与未接菌相比,油菜抗病品种6M80和感病品种中双11号接菌后第3、6、9、12天共分别存在6 607个和2 499个差异表达基因(differentially expressed genes,DEGs)。功能注释结果表明,大多数DEGs参与信号传导、生物代谢、转录过程及防御机制。油菜品种6M80和中双11号中分别存在82个和53个防御酶相关的DEGs。研究表明,根肿菌侵染油菜抗病品种早期防御酶活性较高,其相关差异表达基因参与木质素生物合成和过氧化氢代谢,并在抗根肿病过程中发挥着重要作用。     

Comparison of an in vitro and a damping-off assay to test soils for suppressiveness to Pythium aphanidermatum

Testing of soil samples in greenhouse assays for suppressiveness to soilborne plant pathogens requires a considerable investment in time and effort as well as large numbers of soil samples. To make it possible to process large numbers of samples efficiently, we compared an in vitro growth assay with a damping-off assay using Pythium aphanidermatum as the test organism on tomato seedlings. The in vitro test compares the radial growth or relative growth of the fungus in soil to that in autoclaved soil and reflects suppressiveness of soils to the pathogen. We used soils from a field experiment that had been farmed either organically or conventionally and into which a cover crop (oats and vetch in mixture) had been incorporated 0, 10, 21, and 35 days previously. We obtained a significant, positive correlation between damping-off severities of tomato seedlings in damping-off assays and both relative and radial growth in vitro. In addition, radial and relative growth of P. aphanidermatum in the in vitro assay were positively correlated with several carbon and nitrogen variables measured for soil and incorporated debris. We did not find differences between the two farming systems for either growth measures of P. aphanidermatum or disease severities on tomato at different stages of cover crop decomposition. The in vitro assay shows potential for use with any fungus that exhibits rapid saprophytic growth, and is most suitable for routine application in suppressiveness testing.     

Short-term Cover Crop Decomposition in Organic and Conventional Soils: Soil Microbial and Nutrient Cycling Indicator Variables Associated with Different Levels of Soil Suppressiveness to Pythium aphanidermatum

Stages of oat–vetch cover crop decomposition were characterized over time in terms of carbon and nitrogen cycling, microbial activity and community dynamics in organically and conventionally managed soils in a field experiment and a laboratory incubation experiment. We subsequently determined which variables describing soil microbial community dynamics, C and N cycling could be used as predictors of Pythium aphanidermatum damping-off incidence and relative growth. Disease incidence and relative growth of P. aphanidermatum were measured in growth chamber assays and in vitro growth tests. No significant differences were detected between the conventional and organic farming system with respect to either relative growth or disease incidence. Stepwise discriminant analysis on three classes of disease incidence or relative growth led to selection of qualitatively similar variables. Only one soil microbial variable, total biomass of actinomycetes, was selected. Total C and N content of debris extracted from soil as well as NH₄-N content of soil were selected most consistently and show promise for assessment of potential damping-off incidence by P. aphanidermatum for young seedlings.     

Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species

An inoculation method for Exobasidium splendidum and Exobasidium vaccinii was developed on the dwarf shrub Vaccinium vitis-idaea. Using inoculated ramets, we investigated whether there are differences between V. vitis-idaea populations in the susceptibility to Exobasidium infections and whether the defence reaction of V. vitis-idaea is visible at a molecular level. Sixteen V. vitis-idaea clones from four populations were propagated in tissue cultures and the ramets were inoculated with E. splendidum or E. vaccinii fungi. The expression of three flavonoid biosynthetic genes (chalcone synthase, dihydroflavonol 4-reductase and anthocyanidin synthase) and the accumulation of flavonoids and hydroxycinnamic acids were determined in response to E. splendidum infection. A pathogenesis-related (PR 4) gene was isolated and its expression was studied in host ramet leaves. To our knowledge, this was the first successful artificial infection reported with E. splendidum. Disease frequencies of the inoculated ramets were between 32% and 47% for E. splendidum and 33% for E. vaccinii, but below 10% in uninoculated control ramets. There were no differences in disease frequencies between V. vitis-idaea populations. Both symptomatic leaves and healthy leaves of diseased ramets showed activation of flavonoid biosynthesis at the gene level, whereas expression of PR 4 was observed only in symptomatic leaves. The increase of flavonoid biosynthesis in healthy leaves of diseased ramets may represent a general response to stress or a role in defence against the pathogen E. splendidium. Ability of V. vitis-idaea to defend chemically against Exobasidium fungi and the heterogeneity of genotypes, age, size, and growth rates in host plant populations might be reasons for the low infection incidence of Exobasidia in nature.     

苦豆子内生真菌诱导子促进宿主生物碱合成积累的生理防卫反应

分别以4株苦豆子内生真菌菌液浓缩物为诱导子,研究不同诱导时间下,各诱导子对苦豆子组培苗中氧化苦参碱（OMA）含量以及防御酶苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）和过氧化氢酶（CAT）活性的影响。结果显示：4种苦豆子内生真菌的菌液浓缩物均可提高宿主中OMA的积累,其中诱导效果最佳的是内生真菌诱导子XKYKDF₄₀,在0~15 d诱导期间宿主中OMA含量持续增长且始终高于对照,在诱导第15 d达到最高,为3.616 mg·g^-1,是同时期对照组的2.09倍。4种内生真菌诱导子还可诱发宿主中生物碱合成关键酶PAL和3种抗氧化酶（POD、APX和CAT）活性升高。诱导子XKYKDF₄₀激发PAL活性的作用最佳,在诱导第6 d PAL活性达最大值,为61.92 U·g^-1,是同时期对照组的1.42倍;与其他诱导子相比,诱导子XKZKDF₂₇能够明显诱发POD的活性,在诱导第12 d达到最大值,为62.30 U·mg^-1,是同时期对照组的2.70倍;诱导子XKZKDF₁₁可诱导宿主中的APX和CAT活性大幅度提高,在诱导第12 d酶活性达到最高,分别为0.5538 U·mg^-1和19.82 U·mg^-1,是同时期对照组的4.97倍和3.00倍。结果表明,苦豆子内生真菌诱导子的加入不仅激活了宿主组培苗的防御性应答反应,还促进了OMA的合成。     

小菜蛾对高效氯氰菊酯和阿维菌素的抗药性监测

用浸叶法监测了青岛地区2003-2009年小菜蛾对高效氯氰菊酯和阿维菌素的抗药性。结果表明,小菜蛾对高效氯氰菊酯和阿维菌素的抗药性在2007年达到最高,分别为极高抗和中抗水平。2009年,两种农药的抗药性均大幅下降,分别为中抗和低抗水平。     

Induction of hypericins and hyperforin in Hypericum perforatum L. in response to biotic and chemical elicitors

Hypericum perforatum L. produces hyperforins, a family of antimicrobial acylphloroglucinols; and hypericins, a family of phototoxic anthraquinones exhibiting anti-microbial, anti-viral, and anti-herbivore properties in vitro. To determine whether these secondary metabolites are part of the specific plant defense systems that are mediated by methyl jasmonate or salicylic acid, we used meristem cultures to assess the effects of exposure to exogenous application of these chemical elicitors. Levels of hypericins in plant tissue increased in response to both elicitor treatments; total hypericin levels increased as much as 3.3 times control levels when treated with 200 μ methyl jasmonate for 14 days. Increased hyperforin concentrations were detected when plantlets were treated with 1 m salicylic acid or 50 μ methyl jasmonate. For assessing responses to a biotic elicitor, greenhouse-grown plant materials were inoculated with the plant pathogen, Colletotrichum gloeosporioides. Levels of hypericins increased twice as much as the control when inoculated with 1 × 10⁴ spores per ml; higher doses of spores overwhelmed the plant defenses. The elevation of hypericins and hyperforin in response to chemical and biotic elicitors suggests that these secondary metabolites are components in the inducible plant defense responses of H. perforatum.     

Identification of differentially expressed genes in tolerant and susceptible potato cultivars in response to Spongospora subterranea f. sp. subterranea tuber infection

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) has recently become one of the most devastating potato diseases of economic importance in South Africa. The use of resistant cultivars has long been considered the most effective and sustainable strategy to manage the pathogen. However, little is known about the molecular mechanisms underlying resistance of potato tubers to Sss. Using RNA-sequencing (RNA-seq), 2058 differentially expressed genes (DEGs) were identified from two potato cultivars (tolerant and susceptible) in response to Sss infection. Analysis of the expression patterns of 10 selected defence-response genes was carried out at two different stages of tuber growth using RT-qPCR to validate the RNA-seq data. Several defence-related genes showed contrasting expression patterns between the tolerant and susceptible cultivars, including marker genes involved in the salicylic acid hormonal response pathway (StMRNA, StUDP and StWRKY6). Induction of six defence-related genes (StWRKY6, StTOSB, StSN2, StLOX, StUDP and StSN1) persisted until harvest of the tubers, while three other genes (StNBS, StMRNA and StPRF) were highly up-regulated during the initial stages of disease development. The results of this preliminary study suggest that the tolerant potato cultivar employs quantitative resistance and salicylic acid pathway hormonal responses against tuber infection by Sss. The identified genes have the potential to be used in the development of molecular markers for selection of powdery scab resistant potato lines in marker-assisted breeding programmes.     

Localization of phytoalexin accumulation and determination of changes in lignin and carbohydrate composition in carnation (Dianthus caryophyllus L.) xylem as a consequence of infection with Fusarium oxysporum f. sp. dianthi,by pyrolysis-mass spectrometry

Minute pieces of xylem and other tissues from stems of healthy and fungus-infected plants of two carnation cultivars Novada and Lena were investigated for lignification (lignin/polysaccharide ratios) and lignin composition by means of pyrolysis mass spectrometry and pyrolysis gas chromatography mass spectrometry. This technique proved also very effective for the localization of dianthramide phytoalexins which accumulate in carnation after infection withFusarium oxysporum f.sp.dianthi. The composition of healthy tissues from both cultivars was practically the same. In the resistant cultivar Novada, infection induced a change from guaiacyl-syringyl lignin into a mainly guaiacyl lignin in the gum-occluded parts of the xylem. Considerable amounts of the phenolic amide dianthalexin and of other dianthramide phytoalexins were present in occluded xylem, but not in adjacent phloem, medulla or unoccluded xylem. Xylem from susceptible Lena suffering degradation was characterized by a loss of syringyl groups from the lignin and by demethylation of pectin in an early stage of infection. Small quantities of dianthalexin and other dianthramide phytoalexins were found in Lena when local defense responses (particularly occlusion) had occurred. In both cultivars evidence for degradation of hemicellulose was found.Samenvatting Kleine stukjes xyleem en andere weefsels van de stengels van gezonde en geïnoculeerde planten van een tweetal anjercultivars, Novada en Lena, werden onderzocht op mate van lignificatie (lignine/polysaccharide verhouding) en ligninesamenstelling met behulp van pyrolyse-massaspectrometrie en pyrolyse-gaschromatografie massaspectrometrie. Deze techniek bleek ook bruikbaar voor het localiseren van fytoalexinen van het dianthramide-type, welke in anjer accumuleren na infectie metFusarium oxysporum f.sp.dianthi. De samenstelling van gezonde weefsels was bij beide cultivars praktisch gelijk. Bij de resistente cultivar Novada leidde infectie tot een verandering van guaiacyl-syringyllignine in guaiacyl-lignine in door gommen afgesloten delen van het xyleem. Xyleem met gommen bevatte aanzienlijke hoeveelheden van het fenolamide dianthalexine en van de andere fytoalexinen van het dianthramide type. De fytoalexinen werden niet aangetroffen in aangrenzend floeem, merg of gezond ogend xyleem. Bij de vatbare cultivar Lena trad afbraak van het xyleem op, dat daarbij armer aan syringyllignine werd, terwijl tevens in een vroeg stadium pectine werd gedemethyleerd. In Lena werden slechts kleine hoeveelheden fytoalexinen gevonden, met name bij locaal opgetreden afweerreacties zoals gomvorming. Bij beide cultivars werden aanwijzingen voor afbraak van hemicellulosen gevonden.     

Combined effects of salt stress and cucurbit downy mildew (Pseudoperospora cubensis Berk. and Curt. Rostov.) infection on growth,physiological traits and antioxidant activity in cucumber (Cucumis sativus L.) seedlings

Abiotic and biotic stresses both decrease quality and quantity of cultivated plants. We compared responses of Cucumis sativus L. cvs Sardes and Beith alpha which were treated with 50 or 100 mM NaCl and/or infected by Pseudoperospora cubensis Berk. and Curt. Rostov.. Both cultivars were affected remarkably when fungus infection was combined with salt stress however; cv Sardes was found out to be relatively tolerant with sustainable growth performance, better leaf water status and lower lipid peroxidation levels under this condition. This relative tolerance could be related to the fine tuning of the activities of the antioxidative enzymes and proline levels.     

Multiple resistance of Papaver rhoeas L. to 2,4‐D and acetolactate synthase inhibitors in four European countries

The issue of cross‐ or multiple resistance to acetolactate synthase (ALS) inhibitors and the auxinic herbicide 2,4‐D was investigated in Papaver rhoeas L., a common and troublesome weed in winter cereals, in a broad‐scale study across four European countries. A combination of herbicide sensitivity bioassays and molecular assays targeting mutations involved in resistance was conducted on 27 populations of P. rhoeas originating from Greece (9), Italy (5), France (10) and Spain (3). Plants resistant to the field rate of 2,4‐D were observed in 25 of the 27 populations assayed, in frequencies ranging from 5% to 85%. Plants resistant to ALS‐inhibiting herbicides (sulfonylureas) were present in 24 of the 27 populations, in frequencies ranging from 4% to 100%. Plants resistant to 2,4‐D co‐occurred with plants resistant to sulfonylureas in 23 populations. In four of these, the probability of presence of plants with cross‐ or multiple resistance to 2,4‐D and sulfonylureas was higher than 0.5. ALS genotyping of plants from the field populations or of their progenies, identified ALS alleles carrying a mutation at codon Pro197 or Trp574 in 2,4‐D‐sensitive and in 2,4‐D‐resistant plants. The latter case confirmed multiple resistance to 2,4‐D and ALS inhibitors at the level of individual plants in all four countries investigated. This study is the first to identify individual plants with multiple resistance in P. rhoeas, an attribute rarely assessed in other weed species, but one with significant implications in designing chemical control strategies.     

Systemicity of Xanthomonas campestris pv. musacearum and time to disease expression after inflorescence infection in East African highland and Pisang Awak bananas in Uganda

Banana xanthomonas wilt (XW) caused by Xanthomonas campestris pv. musacearum (Xcm) attacks all banana cultivars. Xcm in inflorescence‐infected Pisang Awak plants with wilting male bud bracts is restricted to the upper parts of the true stem; therefore, cutting these plants at the pseudostem base has been recommended to prevent further Xcm spread. In order to fine‐tune existing control strategies, this study examined the movement of Xcm into plants and mats, in relation to disease incubation period. Mature Pisang Awak and East African highland (AAA‐EA) plants were inoculated with Xcm through abscission wounds of female bracts, male bud bracts, male flowers, a combination of male bud bracts and flowers, and by cutting male buds with a contaminated machete. Thirty plants per genotype and treatment were monitored for 24 months for disease symptoms. An additional 68 AAA‐EA and 33 Pisang Awak plants were sampled weekly to assess the rate of Xcm spread within the plants. All floral entry points resulted in disease, with the highest incidence in combined male bract and male flower abscission wound inoculations. The study confirmed the systemicity of Xcm, with the pathogen able to live within the mat for long periods (5–16 months) without causing disease. Reliance on disease symptom expression to manage XW is therefore not sufficient. The long incubation period in lateral shoots may explain the current resurgence of the disease in locations where the disease was thought to have been successfully eradicated.     

Effects of three esca-associated fungi on Vitis vinifera L.: I. Characterization of secondary metabolites in culture media and host responses to the pathogens in calli

Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) produced scytalone, isosclerone and pullulans in liquid cultures, as well as in calli. Secondary metabolites and host defense compounds were shown to occur in calli of Vitis vinifera cv. Italia and cv. Matilde infected by the esca-associated fungi Pch, Tmi and Fomitiporia mediterranea (Fme). Calli of both cvs. were grown as dual cultures with Pch, Tmi and Fme. The fungi grew well in the presence of calli of both cvs., but callus growth was reduced. Accumulation and changes of total phenolics and recurring phenolics, and of two phytotoxic pentaketides and pullulans were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that concentrations of phenolics are strongly related to the cv. The paper discusses the possible relationship between melanin biosynthesis in Pch and Tmi, which utilize pentaketide metabolites as intermediates and their pathogenicity related to phytotoxity of scytalone and isosclerone.