Inoculum potential of Sclerotinia sclerotiorum sclerotia depends on isolate and host plant

The soilborne fungus Sclerotinia sclerotiorum infects many important crop plants. Central to the success of this pathogen is the production of sclerotia, which enables survival in soil and constitutes the primary inoculum. This study aimed to determine how crop plant type and S. sclerotiorum isolate impact sclerotial production and germination and hence inoculum potential. Three S. sclerotiorum isolates (L6, L17, L44) were used to inoculate plants of bean, carrot, lettuce, oilseed rape (OSR) and potato, and the number and weight of sclerotia per plant quantified. Carpogenic germination of sclerotia collected from different hosts was also assessed for L6. Production of sclerotia was dependent on both crop plant type and S. sclerotiorum isolate, with OSR and lettuce supporting the greatest number (42–122) and weight (1.6–3.0 g) of sclerotia per plant. The largest sclerotia were produced on OSR (33–66 mg). The three S. sclerotiorum isolates exhibited a consistent pattern of sclerotial production irrespective of crop type; L6 produced large numbers of small sclerotia while L44 produced smaller numbers of large sclerotia, with L17 intermediate between the two. Germination rate and percentage was greatest for larger sclerotia (4.0–6.7 mm) and also varied between host plants. Combining sclerotial production data and typical field crop densities suggested that infected carrot and OSR could produce the greatest number (3944 m^?2) and weight (73 g m^?2) of S. sclerotiorum sclerotia, respectively, suggesting these crops potentially contribute a greater increase in inoculum. This information, once further validated in field trials, could be used to inform future crop rotation decisions.     

Interaction betweenSclerotinia sclerotiorum andConiothyrium minitans strains with different aggressiveness

The effects ofSclerotinia sclerotiorum live and autoclaved sclerotia, and sclerotial exudates, and commercial oxalic acid were testedin vitro on sevenConiothyrium minitans strains differing in aggressiveness towardsS. sclerotiorum. Only sclerotial exudates and autoclaved sclerotia affected the mycelial growth rate of almost all the strains tested, whereas a change in theC. minitans mycelial growth pattern was observed in the presence of autoclaved sclerotia and live sclerotia germinating by the myceliogenic eruptive germination. In addition, sclerotial exudates had a stimulatory effect on spore germination. These findings indicate that the various treatments could influence theC. minitans strains regardless of their aggressiveness.     

Effect of Combined Treatment of Pasteurisation and <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> on Sclerotia of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Soil

Integrated control of soil-borne plant pathogens such as Sclerotinia sclerotiorum is becoming more important as the soil fumigant methyl bromide is being phased out of use. Two alternative methods of control that have been found to reduce viability of sclerotia are steam sterilisation (pasteurisation) of soil or the application of the mycoparasite Coniothyrium minitans. This work investigated the possibility of integrating these two control measures. Soil was pasteurised in an autoclave, using a temperature of 80 °C for 3 min to simulate the possible temperatures reached by soil steaming machines for field use. Coniothyrium minitans was subsequently applied to the pasteurised soil to assess the effects of the combination of control measures in reducing sclerotial viability of S. sclerotiorum. Similar results were found in two soil types. Either method used individually was effective in decreasing the number of viable sclerotia, but no further reduction in sclerotial viability was seen when the two methods were combined. Coniothyrium minitans was found to colonise pasteurised sclerotia significantly quicker than untreated sclerotia, and it was seen that there was an increase in number of C. minitans in pasteurised soil in the presence of sclerotia. Experiments were also conducted to investigate the effect of application timing of the biocontrol agent to soil following pasteurisation, in relation to sclerotial infection. Here, two different isolates of S. sclerotiorum were used, with similar results. Application of C. minitans to soil immediately following pasteurisation resulted in sclerotial infection by the mycoparasite, but application 7 days or more after soil pasteurisation resulted in low recovery of the biocontrol agent from sclerotia, possibly due to the mycoparasite being masked by the presence of other fungi which colonised the sclerotia first.     

Production,survival and evaluation of solid-substrate inocula ofConiothyrium minitans againstSclerotinia sclerotiorum

Coniothyrium minitans grew on all ten solid-substrates (barley, barley-rye-sunflower, bran-vermiculite, bran-sand, maizemed-perlite, millet, oats, peat-bran, rice and wheat) tested, producing high numbers of germinable pycnidiospores (1.9–9.3×10⁸ g^–1 air dry inocula). All solid substrate inocula survived better in the laboratory at 5 and 15 °C than at 30 °C for at least 64 weeks.In pot bioassays carried out in the glasshouse and field, soil incorporations of each inoculum almost completely inhibited carpogenic germination ofS. sclerotiorum. In the field bioassay, no sclerotia were recovered after 38 weeks fromC. minitans-treated pots compared to 56% from control pots. In the glasshouse bioassay, 9–30% of sclerotia were recovered after 20 weeks fromC. minitans-treated pots, but 88–100% of these were infected by the antagonist. The antagonist also spread to infect sclerotia in control pots.In larger scale glasshouse trials, single preplanting soil-incorporations of five inocula (barley-ryesunflower, maizemeal-perlite, peat-bran, rice and wheat) controlled Sclerotinia disease in a sequence of lettuce crops, with only small differences between the types of inocula tested. At harvest,C. minitans reduced sclerotial populations on the soil surface and over 74% of sclerotia recovered fromC. minitans-treated plots were infected by the antagonist.C. minitans survived in soil in all solid-substrate inocula-treated plots for at least 39 weeks at levels of 10⁴–10⁵ colony forming units cm^–3 soil and spread to infect over 36% of sclerotia recovered from control plots.     

Quantitative Aspects of Infection of Sclerotinia sclerotiorum Sclerotia by Coniothyrium minitans – Timing of Application, Concentration and Quality of Conidial Suspension of the Mycoparasite

White mould disease leads to production of sclerotia, which subsequently survive in soil and may be responsible for future epidemics. The effect of the mycoparasite Coniothyrium minitans in decreasing survival of sclerotia of Sclerotinia sclerotiorum was studied. Infection of sclerotia of S. sclerotiorum by C. minitans can be achieved by a single conidium. Under optimal conditions, 2 conidia per sclerotium produced 63% of the maximum infection (ca. 90%) of sclerotia produced by up to 1000 conidia. Similar results were observed on the infection of stem pieces infected by S. sclerotiorum. In field trials, application of conidial suspensions of C. minitans to a bean crop soon after white mould outbreak led to a higher percentage of sclerotial infection than later applications. Ninety per cent infection of sclerotia was obtained within 3 weeks of application by C. minitans suspensions in the range of 5 × 10⁵ and 5 × 10⁶ conidia ml^–1 at 1000 l ha^–1. The concentration of the conidial suspensions and the isolate used were of less importance. The result was marginally affected by the germinability of the conidia (75% against 61% infected sclerotia at 91% and 16% viability of isolate IVT1, respectively). Less apothecia of S. sclerotiorum developed in soil samples collected after 2 months from plots sprayed immediately after disease outbreak than from those treated 11–18 days later. It is concluded that a suspension of 10⁶ conidia ml^–1 in 1000 l ha^–1 (= 10¹² conidia ha^–1) sprayed immediately after the first symptoms of disease are observed, results in > 90% infection of sclerotia of S. sclerotiorum. The infection of sclerotia, which prevents their carry-over, occurs within a broad range of inoculum quality.     

Efficiency of isolates ofConiothyrium minitans as mycoparasites ofSclerotinia sclerotiorum,Sclerotium cepivorum andBotrytis cinerea on tomato stem pieces

Summary Twenty five isolates ofConiothyrium minitans were screened for antagonism toSclerotinia sclerotiorum in a Petri dish bioassay using tomato stem segments placed on sterile sand. The antagonistic activity of 23 isolates was quite uniform and only two less antagonistic isolates were identified. Antagonism, expressed as a reduction in the rate of tissue colonization byS. sclerotiorum, occurred, whetherC. minitans was co-inoculated at the same time, one day before or one day afterS. sclerotiorum, but was slightly restricted whenS. sclerotiorum was given a lead of one day. On average, 50–80% of sclerotia of S.sclerotiorum formed on the stem pieces were infected byC. minitans two weeks after inoculation. Excluding the less antagonistic isolates,Coniothyrium minitans was recovered from over 80% ofS. sclerotiorum-infected stem segments when co-inoculated but from a maximum of only 7% of stem pieces when exposed toC. minitans alone. When the experiments were carried out on non-sterile soil instead of sterile sand, infection of stem pieces byS. sclerotiorum was reduced and recovery ofS. sclerotiorum andC. minitans from stem segments was decreased. SevenC. minitans isolates were also screened againstSclerotium cepivorum andBotrytis cinerea and, whereas the effect ofC. minitans onS. cepivorum-infected tissue and sclerotia was essentially similar to that observed withS. sclerotiorum, B. cinerea infected tissue and sclerotia were not invaded by the antagonist.     

Effect of Inoculum Rates and Sources of Coniothyrium minitans on Control of Sclerotinia sclerotiorum Disease in Glasshouse Lettuce

Coniothyrium minitans isolate Conio grew on both maizemeal-perlite and ground maizemeal-perlite, producing high numbers (1.6×10⁷ conidiag^–1 inoculum) of germinable conidia. Coniothyrium minitans isolate Conio applied as a preplanting soil incorporation of maizemeal-perlite inoculum at full application rate (0.6lm^–2; 10¹¹ colony forming units (cfu)m^–2) significantly reduced Sclerotinia disease in a sequence of three lettuce crops grown in a glasshouse. No reduction in disease was achieved with any of the reduced rate treatments (10⁸cfum^–2) of a range of C. minitans isolates (Conio ground maizemeal-perlite at reduced rate, Conio and IVT1 spore suspensions derived from maizemeal-perlite, IVT1 spore suspension derived from oats and Contans® WG spore suspension). After harvest of the second and third crops, C. minitans maizemeal-perlite at full rate reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore suspension and reduced rate maizemeal-perlite inocula. Coniothyrium minitans was recovered from the soil throughout the trial, between 10⁵ and 10⁷cfucm^–3 in maizemeal-perlite inoculum full rate treated plots and 10¹–10⁴cfu cm^–3 in all other inoculum treated plots.Pot bioassays were set up corresponding to the inoculum used in the glasshouse, with the addition of Conio ground maizemeal-perlite at a rate corresponding to the full rate maizemeal-perlite. Coniothyrium minitans maizemeal-perlite and ground maizemeal-perlite at full rate significantly decreased carpogenic germination, recovery and viability of sclerotia and increased infection of sclerotia by C. minitans in comparison with spore suspension treatments, reflecting results of the glasshouse trials. Additionally, reduced maizemeal-perlite treatment also decreased apothecial production, recovery and viability of sclerotia compared with the spore suspension treatment, despite being applied at similar rates. Simultaneous infection of sclerotia by several isolates of C. minitans was demonstrated. Inoculum level in terms of colony forming unitscm^–3 of soil appears to be a key factor in both control of Sclerotinia disease and in reducing apothecial production by sclerotia.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

Use of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> as a biocontrol agent and some molecular aspects of sclerotial mycoparasitism

The use of the sclerotial mycoparasite Coniothyrium minitans as a biological control agent of diseases caused by sclerotium-forming pathogens especially Sclerotinia sclerotiorum is briefly reviewed. A number of studies have examined production and application methods, integrated control, ecology, and modes of action in order to understand the biology of the mycoparasite and enhance activity and reproducibility of use. Recently, development of a number of molecular-based techniques has begun to allow the examination of genes involved in mycoparasitism. Some of these procedures have been applied to identify pathogenicity genes involved in the infection of sclerotia of S. sclerotiorum by C. minitans and this work is discussed.     

Histopathology of Sclerotinia sclerotiorum infection and oxalic acid function in susceptible and resistant soybean

The success of the necrotrophic fungus Sclerotinia sclerotiorum is largely dependent on its major virulence factor, oxalic acid (OA). Virulence is lost in transgenic plants that express OA degrading enzymes, e.g. oxalate oxidase (OxO). The histopathology of S. sclerotiorum infection and OA accumulation was examined in a transgenic soybean line over‐expressing OxO (OxO‐OE) and its isogenic parent (WT). In situ flower inoculation showed that the OxO‐OE plants were highly resistant to the pathogen while the WT parents were susceptible. This difference in resistance was not apparent in the floral tissues, as aggressive hyphal activity was similar on both hosts, showing that high OxO activity and low OA accumulation in OxO‐OE was not a deterrent. However, the process of fungal infection on excised leaf tissue differed on the two hosts. Primary lesions developed and showed similar severe ultrastructural damage on both hosts but rapid lesion expansion (colonization) proceeded only on the WT, concomitant with OA accumulation. Oxalic acid rose in OxO‐OE 1 day post‐inoculation and did not change over the following 3 days, showing that colonization can be blocked by maintaining low levels of OA. However, OxO degradation of OA did not deter initial host penetration and primary lesion formation. This shows that OA, the major virulence factor of S. sclerotiorum, is critical for host colonization but may not be required during primary lesion formation, suggesting that other factors are contributing to the establishment of the primary lesion.     

Use of fungal antagonists for biocontrol of damping-off and sclerotinia diseases

Two mycoparasites, Pythium oligandrum and Coniothyrium minitans, have been tested for their ability to act as disease biocontrol agents. P. oligandrum oospores, grown in a cane molasses liquid medium and coated onto cress and sugar-beet seeds using commercial thin-film or pelleting techniques, gave significant control of damping-off in cress and sugar-beet caused by Pythium ultimum and Aphanomyces cochlioides respectively, in glasshouse pot trials. In some cases, the control was equivalent to fungicide drenches or standard fungicide seed treatments, but little control was achieved with any treatment when the pathogen inoculum potential in the soil was high. Pre-planting application of a solid substrate preparation of C. minitans gave reproducible control of sclerotinia disease in the glasshouse. The degree of control was equivalent to that achieved with regular foliar sprays of vinclozolin, when there was less than 40% disease in the control plots naturally infested with Sclerotinia sclerotiorum. At higher disease levels biocontrol was lost. Nevertheless, C. minitans survived in the soil for over one year and continued to degrade sclerotia and reduce apothecial production. The commercial potential of these biocontrol agents is discussed.     

Cultivar‐dependent partial resistance and associated defence mechanisms in wheat against Zymoseptoria tritici

Septoria tritici blotch caused by the fungus Zymoseptoria tritici is one of the most devastating foliar diseases of wheat. Knowledge regarding mechanisms involved in resistance against this disease is required to breed durable resistances. This study compared the expression of defence and pathogenicity determinants in three cultivars in semicontrolled culture conditions. The most susceptible cultivar, Alixan, presented higher necrosis and pycnidia density levels than Altigo, the most resistant one. In Premio, a moderately resistant cultivar, necrosis developed as in Alixan, while pycnidia developed as in Altigo. In noninfectious conditions, genes coding for PR1 (pr1), glucanase (gluc) and allene oxide synthase (aos) were constitutively expressed at a higher level in both Altigo and Premio than in Alixan, while chitinase2 (chit2), phenylalanine ammonia‐lyase (pal), peroxidase (pox2) and oxalate oxidase (oxo) were expressed at a higher level in Premio only. Except for aos, all genes were induced in Alixan during the first steps of the symptomless infection phase. Only pox2, oxo, gluc and pal genes in Altigo and pal, chs and lox genes in Premio were up‐regulated at some time points. Basal cultivar‐dependent resistance against Z. tritici could therefore be explained by various gene expression patterns rather than high expression levels of given genes. During the necrotrophic phase, Z. tritici cell wall‐degrading enzyme activity levels were lower in Altigo and Premio than in Alixan, and were associated more with pycnidia than with necrosis. Similar tissue colonization occurred in the three cultivars, suggesting an inhibition of the switch to the necrotrophic lifestyle in Altigo.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1‐α (EF1‐α) and β‐tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania.     

Piriformospora indica reduces fusarium head blight disease severity and mycotoxin DON contamination in wheat under UK weather conditions

Piriformospora indica (Sebacinaceae) is a cultivable root endophytic fungus. It colonizes the roots of a wide range of host plants. In many settings colonization promotes host growth, increases yield and protects the host from fungal diseases. Evaluation was made of the effect of P. indica on fusarium head blight (FHB) disease of winter (cv. Battalion) and spring (cv. Paragon, Mulika, Zircon, Granary, KWS Willow and KWS Kilburn) wheat and consequent contamination by the mycotoxin deoxynivalenol (DON) under UK weather conditions. Interactions of P. indica with an arbuscular mycorrhizal fungus (Funneliformis mosseae), fungicide application (Aviator Xpro) and low and high fertilizer levels were considered. Piriformospora indica application reduced FHB disease severity and incidence by 70%. It decreased mycotoxin DON concentration of winter and spring wheat samples by 70 and 80%, respectively. Piriformospora indica also increased aboveground biomass, 1000‐grain weight and total grain weight. Piriformospora indica reduced disease severity and increased yield in both high and low fertilizer levels. The effect of P. indica was compatible with F. mosseae and foliar fungicide application. Piriformospora indica did not have any effects on plant tissue nutrients. These results suggest that P. indica might be useful in biological control of Fusarium diseases of wheat.     

Differential behaviour of sheath blight pathogen Rhizoctonia solani in tolerant and susceptible rice varieties before and during infection

This study characterized the early infection and establishment of the sheath blight pathogen Rhizoctonia solani on a tolerant rice variety, Swarnadhaan (IET 5656), and a susceptible variety, Swarna (MTU 7029). Assays using whole plants showed that disease severity was higher in Swarna than Swarnadhaan. In a detached leaf assay, Swarnadhaan showed a disease index that was 50% less than that with Swarna. Rhizoctonia solani exhibited different growth behaviour in the tolerant and susceptible varieties. The pathogen showed more hyphal growth in the susceptible host than in the tolerant variety. It also showed profuse branching, making intimate contact with the host surface to form more inter‐ and intracellular structures, and greater sclerotial development in the susceptible host compared to the tolerant one. Using light and scanning electron microscopy, it was observed for the first time that the pathogen could intercept host surface structures and use these for anchorage or penetration. Transformed R. solani, expressing green fluorescent protein, was observed using confocal laser scanning microscopy to investigate pathogen behaviour, including the formation of infection cushions and subsequent colonization of the host tissues. This is the first ultrastructural report to characterize the differential behaviour of the sheath blight pathogen in the vicinity and within tolerant and susceptible rice plants.     

Comparative pathogenicity of sexual and asexual spores of Zymoseptoria tritici (septoria tritici blotch) on wheat leaves

Zymoseptoria tritici ascospores and pycnidiospores are considered the main forms of primary and secondary inoculum, respectively, in septoria tritici blotch epidemics. The pathogenicity of the two types of spores of the same genotypic origin were compared through a two‐stage inoculation procedure in controlled conditions. Adult wheat leaves were inoculated with ascospores collected from field sources, yielding 119 lesions; pycnidiospores collected from 12 lesions resulting from these ascospore infections were then used for inoculation. Lesion development was assessed for 5 weeks; latent period, lesion size, and pycnidium density were estimated for different isolates. The latent period was calculated as the maximum likely time elapsed between inoculation and either the appearance of the majority of the sporulating lesions (leaf scale) or the appearance of the first pycnidia (lesion scale). The latent period was significantly longer (c. 60 degree‐days, i.e. 3–4 days) after infection with ascospores than with pycnidiospores. No difference was established for lesion size and density of pycnidia. A comparison with other ascomycete fungi suggested that the difference in latent period might be related to the volume of spores and their ability to cause infection. Fungal growth before the appearance of lesions may be slower after inoculation with an ascospore than with a pycnidiospore. The mean latent period during the very beginning of epidemics, when first lesions are mainly caused by ascospores, may be longer than during spring, when secondary infections are caused by pycnidiospores. Disease models would be improved if these differences were considered.     

Development of three fusarium crown rot causal agents and systemic translocation of deoxynivalenol following stem base infection of soft wheat

Fusarium pseudograminearum, F. culmorum and F. graminearum are the most important fusarium crown rot (FCR) causal agents. They have the common ability to biosynthesize deoxynivalenol (DON). To elucidate the behaviour of each of the three species, a comparative study was carried out to investigate symptom progression, fungal systemic growth and translocation of DON following stem base inoculation of soft wheat. FCR symptoms were mainly localized in the inoculated area, which extended up to the second node for all inoculated species. Only the most aggressive strains caused symptoms up to the third node. Real‐time quantitative PCR showed that fungal colonization reached the third node for all the tested species, but a low percentage of plants showed colonization above the third node following inoculation with the most aggressive strains. Fungal growth was detected in symptomless tissues but none of the three species was able to colonize as far as the head tissues. However, even if the pathogens were not detected in the heads, DON was detected in head tissues of the plants inoculated with the most aggressive strains. These results demonstrate that F. pseudograminearum, F. culmorum and F. graminearum, under the same experimental conditions, follow a similar pattern of symptom progression, fungal colonization and DON translocation after stem base infection. Differences in the extent of symptoms, fungal colonization and mycotoxin distribution were mainly attributable to strain aggressiveness. These findings provide comparative information on the events following infection of the stem base of wheat by three of the most important FCR casual agents.     

Overwintering of Ampelomyces mycoparasites on apple trees and other plants infected with powdery mildews

Apple shoots and aerial parts of 13 other plant species infected with powdery mildews during the previous season were collected in late winter and early spring between 1998 and 2003 at a total of 34 sample sites in Hungary. Samples were examined for the presence of overwintering structures of Ampelomyces, common mycoparasites of powdery mildews. Pycnidia and resting hyphae resembling those of Ampelomyces were found on six plant species, including apple. Their viability and subsequent mycoparasitic activity of the hyphae emerging from the overwintered fungal structures were studied in vitro to determine whether they can serve as sources of primary inocula of Ampelomyces in the spring. Overwintered pycnidia of Ampelomyces collected in the spring, and produced in both the ascomata and the conidiophores of powdery mildews during the previous season, initiated the life cycle of these mycoparasites when placed close to fresh powdery mildew colonies in vitro. Similarly, thick-walled resting hyphae, found in the dried powdery mildew mycelia which covered the overwintered aerial parts of the host plants, also germinated and gave rise to new intracellular pycnidia of Ampelomyces when powdery mildew colonies were inoculated with them in vitro. On apple trees, Ampelomyces mycoparasites overwintered as resting hyphae in the dried powdery mildew mycelia covering the shoots and in the parasitized ascomata of Podosphaera leucotricha on the bark and the scales of the buds. Approximately 31% of the field samples collected from apple trees in spring between 1998 and 2003 contained overwintered structures of Ampelomyces. Artificial bursting of apple buds in the laboratory showed that both P. leucotricha and Ampelomyces start their life cycle during or soon after bud burst, but Ampelomyces can only slowly follow the spread of its mycohost on infected leaves. Most probably, the mycoparasites did not overwinter in the dormant hyphae of P. leucotricha in the buds, but only on the bark and the bud scales, as their hyphae were not found in the young hyphae of apple powdery mildew that appeared on the leaf tissues during bud burst. This study demonstrated that Ampelomyces mycoparasites can survive the winter in the field as pycnidia and as resting hyphae in the dried mycelia of their mycohosts.     

Biological control of sclerotinia stem rot of canola using antagonistic bacteria

Stem rot caused by Sclerotinia sclerotiorum is a major fungal disease of canola worldwide. In Australia the management of stem rot relies primarily on strategic application of synthetic fungicides. In an attempt to find alternative strategies for the management of the disease, 514 naturally occurring bacterial isolates were screened for antagonism to S. sclerotiorum. Antifungal activity against mycelial growth of the fungus was exhibited by three isolates of bacteria. The bacteria were identified as Bacillus cereus (SC‐1 and P‐1) and Bacillus subtilis (W‐67) via 16S rRNA sequencing. In vitro antagonism assays using these isolates resulted in significant inhibition of mycelial elongation and complete inhibition of sclerotial germination by both non‐volatile and volatile metabolites. The antagonistic strains caused a significant reduction in the viability of sclerotia when tested in a greenhouse pot trial with soil collected from the field. Spray treatments of bacterial strains reduced disease incidence and yielded higher control efficacy both on inoculated cotyledons and stems. Application of SC‐1 and W‐67 in the field at 10% flowering stage (growth stage 4·00) of canola demonstrated that control efficacy of SC‐1 was significantly higher in all three trials (over 2 years) when sprayed twice at 7‐day intervals. The greatest control of disease was observed with the fungicide Prosaro^® 420SC or with two applications of SC‐1. The results demonstrated that, in the light of environmental concerns and increasing cost of fungicides, B. cereus SC‐1 may have potential as a biological control agent of sclerotinia stem rot of canola in Australia.