Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum

Pathogen development and host responses in wheat spikes of resistant and susceptible cultivars infected by Fusarium culmorum causing Fusarium head blight (FHB), were investigated by means of electron microscopy as well as immunogold labelling techniques. The studies revealed similarities in the infection process and the initial spreading of the pathogen in wheat spikes between resistant and susceptible cultivars. However, the pathogen’s development was obviously more slow in the resistant cultivars as in comparison to a susceptible one. The structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of the resistant cultivars, than in the susceptible one. β -1,3-glucan was detected in the appositions and papillae. Furthermore, immunogold labelling of lignin demonstrated that there were no differences in the lignin contents of the wheat spikes between susceptible and resistant cultivars regarding the uninoculated healthy tissue, but densities of lignin in host cell walls of the infected wheat spikes differed distinctly between resistant and susceptible cultivars. The lignin content in the cell walls of the infected tissues of the susceptible wheat cultivar increased slightly, while the lignin accumulated intensely in the host cell walls of the infected wheat spikes of the resistant cultivars. These findings indicate that lignin accumulation in the infected wheat spikes may play an important role in resistance to the spreading of the pathogen in the host tissues. Immunogold labelling of the Fusarium toxin DON in the infected lemma showed the same labelling patterns in the host tissues of resistant and susceptible cultivars. However, there were distinct differences in the toxin concentration between the tissues of the susceptible and resistant cultivars. At the early stage of infection, the labelling densities for DON in resistant cultivars were significantly lower than those in the susceptible one. The present study indicates that the FHB resistant cultivars are able to develop active defence reactions during infection and spreading of the pathogen in the host tissues. The lower accumulation of the toxin DON in the tissues of the infected spikes of resistant cultivars which results from the host’s defence mechanisms may allow more intensive defence responses to the pathogen by the host.     

Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew

Levels of resistance to Plasmopara viticola, from susceptible to highly resistant, of different grapevine cultivars were observed in vineyards and confirmed by the symptoms developed after inoculations. On the abaxial surface of infected leaves, P. viticola developed abundant sporangiophores on susceptible cultivars (Chasselas, Gamay, Gamaret and Pinot Noir), whereas on less susceptible cultivars (Seyval Blanc and Johanniter) the parasite produced few sporangiophores and some necrotic spots at the site of infection. On resistant cultivars (Bronner, Solaris, IRAC 2091), P. viticola induced a hypersensitive response and only necrotic spots were visible and the disease ceased to develop. Stilbenes were analyzed 4, 7, 24, 48 h post-infection (hpi) on small leaf samples cut from the site of infection. Large differences were observed between the cultivars at 24 and 48 hpi. Susceptible grapevines produced resveratrol and its glycoside, piceide. In contrast, resistant plants produced high concentration of ε- and δ-viniferin. Resveratrol and piceide have little or no toxicity activity against P. viticola, whereas δ-viniferin is highly toxic and can be considered an important marker for resistance of grapevine to downy mildew. The importance of oxidative dimerization of resveratrol in comparison to the extent of its glycosylation in defense reaction of grapevines against P. viticola is discussed.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression

Large-scale cDNA-AFLP profiling identified numerous genes with increased expression during the resistance response of wheat to the Septoria tritici blotch fungus, Mycosphaerella graminicola. To test whether these genes were associated with resistance responses, primers were designed for the 14 that were most strongly up-regulated, and their levels of expression were measured at 12 time points from 0 to 27 days after inoculation (DAI) in two resistant and two susceptible cultivars of wheat by real-time quantitative polymerase chain reaction. None of these genes was expressed constitutively in the resistant wheat cultivars. Instead, infection of wheat by M. graminicola induced changes in expression of each gene in both resistant and susceptible cultivars over time. The four genes chitinase, phenylalanine ammonia lyase, pathogenesis-related protein PR-1, and peroxidase were induced from about 10- to 60-fold at early stages (3 h–1 DAI) during the incompatible interactions but were not expressed at later time points. Nine other genes (ATPase, brassinosteroid-6-oxidase, peptidylprolyl isomerase, peroxidase 2, 40S ribosomal protein, ADP-glucose pyrophosphorylase, putative protease inhibitor, methionine sulfoxide reductase, and an RNase S-like protein precursor) had bimodal patterns with both early (1–3 DAI) and late (12–24 DAI) peaks of expression in at least one of the resistant cultivars, but low if any induction in the two susceptible cultivars. The remaining gene (a serine carboxypeptidase) had a trimodal pattern of expression in the resistant cultivar Tadinia. These results indicate that the resistance response of wheat to M. graminicola is not completed during the first 24 h after contact with the pathogen, as thought previously, but instead can extend into the period from 18 to 24 DAI when fungal growth increases dramatically in compatible interactions. Many of these genes have a possible function in signal transduction or possibly as regulatory elements. Expression of the PR-1 gene at 12 h after inoculation was much higher in resistant compared to susceptible recombinant-inbred lines (RILs) segregating for the Stb4 and Stb8 genes for resistance. Therefore, analysis of gene expression could provide a faster method for separating resistant from susceptible lines in research programs. Significant differential expression patterns of the defense-related genes between the resistant and susceptible wheat cultivars and RILs after inoculation with M. graminicola suggest that these genes may play a major role in the resistance mechanisms of wheat.     

The role of seedling infection in epiphytotics of ascochyta blight on chickpea

Didymella rabiei, the causal agent of ascochyta blight, survives on infected seeds and seedlings. Diseased seedlings originating from infected seeds occasionally serve as the source for primary infection in chickpea crops. Experiments carried out independently in Australia and in Israel provided quantitative information on the temporal and spatial distribution of ascochyta blight from initial infections and on the relationship between the amount of initial infection and the intensity of subsequent epiphytotics for cultivars differing in susceptibility to the pathogen. Disease spread over short distances (<10 m) from individual primary infections, was governed by rain and wind, and was up to five times greater down-wind than up-wind. Cultivar response to D. rabiei significantly affected the distance and area over which disease spread and the intensity of the disease on infected plants. At onset of the epiphytotic, the relationship between disease spread and time was exponential (P < 0.05; R ² > 0.95) and the area of the resulting foci was over 10 times greater in susceptible cultivars than in resistant cultivars. Regression equations showed the relationship between disease severity and the distance from the focus-plants was inverse-linear for all cultivars tested (P < 0.05). A simulation model based on the experimental data revealed that even if primary infection is infrequent (less than 1% of plants), the consequences are potentially devastating when susceptible cultivars are used. The epidemiological information and simulation model generated by this study provide an increased understanding of the development of an epiphytotic in which the primary foci of disease originate from infected chickpea seedlings.     

Effectiveness and profitability of the <Emphasis Type="Italic">Mi</Emphasis>-resistant tomatoes to control root-knot nematodes

Experiments were conducted to determine the effectiveness and profitability of the Mi-resistance gene in tomato in suppressing populations of Meloidogyne javanica in a plastic-house with a natural infestation of the nematode. Experiments were also conducted to test for virulence and durability of the resistance. Monika (Mi-gene resistant) and Durinta (susceptible) tomato cultivars were cropped for three consecutive seasons in non-fumigated or in soil fumigated with methyl bromide at 75 g m^–2 and at a cost of 2.44 euros m^–2. Nematode densities were determined at the beginning and end of each crop. Yield was assessed in eight plants per plot weekly for 6 weeks. The P_f/P_i values were 0.28 and 21.6 after three crops of resistant or susceptible cultivars, respectively. Growth of resistant as opposed to susceptible tomato cultivars in non-fumigated soil increased profits by 30,000 euros ha^–1. The resistant Monika in non-fumigated soil yielded similarly (P > 0.05) to the susceptible Durinta in methyl bromide fumigated soil but the resistant tomato provided a benefit of 8800 euros ha^–1 over the susceptible one because of the cost of fumigation. Selection for virulence did not occur, although the nematode population subjected to the resistant cultivar for three consecutive seasons produced four times more eggs than the population on the susceptible one. Such a difference was also shown when the resistant cultivar was subjected to high continuous inoculum pressure for 14 weeks. The Mi-resistance gene can be an effective and economic alternative to methyl bromide in plastic-houses infested with root-knot nematodes, but should be used in an integrated management context to preserve its durability and prevent the selection of virulent populations due to variability in isolate reproduction and environmental conditions.     

Analysis of head and leaf reaction towards <Emphasis Type="Italic">Microdochium nivale</Emphasis>

This research examined the variation in the response of eight commercial wheat cultivars to Microdochium nivale isolates using both in vivo FHB tests (AUDPC and RHW measurements) and in vitro detached leaf assays (LGR). Irrespective of fungal variety, the two Italian cvs Fortore and Norba exhibited the greatest amount of visual disease symptoms (mean AUDPC=2.2 and 2.3, respectively), being significantly more susceptible than the other six cultivars (AUDPC 1.24) (P < 0.05). Irrespective of fungal variety, the Italian cv. Norba and the Irish cv. Falstaff were more susceptible than the other cultivars (except Fatima 2) in terms of RHW (P < 0.05), while the cvs Fortore, GK Othalom and Consort were more resistant than the other five cultivars (P < 0.05). In the detached leaf assay, the Hungarian cv. GK Othalom and the Italian cv. Norba were more susceptible (mean LGR=0.79 and 0.81 mm day^–1, respectively) to M. nivalethan the other six cultivars (mean LGR=0.51–0.72) (P < 0.05). Analysis of the relationship between head and leaf reaction to M. nivaleinfection revealed no significant correlation.     

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.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

The Effect of Different Levels of Beet Cyst Nematodes (Heterodera schachtii) and Beet Necrotic Yellow Vein Virus (BNYVV) on Single and Double Resistant Sugar Beet Cultivars

Beet cyst nematode-resistant cultivars, which were introduced recently, originated from the homozygous inbred line B883. This translocation product was unstable and the transmission of resistance when crossed with a susceptible cultivar did not exceed 94%. Tests with the resistant cultivars in climate cabinets showed a wide variety of resistance against Heterodera schachtii and beet necrotic yellow vein virus (BNYVV), expressed as average numbers of infective units per plant and percentages of resistant plants. In a series of field trials at different levels of infection of H. schachtii, their multiplication rates on all resistant cultivars depended on the initial density, which was caused by the presence of small numbers of susceptible plants. Since tolerance to wilting was also incorporated in B883, reasonable yields were obtained in the presence of H. schachtii. However, at increasing initial densities of H. schachtii, yields decreased considerably, since penetrating juveniles cause a hypersensitivity reaction in resistant plants. Based upon the results of three series of field trials, it was concluded that resistant cultivars should preferably be applied at population densities between 500 and 2000 eggs and juveniles of H. schachtii per 100ml of soil. Cultivars with double resistance against H. schachtii and BNYVV behaved like those with H. schachtii resistance in soils infected with beet cyst nematodes, but not with BNYVV. In soils with a combined infection of H. schachtii and BNYVV double resistant cultivars were far superior to single resistant ones, since damage caused by BNYVV was far more serious than damage caused by H. schachtii. No substantial interaction between soil pathogens nor types of resistance could be detected.     

Spread of potato virus Y in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y^NTN (PVY^NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY^NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.     

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.     

Cacao leaf procyanidins increase locally and systemically in response to infection by Moniliophthora perniciosa basidiospores

Flavan-3-ol monomers and oligomers from dimers to decamers were quantitatively determined by HPLC-FLD in leaves of four Theobroma cacao varieties. Cacao flush leaves had higher procyanidin content than mature leaves. Moniliophthora perniciosa basidiospore infection results in an increase in cacao leaf procyanidins in the inoculated leaves, as well as in the uninoculated, adjacent, older leaves on an actively growing branch, resulting in both a local and systemic increase in procyanidin content in response to infection. Procyanidin oligomers increased significantly in leaves of the less susceptible genotype Scavina 6 upon infection, and total procyanidin levels were higher than in Cacao Comum a more disease susceptible genotype. Selection of genotypes with high constitutive and/or inducible content of procyanidins in young flush leaves may be a strategy to develop witches’ broom disease resistant cultivars.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

Emmer wheat, a potential new host of Tilletia indica

Two representative Italian emmer wheat (Triticum dicoccum) landraces, two selected lines and three improved emmer wheat cultivars, derived from crosses with durum wheat (Molisano landrace × ‘Simeto’), were tested for their susceptibility to Tilletia indica, the cause of Karnal bunt of wheat. Plants of emmer wheat were inoculated by injecting allantoid sporidial suspensions into the boot cavity of plants, just prior to ear emergence. A highly susceptible Indian spring wheat cultivar (Triticum aestivum) was used as a comparative control. At maturity of the plants, the seeds were harvested and assessed for incidence and severity of disease. All emmer wheat genotypes tested were infected but showed differing levels of susceptibility. The percentage of infected seeds for individual genotypes ranged from 5.4 to 75.0% compared with 99.1% for WL-711. The severity of infection was less in the old landraces, but it was higher in all the improved emmer wheat cultivars. In conclusion, Italian cultivars of emmer wheat were found to be highly susceptible to T. indica, and are potentially able to support the establishment of the pathogen.Authors L. Riccioni and M. Valvassori contributed equally to this work and should both be considered as first author.     

Growth inhibitors associated with fusarium wilt of tomato

The localization of vascular infections by a process of vascular occlusion is a primary basis for single dominant gene resistance of tomato to fusarium wilt. Failure of this response process to prevent systemic spread of fusarium in susceptible tomato plants is associated with an inhibition of tylose development, a key factor in the overall walling off process. This research was designed to detect, isolate and characterize inhibitory metabolites that accumulate during the course of the tomato/ Fusarium interaction which could inhibit tylose development. Isolation of inhibitors from chloroform extracts of systemically infected tissues was achieved by a series of successive chromatographic separations and bioassays. R_FValues of inhibitors in the several solvent systems employed and their respective colour reactions with chromogenic reagents were recorded. The accumulation of a highly inhibitory compound in chloroform extracts during the early stages of Fusarium infection was determined quantitatively by colourimetric assay in a subsequent experiment. This compound, tentatively identified as rishitin, was isolated from the region of initial infection in stem vascular tissues 1–11 days after inoculation. This inhibitor accumulated rapidly in near-isolines of both resistant and susceptible tomato cultivars in 1–4 days and then continued to accumulate in susceptible, but declined in resistant, plants from 5 to 11 days. The relative phytotoxicity of this compound at low concentrations was determined by assessing its effects on the viability of cultured tomato cells. These data, together with measurements of its accumulation and determinations of its distribution in infected tissues, indicates that rishitin, a host product that accumulates in response to infection, could account for the inhibition of tylose development observed in susceptible tomato plants following infection by Fusarium. Thus we are faced with a paradox in that a substance that presumably functions in defence can, under certain circumstances, interfere with the defence process. A likely resolution of this paradox is discussed.     

Responses of cucumber cultivars to induction of systemic resistance against anthracnose by plant growth promoting fungi

Initial experiment on the reactions of five Japanese cultivars of cucumber toColletotrichum orbiculare infection in the greenhouse revealed that cv Suyo and Gibai were susceptible and moderately susceptible, respectively, while cv Shogoin fushinari and Sagami hanjiro were resistant to infection byC. orbiculare; cv Ochiai fushinari was moderately resistant. The ability of 16 plant growth promoting fungi (some isolates belonged to species ofPhoma and some non-sporulating isolates) isolated from zoysiagrass rhizospheres to induce systemic resistance in the above five cucumber cultivars was tested by growing plants in potting medium infested with barley grain inocula of PGPF in the greenhouse. The second true leaves of 21-day-old plants were challenge inoculated withC. orbiculare and disease assessed. Nine, out of 16 isolates, caused significant reduction of disease caused byC. orbiculare in at least two cultivars.Phoma isolates (GS8-1 and GS8-2) and non-sporulating isolates (GU21-2, GU23-3, and GU24-3) significantly reduced the disease in all the five cultivars. The disease suppression in cucumber was due to the induction of systemic resistance, since the inducer(s) and the pathogen were separated spatially and that the inducer did not colonize aerial portions. The resistance induced by certain isolates in a susceptible cultivar was less than that in a resistant cultivar. Disease suppression caused by isolate GU21-2 was similar to theC. orbiculare induced control in certain cultivars. The average rate of expansion of lesion diameter on leaves due toC. orbiculare was slower due to induction with the selected plant growth promoting fungi compared to the uninduced control plants. Roots of four cultivars were colonized by only three isolates, however, roots of one cultivar (Suyo) was colonized by five isolates suggesting the cultivar-specific root colonization ability.Abbreviations cv cultivar(s) - PGPF plant growth promoting fungal isolates - PGPR plant growth promoting rhizobacteria