Life at the edge – the cytology and physiology of the biotroph to necrotroph transition in Hymenoscyphus fraxineus during lesion formation in ash

The progress of colonization of ash stems from ascospore inocula of Hymenoscyphus fraxineus was examined by light and electron microscopy. The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during lesion formation. Following direct penetration into epidermal cells, the fungus produced intracellular hyphae that invaded up to five cells before plant cells died. A lack of close attachment between the hyphal cell wall and plant cell membrane was revealed by plasmolysis of epidermal cells. Plant cells died at the centre of the infection but hyphae at the edge were typically found in living plant cells even around large lesions. During biotrophic invasion, the cytoplasm of penetrated plant cells showed very little response despite the plant cell membrane being in direct contact with the fungal cell wall. Before plant cell death, dark staining of the cytoplasm and proliferation of small vesicles was noted, but organelles retained normal ultrastructure. Dead plant cells contained dark brown, osmiophilic droplets. Penetration between epidermal or collenchyma cells was usually targeted to shared pits and involved constriction of hyphae. The transition to necrotrophy was not associated with a clear change in hyphal morphology. Biotrophic intracellular hyphae contained dense cytoplasm but hyphae in dead plant cells were more vacuolated. Remarkably little plant cell wall degradation was observed despite the fungus penetrating up to 18 cells deep into stem tissue. Features of the development of the ash dieback fungus are compared with other hemibiotrophic pathogens.     

Evaluation of Induction of Systemic Resistance in Pepper Plants (Capsicum Annuum) to Phytophthora capsici Using Trichoderma harzianum and its Relation with Capsidiol Accumulation

The effect of pepper seed and root treatments with Trichoderma harzianum spores on necrosis caused in stems by Phytophthora capsici inoculation and on the course of capsidiol accumulation in the inoculated sites were studied. The results indicate that seed treatments significantly reduced stem necrosis, which fell by nearly a half compared with the values observed in plants grown from non-treated seeds. Necrosis was also reduced in plants whose roots were drenched with various doses of T. harzianum spores, although the extent of necrosis was not correlated with the dose used. Attempted isolation of P. capsici and T. harzianum from the zones immediately contiguous with the necrotic zones revealed the presence of the former but not of the latter, suggesting that there was no direct contact between them in the zones of isolation, which means that there was no competition for space. The percentage of P. capsici isolated 9 days after inoculation was greater in non-treated inoculated plants than in treated inoculated plants. These results suggest that T. harzianum, introduced into the subterranean part of the plant, induces a systemic defense response against P. capsici in the upper part of the plant. Analysis of capsidiol in the stems of treated inoculated plants by the end of the sixth day after inoculation, revealed that its concentration was more than seven-fold greater than in non-treated and inoculated plants, while after 9 days, the concentration of capsidiol decreased in the treated inoculated plants and increased in the non-treated inoculated plants. The high concentration of capsidiol detected in treated and inoculated stems after 6 days might be one of the contributing factors, but not necessarily the main factor, in delaying lesion development in the stems of pepper plants.     

Histological and ultrastructural comparisons of compatible, incompatible and -β-amino- n -butyric acid-induced resistance responses of pepper stems to Phytophthora capsici

The compatible interaction of pepper stems with Phytophthora capsici showed more rapid and severe disease development than did the incompatible interaction, although pathogen penetration styles of host cells in compatible and incompatible interactions were similar to each other. Treatment with -β-amino- n -butyric acid (BABA) protected the pepper plants against P. capsici infection. Reduced hyphal growth and sporangial formation were found after P. capsici infection in BABA-induced resistant and incompatible reactions. One of the most noticeable ultrastructural features of the BABA-induced resistant reaction was the formation of electron-dense wall appositions. The thick and dense wall appositions that encased the haustoria restricted haustorial development, thus leading to limitation of further pathogen penetration into inner plant tissues. A main host response in the incompatible interaction was the occlusion of cortical cells with an amorphous material. Plugging of the intercellular spaces in the cortical cells with electron opaque material was frequently observed in the incompatible interaction, but not in the compatible interaction. Another common feature of the BABA-induced resistant and incompatible reactions was degeneration of mitochondrial structure within penetrating hyphal cytoplasm. The mitochondrial structure in the BABA-induced resistant or incompatible reactions had no distinct double membrane layer and well-shaped cristae.     

Effect of phenology on susceptibility of Norway spruce (Picea abies) to fungal pathogens

Ceratocystis polonica and Heterobasidion parviporum are important fungal pathogens in Norway spruce (Picea abies). Tree susceptibility to these pathogens with respect to phenology was studied using artificial fungal inoculations at six stages of bud development, and assessed by measuring phloem necroses in the stems of 2‐ and 8‐year‐old trees. Tree capacity for resistance was assessed by measuring phloem nonstructural carbohydrates at each stage. Phloem necroses were significantly larger in trees with fungal versus control inoculations and increased significantly over time. Changes in nonstructural carbohydrates occurred in the trees; a significant decline in starch and a slight but significant increase in total sugars occurred over time. These results suggest that susceptibility to fungal pathogens and carbohydrate levels in the stems of the trees were related to fine‐scale changes in bud development. A trade‐off may occur between allocation of starch (the major fraction of the stem carbohydrate pool) to bud development/shoot growth versus defence of the stem. Previous tests of plant defence hypotheses have focused on herbivory on plants growing under different environmental conditions, but the role of phenology and the effect of pathogens are also important to consider in understanding plant resource allocation patterns.     

Cyclical systemic colonization inVerticillium-infected tomato

It is generally believed thatVerticilliummoves slowly up a plant through spore trapping sites in the vascular tissue in a stepwise fashion. In a resistant plant, rapid defence responses around these sites slow pathogen growth and often restrict the fungus to the root and stem base. In a susceptible plant, the fungus escapes and eventually proliferates logarithmically in the upper stem and leaves. In the present study Craigella tomatoes were infected withVerticillium albo-atrum. Amounts of pathogen were monitored in the upper stems by a PCR-based quantitative assay and S₁nuclease protection was used to study expression of a fungal induced phenylalanine ammonia lyase gene (tPAL5) during a 2–21 day time course. Even in resistant plants fungal colonization and PAL gene expression were found to be fully systemic from the earliest sporulation events (i.e. 2–4 days post-inoculation) and to occur in cycles, the PAL gene cycle following the colonization cycle. Peaks of pathogen growth occurred at 2–4 days and 12–15 days, succeeded by intermittent periods of fungal elimination. The results indicated that wilt is more systemic and also more dynamic in terms of fungal population fluctuations than previously thought.     

Ultrastructural localization of chitin in cell walls of Rigidoporus lignosus, the white-rot fungus of rubber tree roots

The distribution of N-acetylglucosamine residues in the cell wall of the white-rot pathogenic fungus, Rigidoporus lignosus, was studied by using gold labelled wheatgerm agglutinin bound to ovomucoid-colloidal gold. Ultrastructural investigation of R. lignosus-infected root tissues of Hevea brasiliensis showed a modification of the fungal cell wall throughout the infection process. Gold particles were found to occur on both thick- and thin-walled hyphae of R. lignosus rhizomorphs at the root surface. Walls of hyphae that had penetrated the roots were only labelled when they were out of the host cell, suggesting that modification of chitin molecules may be related to the excretion of host cell wall degrading enzymes. Variation in the distribution of gold particles was observed over hyphal walls of both colonized phellem and xylem cells. The observation that N-acetylglucosamine residues were released in the host cell cytoplasm suggests that lytic enzymes alter the fungal cell walls. Released chitin oligosaccharides may play a role in the induction of the root's defence system against fungal attack.     

Laboratory studies to assess the risk of development of resistance to zoxamide

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide‐resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide‐amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors ≤2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target‐site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site‐specific fungicide, appropriate precautions against resistance development should be taken. © 2001 Society of Chemical Industry     

Microscopy of invasion of red clover roots by Trichocladium basicola, and effects of benomyl and prochloraz

Roots of red clover seedlings grown on plates of water agar, or water agar containing benomyl or prochloraz, were inoculated with conidia of Trichocladium basicola and examined by light and transmission electron microscopy. Penetration of host epidermal cells occurred from about 16 h after inoculation of untreated or fungicide-treated seedlings. Intracellular hyphae were constricted at septa and had a beaded appearance. They invaginated the host plasmalemma, but had no obvious deleterious effect on the cytoplasm until they had grown to fill much of the lumen, when host cells degenerated and died. As colonization of the cortex progressed, straight, unconstricted hyphae were formed and from these reproductive hyphae developed, which produced endoconidiophores and chlamydospores on the root surface. Penetration of host cell walls appeared to involve localized action of fungal enzymes. Papillae were often found at sites of penetration, but these rarely obstructed fungal development. Seedlings treated with prochloraz had fewer sites of fungal penetration, and fewer cells in the beaded hyphae than untreated seedlings or those treated with benomyl. Both fungicides caused abnormalities in fungal ultrastructure. Hyphae treated with benomyl were often found to contain lomasomes, while those treated with prochloraz had thickened, fragmented walls, and disorganized cell contents.     

Resistance of Phytophthora capsici to metalaxyl in plastic‐house capsicum crops in southern Italy*

In Calabria (southern Italy), control of crown and root rot of capsicum caused by Phytophthora capsici has relied primarily on soil drenches of metalaxyl. However, severe outbreaks occur every year in glasshouse crops, in which the practice of using plastic mulch and furrow irrigation favours the disease. Single‐hypha isolates of P. capsici collected in Calabria in 1992/1998 were tested in vitro for their level of sensitivity to metalaxyl. Isolates of other species of Phytophthora were used as reference. Fungicide sensitivity was determined by plating mycelial plugs onto potato dextrose agar amended with metalaxyl, at final concentrations ranging from 0.1 to 1000μg mL^?1 a.s. Inhibition of radial growth (%) was determined when colonies on unamended medium had covered approximately two‐thirds of the plate. The ED₅₀ values for inhibition of mycelial growth of P. capsici isolates ranged from 1.41 to44.6μg mL^?1 a.s. More than 80% of the P. capsici isolates from commercial plastic‐house crops in Calabria showed a moderate level of resistance as they were inhibited less than 60% at 5 μg mL^?1 but more than 60% at 100μg mL^?1     

Integrated pest management: A strategy to control resistance of Spodoptera exigua and Helicoverpa armigera caterpillars to insecticides on soybean in the Mekong Delta

The antibiotic nucleoside tubercidin produced by Streptomyces viola-ceoniger was evaluated for in-vivo efficacy and in-vitro activity against Phytophthora capsici, Magnaporthe grisea and Colletotrichum gloeosporioides. Tubercidin was more effective against P. capsici and M. grisea than against C. gloeosporioides in inhibiting mycelial growth. The bioassay on TLC plates was the most sensitive method and allowed the evaluation of antifungal activity of tubercidin even at a low concentration of 0.1 μgml^?1. As compared to the systemic fungicide metalaxyl, tubercidin was similar or somewhat higher in inhibition of mycelial growth of P. capsici. When applied to pepper stems, tubercidin was equally as effective as metalaxyl in the control of phytophthora blight in pepper plants, irrespective of application time and concentration. The treatment with 1000 μg ml^?1 tubercidin induced phytotoxicity in pepper plants. No control efficacy of phytophtora blight was observed in pepper plants supplied with a soil drench of tubercidin. Treatment with tubercidin at 500 μg ml^?1 completely protected pepper plants at first branch stage from phytophthora blight until four days after application. The control efficacy of tubercidin drastically declined seven days after application.     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.     

The plant cell wall as a site for molecular contacts in fungal pathogenesis

The plant cell wall, the most external layer of the plant surface, is the site where most pathogenic fungi first make contact with host cells. A plant–fungus interaction therefore commences at the interface between the plant and the spore. Our current research focusing on the plant cell wall has discovered an extracellular ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase/apyrase; EC3.6.1.15) as a key player in plant defense before the onset of PTI (PAMP-triggered immunity). This review focuses on our recent findings, especially the role of the plant cell wall in the extracellular defense against fungi as well as fungal strategies resulting in successful infection.     

Sexual reproduction increases the possibility that Phytophthora capsici will develop resistance to dimethomorph in China

A total of 1511 isolates of Phytophthora capsici were collected from farms with no history of exposure to the carboxylic acid amide (CAA) fungicides in 32 provinces in China during 2006 to 2013. All 1511 isolates were assayed for mating type and 403 were assayed for sensitivity to dimethomorph (DMM) and metalaxyl. The DMM EC₅₀ values ranged from 0·126 to 0·339 μg mL^?1. Both A1 and A2 mating types were detected on the same farms in four provinces and with a 1:1 ratio. Most isolates were sensitive to metalaxyl but a few exhibited intermediate resistance or resistance to metalaxyl. The segregation of DMM resistance and sensitivity among 337 progeny obtained from hybridization or self‐crossing in vitro indicated that the resistance of P. capsici to DMM is controlled by two dominant genes. Eighteen progeny that were derived from hybridization differed in DMM sensitivity and in fitness. Some progeny were as fit as parental isolates. Given the distribution of mating types and therefore the potential for sexual reproduction, the control of resistance by two dominant genes, and the fitness of hybrid progeny, the risk of P. capsici populations developing DMM resistance in China is substantial.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Studies on the Infection Process of Fusarium Culmorum in Wheat Spikes: Degradation of Host Cell Wall Components and Localization of Trichothecene Toxins in Infected Tissue

After single spikelet inoculation, the infection process of Fusarium culmorum and spread of fungal hyphae in the spike tissues were studied by scanning and transmission electron microscopy. While hyphal growth on outer surfaces of the spike was scanty and no successful penetration was observed, the fungus developed a dense mycelium on the inner surfaces and effectively invaded the lemma, glume, palea and ovary by penetration pegs. During the inter- and intracellular spreading of the fungus, marked alterations in the host tissues were observed, including degeneration of cytoplasm, cell organelles, and depositions of electron dense material between cell wall and plasmalemma. Ultrastructural studies revealed that host cell walls in proximity of the penetration peg and in contact with hyphae were less dense or transparent which suggested that cell wall degrading enzymes were involved in colonisation of host tissues by fungal hyphae. Enzyme- and immunogold-labelling investigations confirmed involvement of extracellular enzymes, that is cellulases, xylanases and pectinases, in degradation of cell wall components. Localization studies of trichothecenes indicated that toxins could be detected in host tissues at an early stage of infection.     

Tolerance to a non-host isolate of Verticillium dahliae in tomato

Tolerance to Verticillium spp. is a condition in which a host plant develops few symptoms despite substantial colonization by the pathogen. In the present paper we have shown that Craigella tomatoes are tolerant to a non-host isolate of V. dahliae, Dvd E6. Symptom expression was used to quantify disease and quantitative PCR to assess the amount of fungus in the stems. The classical incompatible and compatible interactions between Craigella resistant or Craigella susceptible near isolines and V. dahliae, race 1 were used for comparative purposes. Additional experiments using cytological assessment and quantitative PCR showed that in the tolerant interactions one plant defence response, vascular coating, was deployed as effectively as in resistant plants, limiting pathogen distribution. However, a second defence response, which causes the cyclical elimination of fungus from the stem in the classical interactions either does not occur or is substantially delayed in tolerant plants. Thus, the Verticillium population remains stable and substantial throughout the studied time course.     

Microscopical studies of the effect of metalaxyl on the interaction between sunflower,Helianthus annuus L. and downy mildew,Plasmopara halstedii

The mode of action of the fungicide metalaxyl againstPlasmopara halstedii, the causal agent of sunflower downy mildew, was studied following its application before, during and after artificial contamination of seedlings. The development of the fungus within the treated seedlings was examined microscopically and compared to that occuring in untreated genetically susceptible or resistant genotypes. Hypersensitive-like reactions and necrotic zones leading to the inhibition of fungus growth within the hypocotyl were observed for the three modes of application. This suggests that sunflower defence mechanisms activated by genetical resistance are also involved in the control of downy mildew by metalaxyl.     

Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum

An isolate of the fungus Fusarium culmorum constitutively expressing green fluorescent protein was used to investigate the infection process and host response of primary seedling roots and stem base leaf sheaths of soft wheat cv. Genio. Disease progress was assessed macroscopically by visual symptoms, microscopically by confocal laser scanning microscopy (CLSM) and via gene expression analysis of fungal and wheat genes by real‐time quantitative RT‐PCR. In the roots, CLSM investigations revealed an initial intercellular and subsequent intracellular colonization by fungal hyphae. The fungus invaded the rhizodermal layer and cortex but was not seen to colonize the stele. The fungus consistently expressed TRI5 (24, 48 and 96 h post‐inoculation), indicating that trichothecenes were being synthesized throughout this phase of infection and colonization. The expression of the six host defence‐associated genes (Wheatwin 1‐2, PR1, Chitinase, PAL, WIR1 and LOX) increased early in infection and decreased during later stages. In the stem base, CLSM observations revealed the fungus sequentially penetrating though the first, second and third basal leaf sheaths. Expression of TRI5 was initiated early in the infection of each leaf sheath. The expression of the host defence‐associated genes varied over time and across leaf sheaths, and all were also expressed in leaf sheaths which had not yet been in contact with the fungus. Expression of LOX and WIR1 were particularly enhanced in the third leaf sheath.     

Induced resistance againstPhytophthora capsici in pepper plants in response to DL-β-amino-n-butyric acid

Treatment of pepper plants with the nonprotein amino acid, DL-ß-amino-n-butyric acid (BABA) induced resistance to subsequent infection byPhytophthora capsici. In contrast, theα-, andγ-isomers of aminobutyric acid were ineffective as inducers of resistance. A relatively high concentration of BABA at 1,000μg ml^?1, which had no antifungal activityin vitro againstP. capsici, was required to induce resistance against Phytophthora blight with a foliar and stem spray, thus leading to complete control of the disease. About 1 day interval between BABA-treatment and challenge inoculation was sufficient to induce resistance in pepper plants. High inoculum levels ofP. capsici caused Phytophthora development slowly in pepper stems treated with BABA, especially at early plant growth stage, which suggests that the induced resistance in pepper plants may be more quantitative rather than qualitative. BABA applied to the root system also protected pepper stems fromP. capsici infection.     

An ultrastructural study, including cytochemistry and quantitative analyses, of the interactions between pseudomonads and leaves of Phaseolus vulgaris L.

Infection of Phaseolus vulgaris cultivars Red Mexican and Tendergreen with Pseudomonas fluorescens, P. syringae pv. coronafaciens, P.s. pv. phaseolicola races 1 and 3, and a mutant of race 3 (race 3 M1) with altered cultivar specific virulence was examined. In addition to qualitative observations of the development of colonies of bacteria and the responses in adjacent plant cells, quantitative analyses were made of the numbers of bacteria within sections of colonies, contact between bacteria and the plant cell wall, the accumulation of fibrillar acidic polysaccharides (which stained with ruthenium red) around bacteria, convolution of the plant cell membrane adjacent to bacteria, deposition of paramural papillae, rupture of the tonoplast and the occurrence of cytoplasmic disorganization. The presence at infection sites of material staining with the periodic acid, thiocarbohydrazide silver proteinate (PATAg) procedure to localize vicinal glycols was also quantified.Cells of P. fluorescens failed to multiply in the plant and seemed tightly bound at junctions between mesophyll cells. The pathovars of P. syringae all multiplied at similar rates during the first 12 h after inoculation and were not closely attached to the plant cell wall. Fibrillar, ruthenium red staining material, considered to be bacterial extracellular polysaccharides, accumulated around cells of the pathovars of P. syringae irrespective of the compatibility of their interaction with cultivars. Amorphous PATAg positive deposits formed around cells of the saprophyte and as condensed aggregates in colonies of P. syringae pathovars in tissue undergoing the hypersensitive reaction (HR) but not during compatible interactions. The PATAg positive material may be involved in the elicitation of responses by plants during the HR. The first response of plant cells to adjacent bacteria was the localized convolution of the plasma membrane; this response was neither race nor species specific. Deposition of paramural papillae was also found to be a non-specific response occurring during compatible and incompatible interactions. Some components of papillae were PATAg positive. Plasmolytic studies demonstrated that, during the HR, irreversible damage to the plasma membrane first occurred 5 and 8 h after initial convolution of the membrane following inoculation of cv. Tendergreen with P.s. pv. coronafaciens and P.s. pv. phaseolicola race 3 respectively. Tonoplast dysfunction appeared to follow irreversible damage to the plasma membrane and preceded loss of compartmentation and cytoplasmic collapse. The ultrastructural study showed that many plant responses were non-specific and therefore could be separated from irreversible damage to the plasma membrane which was the irrevocable event during the HR.