Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes

BackgroundHemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins.MethodsThe three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy.ResultsPvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae.ConclusionThe massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death.General significanceThe histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.     

The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens

The intercellular ascomycetous pathogen Cymadothea trifolii, causing sooty blotch of clover, proliferates within leaves of Trifolium spp. and produces a complex structure called interaction apparatus (IA) in its own hyphae. Opposite the IA the plant plasmalemma invaginates to form a bubble. Both structures are connected by a tube with an electron-dense sheath. Using immunocytochemistry on high-pressure frozen and freeze-substituted samples, we examined several plant and fungal cell wall components, including those in new host wall appositions at the interaction site, as well as a fungal polygalacturonase. Within the tube linking IA and host bubble, labelling was obtained for cellulose and xyloglucan but not for rhamnogalacturonan-I and homogalacturonans. The IA labelled for chitin and beta-1,3-glucans, and for a fungal polygalacturonase. Plant wall appositions reacted with antibodies against callose, xyloglucans and rhamnogalacturonan-I. Cymadothea trifolii partly degrades the host cell wall. Structural elements remain intact, but the pectin matrix is dissolved. A fungal polygalacturonase detected in the IA is probably a key factor in this process. Owing to the presence of chitin and beta-1,3-glucans, the IA itself is considered an apoplastic compartment.     

Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici

Commercial chitosan and laminarin, as well as -glucans, isolated from either Phytophthora megasperma f.sp. glycinea or Saccharomyces cerevisiae, were applied to decapitated tomato (Lycopersicon esculentum Mill.) plants and evaluated for their potential to induce defense mechanisms in root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A significant decrease in disease incidence was monitored in elicitor-treated plants as compared to water-treated plants. No difference was detected in the capacity of the elicitors under study to confer enhanced protection against pathogen attack. Ultrastructural investigations of the infected root tissues from watertreated (control) plants showed a rapid colonization of all tissues including the vascular stele. Fungal ingress was lways associated with marked host cell disorganization and cell wall alteration. In root tissues from elicitortreated plants, restriction of fungal growth to the epidermis and the outer cortex, decrease in pathogen viability, and formation of numerous wall appositions at sites of attempted penetration were the main features of the hostpathogen interaction. The wall appositions were found to vary greatly in their appearance from multi-textured to multi-layered structures, from elongated deposits to hemispherical protuberances. Application of various goldcomplexed probes to root tissue sections revealed that callose, pectin and phenolic-like compounds (likely lignin) were the main components of the newly-formed barriers. By contrast, cellulose appeared confined to outer or intermediate layers resembling the host cell wall in terms of structure and architecture. In the absence of fungal challenge, the cytologically visible consequences of elicitation were restricted to a discrete deposition of electron-opaque substances in the vacuoles of some cells, and wall appositions were not detected. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm ignal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by an exogenous elicitor.Abbreviations AGL Aplysia gonad lectin - FORL Fusariumoxysporum f.sp. radicis-lycopersici The authors wish to thank Sylvain Noël for excellent technical assistance and Drs. J.P. Geiger and Michel Nicole (ORSTOM, Montpellier, France) for providing the purified laccase. This work was supported by a grant from the FCAR-CQVB (Fonds Québécois pour la Formation de Chercheurs et l'Aide à la Recherche and Centre Québécois de Valorisation de la Biomasse) and by a contract from the Company Tourbières Premier Ltée, Rivière-du-Loup, Québec.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. I. Selection from a susceptible cultivar for high and low polysaccharide content

Plant cell walls play a major role in the outcome of host-parasite interactions. Wall fragments released from the plant, and/or the fungal pathogen, can act respectively as endogenous and exogenous elicitors of the defence response, and other wall components, such as callose, lignin, or hydroxyproline-rich glycoproteins, can inhibit pathogen penetration and/or spreading. We have previously demonstrated that calli from tomato cultivars resistant in vivo to Fusarium oxysporum f.sp. lycopersici show a high amount of polysaccharides in vitro. The aim of the present work was to assess the possible role of polysaccharide content and/or synthetic capacity in determining the competence of plant cells for active defence. For this purpose, tomato cell clones with increased and decreased polysaccharide (FL⁺, FL^-) and callose (A⁺, A^-) content have been selected by means of specific stains as visual markers and tested for the effect of these changes on the extent of response to Fusarium. The analysis of several parameters known to be indicative of active defence (cell browning after elicitor treatment, peroxidase and -glucanase induction and inhibition of fungal growth in dual culture) clearly shows that FL⁺ and A⁺ clones have acquired an increased competence for the activation of defence response. The results are thoroughly discussed in terms of an evaluation of the relative importance of constitutive and/or inducible polysaccharide synthetic capacity for plant response to pathogens, and their possible regulation by plant physiological backgrounds.     

Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

贝莱斯芽孢杆菌GDND-2对烟草镰孢根腐病菌生长发育的影响

【背景】尖孢镰孢(Fusariumoxysporum)引起的烟草根腐病在世界烟区普遍发生,严重影响烟草产量和质量,化学农药无法有效防治病害,利用生防菌防治该病成为研究热点。【目的】明确贝莱斯芽孢杆菌(Bacillusvelenzensis)GDND-2对尖孢镰孢生长发育的抑制作用。【方法】制备含10%和20%GDND-2发酵滤液的PDA培养基,涂在玻片上,接种尖孢镰孢分生孢子,观察滤液对尖孢镰孢孢子萌发、菌丝生长、孢子形成和色素产生的影响,应用扫描和透射电镜观察菌体超微结构的变化。【结果】贝莱斯芽孢杆菌GDND-2发酵滤液延迟孢子萌发2h以上,造成芽管膨大畸形,促使菌丝提早分枝,抑制菌丝延伸,使菌丝产生畸形球状结构,10%和20%发酵滤液对菌丝生长抑制率达53.41%和61.58%。滤液延迟病菌产孢,显著抑制产孢量,影响孢子形态,刺激病菌产生色素。10%和20%发酵滤液延迟产孢20h和28h,对产孢量的抑制率为52.11%和78.85%,滤液促使病菌形成小型分生孢子。观察尖孢镰孢的超微结构,部分菌丝膨大、畸形,细胞壁变薄,细胞膜消失,细胞质渗出,胞内呈空腔结构;部分菌丝严重皱缩、扭曲、...     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

内生真菌司氏角担子菌B6对尖孢镰刀菌西瓜专化型菌株的拮抗作用

近年来,尽管西瓜产业不断发展壮大,但轮作土壤种植西瓜易产生枯萎病害导致世界范围内的西瓜严重减产。通过对峙培养实验和抑菌实验探讨了重阳木内生真菌司氏角担子菌(Ceratobasidum stevensii)B6菌株对西瓜枯萎病病原菌尖孢镰刀菌西瓜专化型(Fusarium oxysporum f. sp.Niveum,FON)的拮抗作用,并初步分析了其作用机制。平板拮抗试验的结果表明,内生真菌B6生长过程中不是通过产生抑菌带来抑制FON菌丝,而是利用自身的生长优势将FON完全覆盖。显微观察B6与FON菌丝的接触部位,发现FON菌丝外侧附着B6顶端菌丝形成的胞样结构,表明FON菌丝生长仅受到B6菌丝的抑制。抑菌试验结果显示,B6产生的挥发性物质可以抑制FON的生长和产孢,并使其菌丝分枝明显减少;B6的发酵液对FON的生长和产孢没有抑制作用。因此,推测B6主要通过释放某些挥发性物质产生拮抗作用而抑制FON的生长。     

植物病原菌拮抗性野生艾蒿内生菌的分离、筛选和鉴定

从野生艾蒿的根茎叶部位分离出内生细菌共68株,以棉花枯萎病、稻瘟病、烟草赤星为供试病原菌,采用对峙法对内生菌分别进行抑菌试验,对筛选出的菌株进行了16S rDNA序列测定和系统发育分析。结果表明:从野生艾蒿分离到的内生菌经初筛、复筛,获得抑制病原菌效果最明显的3株菌,结合生理生化特性、菌落特征、细胞形态特征和16S rDNA测序分析结果,菌株L8、S11和R6分别鉴定为Bacillus subtilis,Bacillus cereus,Paenibacillus polymyxa。拮抗实验表明,棉花枯萎病原菌菌丝发生弯曲、打结,烟草赤星的受作用菌丝生长端分枝明显增多,生长端边缘呈珊瑚状分枝,并且出现明显的畸形和萎缩现象。分析表明,可能是由于在培养过程中内生菌产生了化感物质,对病原菌的菌丝产生抑制作用的结果。     

The pleiotropic phenotype of tomato cells selected for altered response to Fusarium oxysporium f. sp. lycopersici cell wall components

Summary With the aim of better understanding in vitro host-parasite interactions, tomato cell lines selected for altered response to Fusarium oxysporum f. sp. lycopersici cell wall components were further characterized. Particularly, their behaviour in dual culture in regard to both fungal inhibition and peroxidase activation was analysed and selected, and control cell clones were screened for esopolysaccharide content and toxin tolerance. Interclonal differences in growth response to 2,4-D and DMSO and the capacity to grow on a medium devoid of hormones (habituation) were taken as parameters representative of physiological variability not directly correlated with the response to pathogens. Significant differences between clones selected for increased (F+) and decreased (F–) response to fungal elicitors were found for pathogen inhibition, peroxidase and esopolysaccharide content, toxin tolerance being reduced in F but not significantly different from the control in F+. As expected, clonal variability for the response to 2,4-D and DMSO, although significant, was not connected with hostparasite interactions. The data reported thus show that selection for a character (response to elicitors), probably critical for the response to pathogens, may lead to the recovery of genotypes showing a set of modifications suggestive of a cascade of events leading to active defense.     

Morphological changes of <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis> induced by CF66I,an antifungal compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The morphological effects of CF66I, an antifungal compound produced by Burkholderia cepacia, on growing hyphae of Fusarium oxysporum were studied by fluorescence microscopy (FM) and transmission electron microscopy (TEM). At 20 μg/ml, CF66I strongly inhibited growth and induced significant changes of the hyphal morphology. These changes included swelling of hyphae with considerable thickening cell wall and abnormal chitin deposition, which was indicative of the alterations in cell wall structure. Furthermore, fluorescein diacetate (FDA) staining indicated the loss of intracellular esterase activity. CF66I probably inhibits fungal growth by interfering with the cell metabolic pathways. At 120 μg/ml, CF66I killed F. oxysporum (accompanied by propidium iodide permeation, intracellular cytoplasm leakage and crushing of hyphal tips), probably by direct damage to the cell membrane. Thus, there are two different antifungal mechanisms of CF66I, depending on its concentration, and further studies on this compound might be useful for us to develop a new class of antifungal agents.     

Interaction between Venturia inaequalis and Apple Callus Tissue Cultures: an Electron Microscopic Study

Untrastructural interactions between Venturia inaequalis and callus cultures from scab susceptible and resistant apple varieties, were similar. Host cell wall changes, appositions, and invagination of host plasmamembrane at sites of close contact with fungal hyphae were regularly observed. The ultrastructural observations are described and discussed. The host cell alterations as well as many fungal structures corresponded to those known in young leaves of susceptible apple varieties.