Histology of Sclerotium cepivorum infection of onion roots and the spatial relationships of pectinases in the infection process

Sclerotium cepivorum (isolate Sc4) hyphae penetrated the epidermis and hypodermis of onion roots and grew into the cortex. Immediately following penetration only the cells through which S. cepivorum grew were lysed, but subsequently cells were killed and cell walls disintegrated ahead of the infection hyphae. Sclerotium cepivorum produced two polygalacturonases (PG) and two pectinesterases (PE) in culture. These isozymes were also found in infected onion root tissues and another PG and a PE were occasionally detected. Two isozymes of PG and three isozymes of PE diffused ahead of the infection hyphae. The spatial distribution of these enzymes was associated with cell death and cell wall degradation. The epidermis, hypodermis, endodermis and vascular tissues were more resistant to hydrolysis than the cortex, but only the endodermis and cells within it retained nuclei following hydrolysis of the surrounding cortical tissues. The cavity within the root cortex became filled with swollen, vacuolate S. cepivorum hyphae.     

Effect of water potential on mycelial growth and perithecial production of Monosporascus cannonballus in vitro

The effects of osmotic water potential (Ψ_s) on mycelial growth and perithecial production of Monosporascus cannonballus , the cause of root rot and vine decline of melons, were examined at 25°C on potato dextrose agar (PDA) amended with KCl, NaCl or sucrose. Patterns of the growth responses of four isolates to decreasing Ψ_s were similar for each of the osmotica. Compared with growth on nonamended PDA (−0·3 MPa), growth of all isolates increased as Ψ_s was reduced to −0·8 MPa. Maximum growth occurred at Ψ_s values of −0·6 to −0·8 MPa. Growth was not reduced below that on nonamended PDA until Ψ_s was reduced to −1·8 MPa, and a 50% reduction in growth did not occur until Ψ_s was reduced to < −2·5 MPa. Reproduction was much more sensitive to reduced Ψ_s than was mycelial growth, and perithecia were produced only at Ψ_s ≥ −0·7 or −0·8 MPa on PDA amended with KCl or NaCl, respectively. Three isolates produced perithecia on PDA amended with sucrose only at Ψ_s ≥ −0·6 MPa, but the fourth isolate produced perithecia at ≥ −1·9 MPa. Colonization of the xylem early in disease development may provide an essential source of water for subsequent reproduction in the root cortex during plant senescence. Postharvest cultivation to expose and desiccate roots may prevent reproduction even when temperatures lethal to hyphae are not attained.     

Biological control of southern blight disease of tomato caused by Sclerotium rolfsii with simplified mycelial formulations of Trichoderma koningii

Two simple formulations of an antagonistic strain of Trichoderma koningii were employed against southern blight disease caused by Sclerotium rolfsii in seedling, potted outdoor and field-grown tomatoes (cvs. Ife No. 1 and Ibadan Local). Corn cob germling inoculum and mycelium powder of T. koningii significantly controlled ( P ≤0·05) symptoms of damping off, blight and wilting in both tomato cultivars. The populations of the antagonist increased from an initial 1 × 10⁴ to about 1 × 10⁶ colony-forming units per g of soil in the protected plants. Moreover, sclerotial counts decreased significantly ( P ≤0·05) in these soils and those sclerotia found had been parasitized by T. koningii. Trichoderma -protected plants were more vigorous than those in the other treatment categories. The significance of these results is discussed in relation to the use of Trichoderma in appropriately simplified formulations.     

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.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Host–parasite relationships of Meloidogyne incognita on spinach

Host–parasite relationships in root-knot disease of spinach caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Nematode-induced mature galls were large and usually contained one or more females and egg masses with eggs. Feeding sites were characterized by the development of giant cells containing granular cytoplasm and many hypertrophied nuclei. The cytoplasm in these giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P _i) in a series from 0–128 eggs and second-stage juveniles per cm³ soil and growth of spinach cv. Symphony F₁ seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^P–T ] was fitted to fresh top- and total plant-weight data for inoculated and control plants. Tolerance limits ( T ) of spinach cv. Symphony F₁ to M. incognita race 1 for fresh top and total plant weights were 0·25 and 0·5 eggs and second-stage juveniles per cm³ soil, respectively. The minimum relative values for fresh top and total plant weights were zero in both cases at P _i ≥ 32 eggs and second-stage juveniles per cm³ soil. Root galling was least at low initial population densities and greatest at 16 eggs and second-stage juveniles per cm³ soil. Maximum nematode reproduction rate was 33·1-fold at the lowest P _i.     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

A new selective medium for Burkholderia caryophylli, the causal agent of carnation bacterial wilt

A new selective medium (APCA medium) was developed for the isolation of Burkholderia caryophylli , the causal agent of carnation bacterial wilt, from both plants and soil. The optimal concentration and combination of antibiotics was investigated to determine the most selective condition for growing B .  caryophylli . The resultant composition of the medium per litre was: 0·79 g (NH₄)₂SO₄, 1·0 g KH₂PO₄, 0·5 g MgSO₄ · 7H₂O, 0·2 g KCl, 2·0 g D-arabinose, 5 mg crystal violet, 50 mg cycloheximide, 50 mg polymyxin B sulphate, 50 mg ampicillin sodium, 10 mg chloramphenicol, 25 mg blue tetrazolium, and 15 g agar. Plating efficiency ranged from 119 to 174% with an average of 141% compared to that of nutrient agar. The bacterium was successfully isolated from contaminated soil and plant tissues with this medium. Moreover, the medium almost completely inhibited the growth of other plant pathogenic bacteria and soil saprophytes. This selectivity was high enough to detect B . caryophylli in contaminated soil.     

Histopathology of Infection and Colonization of Susceptible and Resistant Port-Orford-Cedar by Phytophthora lateralis

ABSTRACT Port-Orford-cedar (POC) root disease, caused by Phytophthora lateralis, continues to kill POC in landscape plantings and natural forests in western North America. POC trees resistant to P. lateralis have been identified and propagated. Cytological observations of P. lateralis in susceptible and resistant roots and stems were made with light and transmission electron microscopy to identify resistance mechanisms. No differences in infection pathway and initial colonization were observed between susceptible and resistant roots, although there were differences in the rate and extent of development. Germ tubes formed appressoria, and penetration hyphae grew either between or directly through epidermal cell walls; inter- and intracellular hyphae colonized the root cortex. In susceptible roots, hyphae penetrated into the vascular system within 48 h of inoculation. In contrast, hyphae in roots of resistant seedlings grew more slowly in cortical cells and were not observed to penetrate to the vascular tissues. In resistant roots, infection was marked by general thickening of cortical cell walls, wall appositions around penetrating hyphae, collapse of cortical cells, and accumulation of osmophillic granules around hyphae. In susceptible stems, hyphae grew inter- and intracellularly in all cells of the secondary phloem except fiber cells, but were concentrated in sieve and parenchyma cells in the functional phloem. The pattern of penetration and colonization of hyphae was similar in the resistant stems, except that hyphae were found in the fiber cells of the xylem. In resistant stems, there were fewer hyphae in the functional phloem, and cytological changes such as damaged nuclei and disintegrated cytoplasm were evident. Structural changes in resistant stems included collapsed cells, wall thickening, secretory bodies, apposition of electron dense materials, and crystals in cell walls.     

Association of Binucleate Rhizoctonia with Soybean and Mechanism of Biocontrol of Rhizoctonia solani

ABSTRACT The association of binucleate Rhizoctonia (BNR) AG-K with soybean and the interaction of BNR, R. solani AG-4, and soybean seedlings were investigated to elucidate the mechanism of biocontrol of R. solani by BNR. Sixty-hour-old seedlings were inoculated and incubated in a growth chamber at 24 degrees C; plants were examined with light microscopy and with scanning and transmission electron microscopy at various times following inoculation. BNR grew over hypocotyls, roots, and root hairs, but only colonized epidermal cells. Hyphae of BNR appeared to attach to the epidermis and, 5.5 h following inoculation, began penetrating cells by means of penetration pegs without forming distinct appressoria or infection cushions. There was evidence of cuticle degradation at the point of penetration. Infection hyphae moved to adjacent epidermal cells by direct penetration of epidermal radial walls. There were epidermal and cortical cell necrosis, beginning with the fragmentation of the tonoplast and followed by the disintegration of cytoplasm, organelles, and plasma membranes. Cell necrosis was also observed in adjacent cells where there was no evidence of BNR hyphae. Cell walls were not destroyed. After 144 h, there was noevidence of BNR hyphae in cortical cells. Attempted penetrations were observed, but papillae formed on the inside of cortical cell walls. Pre-inoculation of soybean seedlings with BNR 24 or 48 h before inoculation with R. solani (1 cm between inocula) affected the growth of R. solani on soybean tissue. There were fewer hyphae of R. solani, the hyphae branched sparingly, and infection cushions were rare when compared with hyphal growth on soybean inoculated only with R. solani. These effects were observed before the BNR hyphae began to intermingle with the hyphae of R. solani on the surface of the inoculated host. Preinoculation of soybean seedlings 24 h before inoculation with R. solani significantly (P = 0.05) reduced disease incidence and severity caused by R. solani AG-4. The lesions caused by R. solani always appeared distally, not proximally, to the BNR inoculum. The interactions of intermingling hyphae of BNR and R. solani were examined in vitro and on the surface of the host. There was no evidence of lysis, mycoparasitism, inhibition of growth, or any other form of antagonism between hyphae. The results of these studies strongly suggest that induced resistance is the mechanism of biocontrol of R. solani on soybean by BNR. The inhibition of hyphal growth of R. solani on the surface of soybean tissue preinoculated with BNR appears to be a novel characteristic of induced resistance.     

Sensitivity of fungi from cereal roots to fluquinconazole and their suppressiveness towards take-all on plants with or without fluquinconazole seed treatment in a controlled environment

The take-all fungus, Gaeumannomyces graminis var. tritici , was highly sensitive to fluquinconazole ( in-vitro EC₅₀ 0·016–0·018 mg L⁻¹), a fungicide developed for use as a seed treatment to control take-all, and to prochloraz (EC₅₀ 0·006 mg L⁻¹). Fungi of other genera that were commonly isolated from cereal roots were sensitive in varying degrees to prochloraz but were relatively insensitive (e.g. Fusarium culmorum , EC₅₀ > 20 mg L⁻¹) or slightly sensitive (e.g. Epicoccum purpurascens , EC₅₀ 0·514 mg L⁻¹) to fluquinconazole. Gaeumannomyces graminis var. graminis and G. cylindrosporus , weak parasites that can protect roots against take-all, and an unnamed Phialophora sp., all closely related to the take-all fungus, were highly or moderately sensitive to fluquinconazole. Alternaria infectoria and E. purpurascens were most consistently effective in suppressing development of take-all on pot-grown wheat plants dual-inoculated with G. graminis var. tritici and the nonpathogen. Take-all was decreased more on dual-inoculated wheat plants grown from seed treated with fluquinconazole or fluquinconazole plus prochloraz than when only an antagonistic fungus ( A. infectoria , E. purpurascens , Fusarium culmorum or Idriella bolleyi ) or a seed treatment was applied. These fungi were less effective in combination with seed treatments on barley. Gaeumannomyces graminis var. graminis and G. cylindrosporus , tested on wheat, suppressed take-all only in the absence of fungicides. It is suggested that the performance of seed treatment containing fluquinconazole against take-all may in some circumstances be enhanced by its partial specificity for the take-all fungus.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Hemibiotrophic infection of Pisum sativum by Colletotrichum truncatum

The infection of pea ( Pisum sativum ) by Colletotrichum truncatum was studied by light and electron microscopy. These investigations were facilitated by use of an Argenteum pea mutant, which has a readily detachable epidermis. Infection pegs emerging from appressoria penetrated epidermal cells directly. Large intracellular primary hyphae formed a dense stromatic mycelium confined within a single epidermal cell. Primary mycelia gave rise to thinner secondary hyphae which radiated into surrounding cells and caused extensive wall dissolution. Melanized sclerotia developed in the centre of chlorotic water-soaked lesions. Acervuli were not observed. Epidermal cells survived initial penetration by primary hyphae, as shown by their ability to plasmolyse and accumulate Neutral red, but all infected cells were dead when the secondary hyphae had formed. Six cultivars of pea were susceptible, but seven other legumes were resistant. A single isoform of polygalacturonase with a pI of 8·3 and apparent M _r of 40000 was purified from culture filtrates and the TV-terminal amino acid sequence determined. The relevance of the results to the taxonomy of C. truncatum and the relationships between infection process and host range are discussed.     

Some properties of a virus-like agent found in Brachypodium sylvaticum in the United Kingdom

A spherical virus–like agent ($$32 nm in diameter) was isolated from a plant of Brachypodium sylvaticum (Huds.) Beauv. growing in a botanic garden in England and showing yellow streaks in the leaves. The agent was readily purified and sedimented as a single component in sucrose density rate gradients. The particles had a sedimentation coefficient at infinite dilution of $$122S and a buoyant density of 1.35 g/cm³ in CsCl and 1·33 in CS₂SO₄. The particles were stable at acid pH but above pH 7·0 in the presence of EDTA dissociated. A protein having a major polypeptide with a molecular weight of $$3·76 × 10⁴ and a species of single stranded RN A with a MW of 1·67 × 10⁶ were detected in the particles. The agent was not transmitted by manual inoculation, by the insects Myzus persicae Sulzer, Rhopalosiphum padi L. or Nephotettix virescens Distant, through soil by leakage from roots or by seed. The particles had physicochemical properties in common with tombus– and sobemoviruses but were not serologically related to 10 members of these groups or to 57 other small spherical RNA plant viruses.     

Effect of water on germination of Plasmopara viticola oospores

The effect of moisture in grape leaf litter holding overwintering Plasmopara viticola oospores was investigated. Oospores were incubated under different regimes of water activity ( a_W 0·991 to 0·123) for 2 to 15 days and their ability to germinate and cause infection was determined using a sensitive leaf disk assay. Reduction of a_W caused a significant shift in the infection dynamics, with maximum effect when a_W  ≤ 0·56. Dynamics of a_W in the leaf litter under natural conditions were estimated from moisture data using a Chen-Clayton equation. Daily patterns of leaf litter moisture (M in % weight) were determined in non rainy periods between mid February and mid June, while the Chen-Clayton equation was calculated using data of a_W and M measured in both sorption and desorption conditions, at different temperatures. Water activity was highest at 08·00 hours, decreased progressively until 14·00 hours, and then increased. Water activity was favourable for oospores to develop in about 25% of the measurements, all made between 18·00 hours and 08·00 hours. A close relationship was found between vapour pressure deficit (VPD in hPa) and a_W of the leaf litter, so that when VPD is lower than 2·13 hPa there is sufficient water for oospores to develop. Results showed that leaf litter moisture due to water from the atmosphere makes oospore development possible during non rainy periods.     

Induction of Terpenoid Synthesis in Cotton Roots and Control of Rhizoctonia solani by Seed Treatment with Trichoderma virens

ABSTRACT Research on the mechanisms employed by the biocontrol agent Trichoderma virens to suppress cotton (Gossypium hirsutum) seedling disease incited by Rhizoctonia solani has shown that mycoparasitism and antibiotic production are not major contributors to successful biological control. In this study, we examined the possibility that seed treatment with T. virens stimulates defense responses, as indicated by the synthesis of terpenoids in cotton roots. We also examined the role of these terpenoid compounds in disease control. Analysis of extracts of cotton roots and hypocotyls grown from T. virens-treated seed showed that terpenoid synthesis and peroxidase activity were increased in the roots of treated plants, but not in the hypocotyls of these plants or in the untreated controls. Bioassay of the terpenoids for toxicity to R. solani showed that the pathway intermediates desoxyhemigossypol (dHG) and hemigossypol (HG) were strongly inhibitory to the pathogen, while the final product gossypol (G) was toxic only at a much higher concentration. Strains of T. virens and T. koningii were much more resistant to HG than was R. solani, and they thoroughly colonized the cotton roots. A comparison of biocontrol efficacy and induction of terpenoid synthesis in cotton roots by strains of T. virens, T. koningii, T. harzianum, and protoplast fusants indicated that there was a strong correlation (+0.89) between these two phenomena. It, therefore, appears that induction of defense response, particularly terpenoid synthesis, in cotton roots by T. virens may be an important mechanism in the biological control by this fungus of R. solani-incited cotton seedling disease.     

Effect of environmental factors and Sclerotium cepivorum isolate on sclerotial degradation and biological control of white rot by Trichoderma

Laboratory assays demonstrated that two isolates of Trichoderma viride and one isolate of Trichoderma pseudokoningii degraded up to 80% of sclerotia of four isolates of Sclerotium cepivorum in a silty clay soil, and also degraded up to 60% of sclerotia in three other soil types. Relationships were defined between the degree of sclerotial degradation by the two T. viride isolates in the silty clay soil and both temperature and soil water potential. Sclerotia were degraded between 10 and 25°C at −0·00012 MPa, but there was little activity of T. viride at 5°C or at −4 MPa. Degradation of S. cepivorum sclerotia also occurred in the absence of Trichoderma at soil water potentials approaching saturation . Experiments using onion seedling bioassays showed that the efficacy of Trichoderma isolates for the control of white rot using the same selection of soils and S. cepivorum isolates was variable, but that there was significant disease control overall. The importance of environmental factors and pathogen isolate in relation to effective biological control of white rot is discussed.     

Integrated control of Allium white rot with Trichoderma viride, tebuconazole and composted onion waste

Two isolates of Trichoderma viride (L4 and S17A) were assessed for biological control of Allium white rot (AWR) with different onion accessions and cultivars, alone and in combination with a tebuconazole-based seed treatment or composted onion waste. In glasshouse tests, 23 new bulb-onion accessions from previous work to detect resistance to Sclerotium cepivorum showed no differences in susceptibility to AWR but, when combined with S17A, disease was reduced by up to two-thirds over all accessions. Trichoderma viride L4 and S17A also reduced the proportion of infected plants for five commercial bulb-onion cultivars and one advanced breeding line by at least one-third. Further glasshouse tests using a salad-onion cultivar showed that L4, S17A, tebuconazole or composted onion waste controlled AWR and at least halved the proportion of diseased plants. Combination treatments of T. viride with either tebuconazole or compost enhanced control and, in some treatments, disease was almost eliminated. In field trials, control of AWR by S17A was significant for 17 out of 18 individual or mixed bulb-onion accessions, with disease reduced overall by more than half. In another field experiment, S17A failed significantly to reduce AWR for two out of three commercial bulb-onion cultivars, while tebuconazole reduced the final proportion of AWR-infected plants over all cultivars from 0·47 to 0·09. Combining S17A and tebuconazole resulted in a similar level of AWR to using tebuconazole alone. The use of T. viride in an integrated strategy with other treatments to enhance control of S. cepivorum is discussed.     

Infection and colonization of strawberry by <Emphasis Type="Italic">Gnomonia fragariae</Emphasis> strain expressing green fluorescent protein

Gnomonia fragariae is a poorly studied ascomycete, which was recently demonstrated to be a cause of severe root rot and petiole blight of strawberry. The pathogen was genetically transformed with the GFP as a vital marker and hygromycin resistance gene. Several stable transformants were obtained, which did not differ in their phenotype from the wild type isolate. Using one of the GFP-tagged isolates the infection process and colonization of roots and petioles of host plant by the pathogen were studied. Fluorescence microscopy examinations of the inoculated plants at different time points showed that plant infection occurs 24 h after inoculation and intensively continues during first 3 days. The specific penetration sites on epidermal cells and preferences in colonization for certain root and petiole tissues were observed. The pathogen intensively colonized and destroyed cortex of roots and petioles and spread rapidly longitudinally within intercellular spaces. The petioles were colonized by the hyphae, which grew mostly in the intracellular spaces of the cortical cells while in the roots the intracellular growth of hyphae occurred only in the later stages of infection. The fungus was also capable to infect the vascular tissues of petioles although these were not the primary tissues colonized by the pathogen. The mature ascomata were formed on the infected petiole bases several weeks after the inoculation. This study presents a genetic transformation method for Gnomonia fragariae and it demonstrates details on infection process and colonization of root, crown and petiole tissues of strawberry by the pathogen.