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.     

Biological Control of Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×10³conidia ml⁻¹), but smaller lesions at high concentration (5×10⁴ conidia ml⁻¹ or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + benomyl (50 μg ml⁻¹) and benomyl (100 μg ml⁻¹) in 2003, and between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + vinclozolin (100 μg ml⁻¹) and vinclozolin (500 μg ml⁻¹) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.     

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.     

<Emphasis Type="Italic">Ulocladium atrum</Emphasis> as a biological control agent for white mold of bean caused by<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m⁻² of 10⁶ spores ml⁻¹ suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha⁻¹ per application in 2001, or Lance (boscalid) at the rate of 750 g ha⁻¹ per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Chemical and biological control of Sclerotinia sclerotiorum in witloof chicory culture

BACKGROUND: Sclerotinia sclerotiorum (Lib.) de Bary is a major pathogen of witloof chicory. For lack of authorised field treatment, post‐harvest sprays with dicarboximide fungicides have been standard practice since the 1970s to prevent root rot and chicory heart decay during the forcing phase. However, the registration of procymidone and vinclozolin has been withdrawn in Europe. The development of organic agriculture and the necessity to reduce fungicide applications in conventional agriculture prompted an assessment of the efficacy of new fungicides and the use of the mycoparasite Coniothyrium minitans (Campbell). RESULTS: A mixture of the fungicides fludioxonil and cyprodinil (Switch^®) applied on chicory roots achieved a very good control of S. sclerotiorum (up to 95%). The use of C. minitans limited root infection, both when applied in the field (50–65% efficacy) and before the forcing period (post‐harvest treatment up to 80%). CONCLUSION: In organic agriculture, two treatments with C. minitans (in field and later at the forcing period) could improve protection against S. sclerotiorum. In conventional agriculture, after the field biological treatment, a post‐harvest chemical treatment could be applied. The addition of other prophylactic methods could lead to a high level of performance in practice against decay caused by S. sclerotiorum. Copyright © 2010 Society of Chemical Industry     

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.     

Suppression of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> by antifungal substances produced by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in MCD (MCD_cm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCD_cm. Sclerotia of Ss soaked in the filtrates of MCD_cm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCD_cm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCD_cm. Treatment of leaves of oilseed rape with cultural filtrates of MCD_cm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.     

核盘菌自噬相关基因SsATG5和SsATG8的功能分析

为探究自噬在核盘菌Sclerotinia sclerotiorum致病过程中的作用,利用酵母Saccharomyces自噬相关基因（autophagy-related gene,ATG）编码的蛋白序列比对核盘菌基因组,获得核盘菌假定ATG,并以核盘菌1980菌株为出发菌株,基于同源重组的原理对假定ATG进行敲除和回补,并测定不同突变体的生长表型和致病能力。结果表明,从核盘菌基因组中比对到2个ATG,分别命名为SsATG5和SsATG8,两者在核盘菌致病过程中均上调表达。SsATG5和SsATG8敲除突变体在菌丝生长、产草酸和侵染垫形成方面与野生型菌株无明显差异,但SsATG5敲除突变体在离体拟南芥Arabidopsis thaliana叶片上的致病力显著下降了约40%,在活体拟南芥植株上的致病力显著下降了约80%,同时SsATG5回补突变体恢复了正常的致病力。表明SsATG5参与了核盘菌的致病过程,证实自噬在核盘菌致病过程中发挥着重要作用。     

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.     

Effectiveness of Coniothyrium minitans and Trichoderma atroviride in suppression of sclerotinia blossom blight of alfalfa

The effects of the mycoparasites Coniothyrium minitans and Trichoderma atroviride on the suppression of alfalfa blossom blight caused by Sclerotinia sclerotiorum were evaluated under indoor and field conditions. When T. atroviride (9·0 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) or C. minitans (9·0 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) were applied to detached young alfalfa florets, T. atroviride effectively inhibited saprophytic growth of S. sclerotiorum, whereas C. minitans showed no inhibition under the same conditions. When T. atroviride (6·9 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) or C. minitans (6·9 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) was applied to young alfalfa petals in vivo just after pollination, the percentage of pod formation was higher for T. atroviride+S. sclerotiorum than that for C. minitans+S. sclerotiorum, and the percentage of pod rot was lower for T. atroviride+S. sclerotiorum than that for C. minitans+S. sclerotiorum. However, when they were applied to senescent petals attached to developing pods of alfalfa at 9·2 × 10⁴ conidia/floret together with S. sclerotiorum at 4·5 × 10³ ascospores/floret at 14 days after pollination, C. minitans was more effective than T. atroviride in suppressing sclerotinia pod rot and seed rot of alfalfa. Field experiments showed that three applications of C. minitans (5·4 × 10⁶ conidia mL⁻¹) or T. atroviride (5·4 × 10⁶ conidia mL⁻¹) at a 7-day interval to blossoms of alfalfa effectively suppressed sclerotinia pod rot in two out of three annual trials. Coniothyrium minitans effectively suppressed sclerotinia seed rot in all three years, whereas T. atroviride was not effective against seed rot in any of the trial years. The efficacy of C. minitans was not significantly different (P > 0·05) from benomyl (250 µg ai mL⁻¹). This study suggests that C. minitans has potential as a biocontrol agent to control blossom blight of alfalfa caused by S. sclerotiorum.     

Single and combined colonization of Sclerotinia sclerotiorum sclerotia by the fungal mycoparasites Coniothyrium minitans and Microsphaeropsis ochracea

Potential enhancement of mycoparasitic efficacy of Coniothyrium minitans and Microsphaeropsis ochracea through concomitant colonization of Sclerotinia sclerotiorum sclerotia was investigated, following observation that the two mycoparasites did not exhibit any mutual antagonism in dual culture assays. Simultaneous application of both mycoparasites increased sclerotia mortality in a temperature range from 16 to 26°C compared to single application, indicating a predominantly additive interaction. With increasing temperature the efficacy of M. ochracea decreased, but C. minitans was unaffected. Degradation of sclerotia by C. minitans proceeded slightly faster than with M. ochracea. Simultaneous colonization of sclerotia was studied at the histopathological level with mycoparasite strains transformed via Agrobacterium tumefaciens‐mediated transformation (ATMT) with reporter genes encoding for DsRed and GFP. Sclerotia colonization followed by fluorescence microscopy revealed effective penetration of the sclerotial rind, growth and formation of pycnidia in the cortex and medulla by both antagonists, resulting in complete degradation of sclerotia within 25 days after single inoculation. Upon simultaneous inoculation, both antagonists concomitantly colonized the sclerotial tissue and independently formed pycnidia in the sclerotial medulla and on the sclerotial rind, demonstrating their ability to co‐colonize the same host fungus. Although the individual growth of the two mycoparasites in dual inoculations was slightly delayed, the sclerotia degrading effects were additive, suggesting a complementary antagonistic interaction. The combined application of two different species of mycoparasites cooperating on the same host fungus and differing in temperature requirements may be advantageous for making biocontrol applications in the field less sensitive to varying environmental and host conditions.     

Growth and survival of the mycoparasiteConiothyrium minitans on lettuce leaves in contact with soil in the presece or absence ofSclerotinia sclerotiorum

Lettuce leaves co-inoculated withSclerotinia sclerotiorum andConiothyrium minitans and controls were placed on, or buried in, soil for a period of two weeks to study development and survival ofC. minitans. OnS. sclerotiorum-infected leaves on the soil surface, the number of colonies ofC. minitans recovered was about 40% of the number of pycnidiospores applied. When buried in the soil there was a reduction to about 2% of the spores applied. WhenC. minitans was applied on healthy lettuce leaves, which were subsequently placed on or in soil, the recovery was about 4%. It is argued that these figures indicate multiplication ofC. minitans onS. sclerotiorum-infected lettuce leaves on the soil, and good survival in all other cases.     

Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and peroxidise

Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

A Polymerase Chain Reaction (PCR) Assay for the Detection of Inoculum of Sclerotinia sclerotiorum

The development of a polymerase chain reaction (PCR) assay for the detection of inoculum of the plant pathogenic fungus Sclerotinia sclerotiorum is described. The PCR primers were designed using nuclear ribosomal DNA internal transcribed spacer sequences. Specific detection of DNA from S. sclerotiorum was possible even in the presence of a 40-fold excess of DNA from the closely related fungus Botrytis cinerea. PCR products were obtained from suspensions of untreated S. sclerotiorum ascospores alone, but DNA purification was required for detection in the presence of large numbers of B. cinerea conidiospores. Specific detection of inoculum of S. sclerotiorum was possible in field-based air-samples, using a Burkard spore trap, and from inoculated oilseed rape petals. The assay has potential for incorporation into a risk management system for S. sclerotiorum in oilseed rape crops.     

Reactive oxygen intermediates and oxalic acid in the pathogenesis of the necrotrophic fungus Sclerotinia sclerotiorum

An effective colonization of the host plant tissue by the necrotrophic fungus Sclerotinia sclerotiorum requires the secretion of the non-host specific toxin oxalic acid (OA), which is known to suppress the generation of reactive oxygen intermediates (ROI). A full-length cDNA coding for an oxalate decarboxylase (TOXDC), which converts OA into CO₂ and formate, was isolated from the basidiomycete Trametes versicolor. It was overexpressed in tobacco plants to study the role of ROI and OA in the interaction between tobacco and S. sclerotiorum. The transgenic plants contained less OA and showed a delayed colonization of S. sclerotiorum; furthermore a strong ROI accumulation and nearly no catalase activity compared to the wild type (WT) plants could be detected. In addition, inoculation experiments with transgenic catalase-deficient plants (CAT1AS) and in vitro studies showed that S. sclerotiorum copes with strong ROI stress. Our results indicate that OA supports the infection process caused by S. sclerotiorum and the fungus itself is able to tolerate high ROI concentrations. The nucleotide sequence data is available from the NCBI Genbank nucleotide-sequence database under the number AY370675     

Sclerotinia rot of blueberry caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

In 2002, rotted flower clusters and blighted shoot tips and leaves were observed on highbush blueberry (Vaccinium corymbosum L.) and rabbiteye blueberry (V. ashei Reade) plants in Chiba, Japan. The causal fungus isolated from the diseased plants was morphologically identified as Sclerotinia sclerotiorum (Libert) de Bary. The fungus reproduced natural symptoms after inoculation, then reisolated from the symptomatic parts. This is the first report of blueberry sclerotinia rot caused by S. sclerotiorum. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB269903#(020501) and AB233346 (020505).     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.