Effects of three esca-associated fungi on Vitis vinifera L.: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines

Secondary metabolites and host defense compounds were shown to occur in xylem sap, and leaves of Vitis vinifera cv. Italia and cv. Matilde naturally infected by the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme). Samples of xylem sap and leaves were collected from healthy vines and from vines showing severe symptoms of brown wood-streaking caused by Pch and Tmi, or from vines with symptoms of both brown wood-streaking and white rot caused by Fme. Xylem sap collection was carried out during the early spring of 2003 and 2004, corresponding to the phenological phases: (A) cotton bud; (B) green tip; (C) leaves out; (D) stretched out leaves; and (E) visible clusters. In the present work we have studied the accumulation of biomolecules (pentaketides and α-glucans), host defense compounds (benzaldehydes, benzoic acid and cinnamic acid derivatives, flavonols, flavanols, flavan-3-ol derivatives and stilbenes) at different stages of grapevine development. Accumulation and changes in total phenolics and recurring phenolics, and of three phytotoxic secondary metabolites (scytalone, isosclerone and pullulan) were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that phenolic concentrations are strongly related to the cv.     

Effects of three esca-associated fungi on Vitis vinifera L.: III. Enzymes produced by the pathogens and their role in fungus-to-plant or in fungus-to-fungus interactions

When the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme) were grown in liquid stationary cultures, it was seen that they were able to live in media containing resveratrol (RES) or tannic acid (TA) as the sole carbon source and that the fungi were able to convert both compounds. Particular attention is paid here to detecting RES and TA conversion. Pch, Tmi and Fme were partially inhibited by RES or TA. Pch, Tmi and Fme produced extracellular tannase, laccase and peroxidase enzymes in liquid or agarized cultures, whether glucose was present or not. When colonies of Pch, Tmi and Fme were confronted, they showed spatially and temporally heterogeneous patterns of laccase and peroxidase activity. The results indicate the non-synergistic, competitive association of Pch and Tmi and the inhibition of Fme growth. Muconic acid, a well-known intermediate in a large number of lignin and phenol oxidative processes, can partly or completely inhibit the lignolytic agent Fme, but is tolerated by Pch and Tmi. An explanation for wood pigmentation patterns by Pch, Tmi and Fme is given.     

Effects of three esca-associated fungi on Vitis vinifera L.: IV. Diffusion through the xylem of metabolites produced by two tracheiphilous fungi in the woody tissue of grapevine leads to esca-like symptoms on leaves and berries

Two naphthalenone pentaketides (scytalone and isosclerone) and α-glucans (pullulan) are produced in vitro and in planta by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi), two tracheiphilous fungi associated with the ‘esca’ disease of grapevines. The possible role of such fungal metabolites in inducing symptoms on leaves and berries was studied in a vineyard of Vitis vinifera cv. Italia located in southern Italy. During early spring, pruning off two to four branches per vine allowed samples of xylem sap to be collected. Vine bleeding, assessed as ml day⁻¹ vine⁻¹, reached its maximum at bud burst and stopped within 28 days. The total amount of sap collected from healthy vines was about a quarter of that from esca-affected vines. During the same period, the leaf water potential of diseased vines increased progressively (i.e. showed less negative values), indicating a dysfunction in water and nutrient supply to the new growth. Both fungi were isolated from the xylem sap and from the woody tissue of branches and the trunk of diseased vines. Conidia isolated from the sap showed a high germination rate (>90%). Bioactive concentrations of the two pentaketides were detected in xylem sap, leaves and berries at various stages of seasonal growth. Exopolysaccharides, including pullulan, were found in the xylem sap. Absorption of culture filtrates of Pch and Tmi, as well as weak solutions of purified preparations of scytalone, isosclerone or pullulan, on detached leaves and berries caused symptoms similar to those shown by the esca-affected vines in the field.     

Effects of three esca-associated fungi on Vitis vinifera L.: V. Changes in the chemical and biological profile of xylem sap from diseased cv. Sangiovese vines

A vineyard of Vitis vinifera cv. Sangiovese was surveyed for incidence of esca and xylem sap collection. Sap samples were collected from healthy vines and from those with dual infection by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) or triple infection by Pch, Tmi and Fomitiporia mediterranea (Fme), during each early spring in a 3-year period (2001–2003). In order to analyse the possible trends in the climatic data, temperature and rainfall were assessed. At sap harvesting, aliquots of sap were assayed for phytotoxicity and extracted with ethyl acetate for phytotoxin recovery. Moreover, the exopolysaccharide (EPS) content was evaluated on several sap samples during the bleeding period. Conidia of Pch and Tmi, mycelium of Fme and their secondary metabolites were found in the sap of the esca-affected vines, indicating that the pathogens and their by-products together with some defence substances were accumulated and then translocated. Bioactivity tests showed toxicity of the sap from esca-affected vines to healthy detached leaves of cv. Sangiovese. The daily amount of sap, the pH, and the volume (J_v) and solute (J_s) fluxes were analysed as a function of the infecting fungi. Pullulan, glucogalactomannan(s) and arabinogalactan(s) are the main EPS in the esca-infected vines, whereas in the sap of healthy vines no traces of pullulan were found. Scytalone and isosclerone usually produced in vitro by Pch and Tmi were also detected in the sap of vines infected by Pch and Tmi or by Pch, Tmi and Fme. The endogenous phytohormone content of healthy vines evaluated by the cutting bioassay was different from that of infected vines. Four phenolics belonging to three classes e.g., benzoic acid derivatives, stilbenes and flavonol-glycosides were separated and identified by HPLC.     

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.     

“超纤渗灌”对摩尔多瓦葡萄生长与果实品质的影响研究

“超细纤维”渗灌是一种通过纤维毛细作用，将水分从超细纤维毛细芯传递到植物根部土壤的节水灌溉方式。试验在宁夏永宁县玉泉营农场的摩尔多瓦（Vitis vinifera L.cv. Moldova）葡萄园中进行，灌溉方式分为环状超细纤维表面渗灌（A*0）、埋深10 cm的环状超细纤维渗灌（A*10）和常规沟灌（CK），每株单次灌水量为30 L，灌水周期为1个月。结果表明，在相同的灌水量下，A*10和A*0的土壤含水量变化较CK更稳定，叶水势总体上高于CK，说明“超细纤维”渗灌能够维持较为稳定的水分状态，延缓了葡萄所遭受的水分胁迫。A*10和A*0的副梢生长量比CK分别提高了92%和57%；A*10的粒重比CK提高了8.1%，且A*10的产量比CK提高46.8%，说明“超细纤维”渗灌对葡萄的生长起到了促进作用。A*0和A*10的果粒可滴定酸和可溶性固形物水平与CK差异不大。成熟期A*0和A*10的果粒脯氨酸含量低于CK，是由于“超细纤维”渗灌能够缓解葡萄所受到的水分胁迫；A*0和A*10的花色苷含量高于CK，分别比CK提高了5.2%和14.3%，表明A*10供水均匀连续，有利于花色苷的累积。收获时，A*0和A*10的可溶性蛋白含量高于CK，其中A*10比CK高29%。，说明“超细纤维”渗灌有助于葡萄可溶性蛋白的合成。因此，“超细纤维”渗灌不仅比沟灌水分利用效率高，且能在一定程度上提高葡萄品质。     

Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants (Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.     

三株芽胞杆菌菌株对松材线虫低温存活与繁殖的影响

为明确细菌在松材线虫生态适应性中的作用,本研究选择与致病相关的松材线虫伴生细菌GD1、马尾松内生细菌GD2以及具有杀松材线虫活性的湿地松内生细菌NJSZ-13为试验对象,测定经这3株芽胞杆菌菌株3个浓度低温驯化10、15和20 d后,在冷冻条件下松材线虫强毒虫株AMA3、中毒虫株AA3和弱毒虫株YW4的存活率和繁殖量。结果表明:低温驯化15 d和20 d后3株菌株对不同毒力线虫的活力影响较驯化10 d后的更显著。在低温驯化15 d、-20℃冷冻处理1 h后,5×10⁶CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为77.22%、83.68%和84.26%,与对照差异显著;5×10⁵CFU/mL浓度菌株GD1处理下,虫株AMA3、AA3和YW4的存活率分别为75.76%、80.67%和81.50%,与对照差异显著。5×10⁶CFU/mL和5×10⁵CFU/mL浓度菌株GD2处理下,与GD1处理组结果相似,菌株NJSZ-13处理组则与菌株GD1和GD2的结果相反。低温驯化15...     

温度诱导对星豹蛛热激蛋白hsp70和hsp90基因表达的影响

为探讨温度诱导对星豹蛛的应激反应,运用实时定量PCR法测定了不同温度对星豹蛛热激蛋白hsp70和hsp90基因表达量的影响。结果表明:经过17、20、23、26℃诱导后,星豹蛛hsp70和hsp90基因表达量与对照组差异不显著,当高于26℃时,随着温度的升高表达量呈先升高后降低的趋势,且在38℃时最高,分别为对照组的716.46和10.44倍,差异显著;低于17℃时,表达量随着温度的降低呈逐渐升高的趋势,其中-1℃时最高,分别为对照组的334.53和9.38倍,差异显著;-1~41℃范围内,在相同诱导温度处理下,星豹蛛hsp70基因表达量显著高于hsp90基因。表明当星豹蛛受到温度高于26℃或低于17℃的刺激后,体内hsp70和hsp90基因表达量均增加,且hsp70基因表达量高于hsp90基因。说明热激蛋白只有在极端高温和低温的条件下才充分表达,从而对机体产生保护作用,且这种保护只在一定温度范围内起作用。     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.