Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae

Interactions between lettuce and a green fluorescent protein (GFP)-expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plant's ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.     

Colonization of roots of subterranean clover cultivars by virulent and avirulent races of Phytophthora clandestina

The colonization of the roots of four cultivars of subterranean clover by isolates representing four races of Phytophthora clandestina was studied. There was a highly significant race × cultivar interaction in the growth of inoculated tap roots and the degree of colonization of roots by the pathogen. While all races were able to infect the roots of all cultivars tested, roots of the susceptible cultivars were colonized more rapidly and extensively than those of the resistant cultivars. In compatible combinations, fungal colonization extended for a few centimetres in the tap root and lateral roots in the moderately susceptible cultivars Trikkala and Meteora, or throughout the whole root system leading to the death of the host in the very susceptible cultivar Woogenellup. In contrast, limited fungal colonization of the tap root and lack of extension of the fungus into lateral roots was typical of incompatible combinations. In all cultivars, lateral roots were as susceptible to infection as tap roots. The number of lateral roots of Woogenellup was significantly reduced by infection. However, neither the rate of lateral root formation nor the total number of lateral roots of Seaton Park, Meteora and Trikkala was reduced by infection with virulent or avirulent races of the pathogen.     

A light and scanning-electron microscopic study of infection of tomato plants by virulent and avirulent races of Cladosporium fulvum

Infection of tomato plants byCladosporium fulvum Cooke was studied using light and scanning-electron microscopy. Races 1.2.3 and 4 ofCladosporium fulvum were used, whereas tomato cultivars, carrying the Cf2 gene (susceptible to race 1.2.3 and immune to race 4) and the Cf4 gene (immune to race 1.2.3 and susceptible to race 4) served as differentials. No differences were observed in growth between compatible and incompatible combinations during germination, subsequent formation of runner hyphae and stomatal penetration. Runner hyphae did not show directional growth towards stomata. Penetration usually occurred on the third or fourth day after inoculation. In compatible combinations the fungus grew intercellularly, often in close contact with spongy mesophyll cells. Under optimal conditions it did not cause visible damage to plant cells during early stages of infection. Under suboptimal conditions in winter, the host cells often reacted with callose deposition, but growth of the fungus did not appear to be inhibited. Ten to twelve days after inoculation conidiophores emerged through the stomata and produced conidia. In incompatible combinations fungal growth was arrested one to two days after penetration and confined to stomata and surrounding cells. Very soon the host cells, in contact with the fungus, deposited extensive amounts of callose. Later these cells turned brown and collapsed. At the surface of the host cells, contacted by fungal hyphae, abundant extracellular material could be observed by scanning-electron microscopy. Removing the epidermis of leaves before inoculation delayed the resistant response. On stripped leaves the rate of fungal growth was equal for both interactions up to ten days after inoculation, but the incompatible combination lacked sporulation.     

Differences in the responses of melon accessions to fusarium root and stem rot and their colonization by Fusarium oxysporum f. sp. radicis‐cucumerinum

Fusarium root and stem rot caused by the fungus Fusarium oxysporum f. sp. radicis‐cucumerinum is a major disease in greenhouse cucumbers. Over the past decade, the disease has been documented in melon greenhouses in Greece, and recently it has been sporadically recorded in greenhouse melons in Israel. Variations in disease response were found among 41 melon accessions artificially inoculated with the pathogen: 10 accessions were highly susceptible (90–100% mortality), 23 exhibited an intermediate response (20–86%) and eight were resistant (0–4%). Two melon accessions – HEM (highly resistant) and TAD (partially resistant) – were crossed with the susceptible accession DUL. The responses of the three accessions and F₁ crosses between the resistant and susceptible parents were evaluated. HEM contributed higher resistance to the F₁ hybrid than TAD. Roots of susceptible and resistant accessions were 100 and 79% colonized, respectively, following artificial inoculation. However, only susceptible plants showed colonization of the upper plant tissues. Microscopic evaluation of cross sections taken from the crown region of the susceptible DUL revealed profuse fungal growth in the intercellular spaces of the parenchyma and in xylem vessels. In the resistant cultivar HEM, very little fungal growth was detected in the intercellular spaces of the parenchyma, and none in the xylem or any other vascular tissue. Finding resistant accessions may create an opportunity to study the genetics of resistance inheritance and to develop molecular markers that will facilitate breeding resistant melon cultivars.     

Bacterial wilt resistance in tomato associated with tolerance of vascular tissues to Pseudomonas solanacearum

High populations of Pseudomonas solanacearum were detected in some, but not all stems of bacterial wilt resistant ('CRA 66', 'Hawaii 7996' and 'Caraibo') and susceptible ('Floradel') tomatoes. Latent infection, i.e. spread of P. solanacearum into xylem vessels, was confirmed in Caraibo, Hawaii 7996 and 'CRA 66' (the resistant parent of Caraibo). None of the plants within the resistant cultivars wilted and those cultivars were characterized by tolerance of the vascular tissues to high bacterial densities. In contrast, plants of cultivar Floradel showed consistent symptoms and wilted rapidly, with higher mean bacterial density than resistant cultivars. Bacterial wilt resistance was not associated with resistance to bacterial root invasion but with the capability of the plant to limit P. solanacearum colonization in the stem. The extent of bacterial colonization is proposed as a criterion to quantify tolerance, complementary to absence of external wilt symptoms used in breeding programmes for resistance.     

Quantification of Root and Stem Colonization of Watermelon by Fusarium oxysporum f. sp. niveum and Its Use in Evaluating Resistance

ABSTRACT Colonization of watermelon root and stem tissues by Fusarium oxysporum f. sp. niveum race 1 and its relationship to the apparent resistance to Fusarium wilt was investigated. In each of 2 years, 17 differentially susceptible watermelon cultivars and one accession were tested in the greenhouse, and 7 cultivars also were tested in the field. Colonization by a chlorate-resistant marked isolate of the fungus was assayed by plating homogenized tissue samples on a selective medium. Six days after inoculation, seedlings of highly resistant, moderately resistant, and susceptible cultivars had F. oxysporum f. sp. niveum race 1 CFU counts in the lower stems of 10(2), 10(3), and 10(4) CFU/g of fresh tissue, respectively. Percent wilt (Y) of the seedlings was positively correlated with colonization (X) by F. oxysporum f. sp. niveum race 1 in roots (Y = 21.2 ln [X + 1] - 140.7, R(2) = 0.85) or lower stems (Y = 17.3 ln [X + 1] - 78.6, R(2) = 0.86). Percent wilt (Y) also was correlated with the ratio (X(r), 0 to 1 values) of lower stem to root colonization (Y = 34 ln X(r) + 112, R(2) = 0.36). Field evaluations confirmed these relationships, and a link between cultivar resistance and a reduced rate of spread of the fungus in primary stems during a season was observed. Fruit yield decreased with increased tissue colonization at linear rates of 9.9 to 12.7 t/ha per ln (CFU/g + 1) (R(2) >/= 0.58). The greenhouse seedling stem colonization assay described may be utilized as a collaborative method to quantify Fusarium wilt resistance in watermelon.     

The infection process of Moniliophthora perniciosa in cacao

The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

Infection of Winter Wheat by a beta-Glucuronidase-Transformed Isolate of Cephalosporium gramineum

ABSTRACT Field-grown winter wheat was inoculated with a beta-glucuronidase-transformed isolate of Cephalosporium gramineum in two field seasons to elucidate the mode of infection in resistant and susceptible cultivars. Colonization of viable root epidermis and cortical cells occurred as soon as 15 days postinoculation and the pathogen was found in the vascular tissues by 20 days postinoculation, well before freezing soil temperatures occurred. Penetration occurred directly through the root epidermis and through wounds adjacent to emerging secondary roots. The pathogen also penetrated through root cap cells and colonized meristematic tissues near root tips to gain access to the vascular system. Lower stem base colonization was observed where the pathogen penetrated directly through the epidermis, wounds, or senescent tissues. Appressorium-like structures, which appeared to aid penetration of cell walls, were often found within cells of both roots and stems after initial colonization. The mechanisms of resistance were not apparent, but less colonization occurred in resistant than in susceptible cultivars.     

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.     

Spatial-Temporal and Quantitative Analysis of Growth and EPS I Production by Ralstonia solanacearum in Resistant and Susceptible Tomato Cultivars

ABSTRACT One susceptible and two resistant cultivars of tomato were tested for differences in infection by Ralstonia solanacearum and for the subsequent multiplication, colonization, and production of the wilt-inducing virulence factor, exopolysaccharide I (EPS I). Bacterial ingress into the taproot was fastest in the susceptible cv. Marion, followed by the resistant cvs. L285 (fivefold slower) and Hawaii 7996 (15-fold slower). Once inside the taproot, R. solanacearum colonized, to some extent, almost all regions of the resistant and susceptible plants. However, colonization occurred sooner in the susceptible than in the resistant cultivars, as measured by viablecell counts of bacteria in the midstems. Rates of multiplication and maximum bacterial cell densities were also greater in the susceptible than in the resistant cultivars. Growth experiments utilizing xylem fluid from infected and uninfected plants indicated that neither antimicrobial activities nor reduced levels of growth-supporting nutrients in the xylem fluids were responsible for the reduced bacterial multiplication in the resistant cultivars. Quantification of EPS I in the infected plants, using an enzyme-linked immunosorbent assay, revealed that the bacterial populations in the susceptible cultivar produced greater amounts of EPS I per plant than those in the resistant cultivars. Immunofluorescence microscopy using antibodies against either EPS I or R. solanacearum cells revealed that bacteria and EPS I were distributed throughout the vascular bundles and intercellular spaces of the pith in the susceptible cultivar, whereas in the resistant cultivars, bacteria and EPS I were restricted to the vascular tissues.     

棉花枯萎病菌接种及粗毒素处理后棉苗维管束病理特征

 利用遗传背景一致而抗病性不同的岱16变异品系和抗病品种中12,研究了接种棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)及用枯萎病菌粗毒素处理后,棉苗维管束的病理变化。研究结果表明,接种枯萎病菌后,病株茎部导管及薄壁细胞中有枯萎菌丝存在。发病严重的感病品种原岱16有菌丝的导管数最多,抗病品种早熟岱16抗最少。所有品种中均出现胶状物或侵填体堵塞导管,这种现象在抗病品种中更为明显。病菌粗毒素处理后,棉苗茎及叶柄导管内也出现侵填体或胶状物堵塞导管,抗病品种茎部导管堵塞快。     

Initial Infection and Colonization of Leaves and Stems of Cling Peach by Tranzschelia discolor

ABSTRACT Initial infection processes and the subsequent colonization of leaves and young stems of peach by Tranzschelia discolor were studied. On leaves where multiple disease cycles of peach rust occur during the growing season, urediniospores germinated after 4 h of wetness. Germ tubes became septate and formed appressoria only over leaf stomata beginning 18 h after inoculation. No appressoria, however, formed over stomata of positive replicas of leaf surfaces indicating nonthigmotropic responses of germ tubes. On young, primary-growth stems (ca. 8 weeks old), stomata were mostly closed, less frequent than on leaves, and recessed from the surface of the cuticle of the epidermis. Although appressoria formation was not observed on inoculated stems, germ tube growth of urediniospores was directional toward stomata. Penetration of stem tissue is apparently a less common event that was reflected by a lower occurrence of stem lesions compared with that of leaf lesions in our potted plant inoculation studies and previous field observations. Still, stem lesions are important as sources of primary inoculum each spring and were reproduced in this study for the first time. Fungal colonization of leaves and stems was subepidermal-intercellular and haustoria were commonly found within mesophyll or cortical cells, respectively. No fungal colonization was observed in cambial stem tissue. Vascular tissue was also not colonized and delimited lesions in leaves and stems. Morphological host responses were not observed in infections on either leaves or young stems. In older stems (>32 weeks old), however, the infection was delimited by a wound periderm after uredinial formation. Furthermore, with continued secondary growth, stems recovered and fungal lesions became part of the bark tissue of woody branches. Thus, the fungus must infect primary-growth branches each year to establish stem lesions.     

Distribution of Pseudomonas solanacearum in the stem tissues of tomato plants with different levels of resistance to bacterial wilt

Ten tomato cultivars, from the USA, Taiwan and French Antilles, were compared for resistance to bacterial wilt as measured by disease and pathogen population invasiveness and density in the stem at the collar and midstem regions. On the basis of disease incidence, cultivars ranked from highly susceptible to totally resistant. By contrast, no significant difference was observed in bacterial population in wilting plants, regardless of the cultivar. All symptomless plants were latently infected at the collar level. Percentage of symptomless plants with bacteria at the midstem level was significantly correlated with the degree of resistance; the more resistant, the lower the stem colonization. Restriction of Pseudomonas solanacearum invasiveness in the vascular tissues of the stem is associated with resistance properties in tomato. This observation may be useful in developing improved criteria for selecting stable resistance to the disease.     

Response ofVerticillium-resistant andVerticillium-susceptible tomato varieties to inoculation with the nematodeMeloidogyne javanica and withVerticillium dahliae

The reaction of two tomato varieties, Gilat 38 (Verticillium resistant) and Rehovot 13 (Verticillium susceptible), to inoculation withMeloidogyne javanica andVerticillium dahliae, was examined. The nematode did not break the resistance of Gilat 38 to the fungus, but it enhanced fungus incidence and symptoms in Rehovot 13.     

Effect of method of inoculation, inoculum density and seedling age at inoculation on the expression of resistance of cocoa (Theobroma cacao L.) to Verticillium dahliae Kleb.

Soil drench and stem puncture inoculation were compared as methods for selecting cocoa cultivars with resistance to Verticillium dahliae. Disease progress was more rapid and induced symptoms were more severe following stem puncture and, under glasshouse conditions, differences between cultivars were detected 15 days after inoculation. Moreover, using stem puncture, inoculum densities of 10⁴ conidia/ml were sufficient to differentiate resistant and susceptible cultivars, whereas with the soil drench method, inoculum densities of 10⁷ conidia/ml were necessary. Although a substantially higher proportion of plants were affected by stem puncture inoculation, the resistance of cultivar Pound-7 remained effective at high inoculum densities of 10⁸ conidia/ml. With either method, older seedlings were more susceptible to V. dahliae than younger ones. However, with stem puncture, 15-day-old seedlings were sufficiently susceptible for a valid disease assessment. In contrast, with soil inoculation, 60-day-old plants were required. In a nursery trial with 15-day-old seedlings, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to Verticillium dahliae , on the basis of root inoculation, were ranked in the same order when stem punctured. Stem puncture inoculation of young seedlings is cost-effective in terms of time and space, and is therefore recommended for screening of cocoa for wilt resistance, especially in large-scale breeding programmes.     

Blue mold of tomato caused by <Emphasis Type="Italic">Penicillium oxalicum</Emphasis> in Japan

In 2006, stem rot and blue-green crusty lesions were found on the stems of tomato plants in Chiba Prefecture, Japan. Penicillium oxalicum was isolated repeatedly from the diseased plants. The causal fungus reproduced natural symptoms after artificial inoculation of tomato plants and was re-isolated from symptomatic plant tissue. P. oxalicum is a new pathogen that causes blue mold on tomato plants in Japan.     

Soluble phenolic acids in Capsicum annuum stems infected with Phytophthora capsici

The soluble phenolic acids of the stems of three Capsicum annuum cultivars associated with differing resistance to Phytophthora capsici showed qualitative and quantitative variation after inoculation, notably an increase in the total phenolic acid content in the resistant and intermediate varieties. The soluble phenolic acids also retarded myeelial growth of P. capsici in culture. The most pronounced inhibitory effect was produced by f-cinnamic acid, followed by p-hydroxybenzoic, vanillic and salicylic acids. Colonization and necrosis spread throughout the stem of the susceptible cultivar whereas the stem of the resistant cultivar was only partially invaded. In the latter, invasion of the tissue adjacent to necrosis occurred in only 20% of the plants.     

Colonization of roots, stolons, tubers and stems of various potato (Solanum tuberosum) cultivars by the black-dot fungus Colletotrichum coccodes

In an attempt to better understand the importance of tuber-borne inoculum in black dot development, several potato cultivars were inoculated with various Colletotrichum coccodes isolates. Symptoms developed first on underground organs (starting 2 weeks after inoculation on roots, and later on stolons and tubers) of inoculated plants; stem infections developed only after 7–10 weeks, depending on the cultivar. Infection with C. coccodes resulted in a reduction in numbers of stolons and tubers in cv. Bintje, but not in the later maturing cv. Roseval. Significant isolate by cultivar interactions were detected from the analysis of root symptoms after inoculation of three potato cultivars (Bintje, Spunta and Desiree) with five C. coccodes isolates. Such an interaction was also detected for stolon/tuber symptoms at the latest scoring date (98 days after inoculation), but not at earlier dates (58, 70 and 84 days after inoculation). These results suggest that protocols based on root colonization might be used for investigating cultivar response to black dot and pathogenicity of C. coccodes isolates, and that some specificity exists in the reaction of potato genotypes to this pathogenic fungus.