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.     

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.     

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.     

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.     

Stimulation of germination of sclerotia of Sclerotium cepivorum at different depths in soil by artificial onion oil

The influence of depth of inoculum on white rot and plant growth, and the response of sclerotia ofSclerotium cepivorum Berk. to artificial onion oil, at various depths in soil, was investigated.Field tests showed that effects of depth of inoculum were apparent 12 weeks after sowing and by week 14, sclerotia buried at 0–2, 10–12 and 18–20 cm reduced onion growth by 75, 56 and 37% in comparison with controls. The results indicate the importance of correct placement of treatments, such as onion oil, in soil to achieve maximum levels of disease control. In vitro tests in closed containers at 15°C showed that between 50–70% of sclerotia ofS. cepivorum germinated on soil treated with artificial onion oil. Production of secondary sclerotia was restricted to 0.5% of those which germinated. Application of oil at 10 cm depths in columns of soil caused germination of 60–70% of sclerotia at 3, 6, 14 and 17 cm over 4 weeks at 15°C. Germination at the soil surface was reduced. In field plots onion oil and one of its components, diallyl disulphide, reduced recovery of introduced sclerotia by 70%.Samenvatting Het effect van de diepte in grond waarop het inoculum vanSclerotium cepivorum berk. zich bevindt op het optreden van witrot in ui en op de ontwikkeling van de uieplant werd onderzocht. Ook werd de uitwerking van in grond geïnjecteerde uienolie op de op verschillende diepten in grond geplaatste sclerotiën bestudeerd.In veldexperimenten werd na 12 weken een duidelijk effect van de diepte van het inoculum op de ontwikkeling van de planten waarneembaar. Na 14 weken bleken sclerotiën die op 0–2, 10–12 en 18–20 cm diepte in grond waren geplaatst de groei van de uieplanten met respectievelijk 75, 56 en 37% te hebben verminderd ten opzichte van de controleplanten. Deze resultaten wijzen er op, dat voor een zo goed mogelijke bestrijding van witrot, de diepte in grond waar middelen zoals uienolie moeten worden toegediend, van grote betekenis kan zijn.Bij in vitro toetsen in gesloten Conway-schalen bij 15°C kiemde 50–70% van de sclerotiën vanS. cepivorum op het oppervlak van grond die behandeld was met synthetische uienolie. De vorming van secundaire sclerotiën beperkte zich tot 0,5% van het aantal gekiemde sclerotiën. Toediening van uienolie op 10cm diepte in grondkolommen veroorzaakte de kieming van 60–70% van de sclerotiën op 3, 6, 14 en 17 cm diepte over een periode van 4 weken bij 15°C. Bij het grondoppervlak was de kieming lager. In veldexperimenten werd na behandeling van grond met uienolie en met een bestanddeel daarvan, diallyl-disulfide, het aantal in grond geïntroduceerde sclerotiën dat kon vorden teruggevonden, met 70% gereduceerd.The senior author was a visiting scientist of the Plant Research Institute, Melbourne, Australia who received a scholarship administered by the International Agricultural Center (IAC) from the Netherlands Ministry of Agriculture.     

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.     

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.     

Suppression of potato cyst nematode root penetration by the endoparasitic nematophagous fungusHirsutella rhossiliensis

The endoparasitic nematophagous fungusHirsutella rhossiliensis was tested for its ability to suppress root penetration and cyst formation by the potato cyst nematode speciesGlobodera pallida. Isolates ofH. rhossiliensis were obtained from infected potato cyst nematode juveniles from different starch potato fields in The Netherlands. The isolates showed no difference in spore adhesion to juveniles on agar plates (adhesion rate: ±90%). The most rapid growing isolate, CBS 108.94, was used for experiments. Vegetative mycelial colonies ofH. rhossiliensis CBS 108.94, grown in potato dextrose broth, were used as soil inoculum. During submerged cultivation the mycelial colonies produced phialides (spore-bearing cells) but no spores. Exposed to the air, however, spores were rapidly formed. The effect of different soil inoculum densities of mycelial colonies on root penetration byGlobodera pallida was examined in an experiment in 250-ml pots. Up to a mycelial colony concentration representing a potential spore density of 10⁴ g^–1 soil no suppression occurred. At approximated densities of 2.5×10⁴ and 10⁵ spores g^–1 soil the numbers of juveniles which penetrated roots were reduced by 30% and 34%, respectively. The distribution of the inoculum could be improved by fragmentation of the mycelial colonies before soil inoculation. Using mycelial fragments, again no suppression of root penetration was observed up to a potential spore density of 10⁴ g^–1 soil, but at densities of 10⁵ and 10⁶ g^–1 a suppression of 54% and 88%, respectively, was measured. In a greenhouse experiment, soil inoculation with mycelial colonies with a potential spore production of 2.5×10⁵ g^–1 soil resulted in a suppression of root penetration of 37% and 51% after 5 and 6 weeks, respectively, but the number of newly formed cysts after 18 weeks in soil was not different for control and inoculated pots. It is concluded thatH. rhossiliensis may be useful for the reduction of root damage caused by juveniles of potato cyst nematodes, but the usefulness for population control is doubtful.     

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.     

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.     

Glasshouse trials of Coniothyrium minitans and Trichoderma species for the biological control of Sclerotinia sclerotiorum in celery and lettuce

Coniothyrium minitans, Trichoderma harzianum (HH3) and Trichoderma sp. (B1) were tested for ability to control disease caused by Sclerotinia sclerotiorum in a sequence of a celery crop and two lettuce crops in the glasshouse. In control plots, over 80% of celery and 90 and 60% of lettuce in first and second crops, respectively, were infected at harvest. Only the C. minitaris treatment in the first lettuce crop decreased disease and increased marketable yield. Nevertheless, C. minitans reduced the number of sclerotia recovered at harvest in the celery and first lettuce crops and decreased sclerotial survival over the autumn fallow periods following the celery and second lettuce crop. C. minitans survived in soil for over 1 year and spread to infect sclerotia in virtually all other plots. C. minitans infected sclerotia at all times of the year but sclerotia still failed to degrade during the summer months when the soil was dry. The Trichoderma species tested had no effect on disease and almost no effect on the survival of the sclerotia. even though they could be recovered from soil for the duration of the experiments.     

Spread of levan-positive populations of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>, the causal agent of olive knot,in central Italy

Mixtures of wet vegetable wastes (Brassica, carrot or onion) and dry onion waste were composted at 50 °C for 7 days. The incorporation of the raw or composted vegetable waste mixtures into sandy loam, silt and peat soils reduced the viability of sclerotia of S. cepivorum in glasshouse pot bioassays. The reduction in viability was dependent on waste type, rate of incorporation, duration of exposure and soil type. Onion waste was the most effective waste type in reducing sclerotia viability in all three soils. The Brassica and carrot wastes were as effective as the onion waste in silt soil but less effective in sandy loam and peat soil. A 50% w/w incorporation rate of the wastes gave the largest reduction in viability, with an increase in reduction over time. Composted onion waste reduced sclerotia viability under glasshouse and field conditions although the effect was smaller in the field. Composted onion waste incorporated into soil at 50% w/w reduced the incidence of Allium white rot on onion seedlings in glasshouse pot tests. Incidence and control of the disease differed with soil type. The most consistent control was achieved in peat soil whereas no control was observed in silt soil. Incorporation of the waste 2 months prior to sowing or transplanting reduced seedling emergence in sandy loam soil and growth in all three soil types. The potential for field application of composted vegetable wastes as a sustainable method for control of Allium white rot and waste disposal is discussed.     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: influence on apothecial production

The effects of different inocula of the mycoparasite Coniothyrium minitans on carpogenic germination of sclerotia of Sclerotinia sclerotiorum at different times of year were assessed. A series of three glasshouse box bioassays was used to compare the effect of five spore-suspension inocula of C. minitans , including three different isolates (Conio, IVT1 and Contans), with a standard maizemeal–perlite inoculum. Apothecial production, as well as viability and C. minitans infection of S. sclerotiorum sclerotia buried in treated soil, were assessed. Maizemeal–perlite inoculum at 10⁷ CFU per cm³ soil reduced sclerotial germination and apothecial production in all three box bioassays, decreasing sclerotial recovery and viability in the second bioassay and increasing C. minitans infection of sclerotia in the first bioassay. Spore-suspension inocula applied at a lower concentration (10⁴ CFU per cm³ soil) were inconsistent in their effects on sclerotial germination in the three box bioassays. Temperature was an important factor influencing apothecial production. Sclerotial germination was delayed or inhibited when bioassays were made in the summer. High temperatures also inhibited infection of sclerotia by C. minitans . Coniothyrium minitans survived these high temperatures, however, and infected the sclerotia once the temperature decreased to a lower level. Inoculum level of C. minitans was an important factor in reducing apothecial production by sclerotia. The effects of temperature on both carpogenic germination of sclerotia and parasitism of sclerotia by C. minitans are discussed.     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.     

Effects of different temperature treatments on dormancy of sclerotia of ten isolates of Sclerotium cepivorum

The variability of dormancy of sclerotia of ten isolates ofSclerotium cepivorum was investigated. Of all isolates tested, the freshly harvested sclerotia were dormant. After drying for 48 hours the sclerotia of six isolates were able to germinate, two isolates stayed dormant and two isolates were infested by hyperparasitic fungi. After storage in soil at 5°C or 20°C, the sclerotia of the different isolates exhibited considerable differences in respect to germination capability, but all isolates showed highest germination after a treatment of 8 weeks at 20°C followed by 8 weeks at 5°C. The sclerotia of all isolates showed an increased capacity to germinate withoutAllium extracts at 10°C after pretreatment at 30°C for 28 days.     

A one-hit model for the infection of clubroot-susceptible cabbage (Brassica oleracea var.capitata) byPlasmodiophora brassicae at various inoculum densities

In two glasshouse and three phytotron experiments, clubroot-susceptible cabbage (Brassica oleracea varcapitata) cv Septa was inoculated with clubroot resting spores at inoculum densities ranging from 0 to 2·10⁷ spores-plant^–1. At densities of 10⁵ spores·plant^–1 and higher all plants developed clubroot symptoms, except in one glasshouse experiment conducted in winter. The proportion of plants developing symptoms plotted against inoculum density showed a sigmoid curve. Although the shape of the curve was similar in all experiments, the inoculum densities required to induce 50% disease incidence varied from 10³ to 10⁵ resting spores·plant^–1. The data of all five experiments could be well described by a generalized one-hit model which involves variation between plants with regard to the probability of infection.Abbreviations cv cultivar - ECD European Clubroot Differential set     

Factors affecting the production of apothecia and longevity of sclerotia of Sclerotinia sclerotiorum

Sclerotia of Sclerotinia sclerotiorum produced naturally on winter oilseed rape and by an isolate from this host on sterile wheat grain were placed in two different soil types in pots, either on the soil surface or 1. 2, 3, 4 or 6 cm deep. Nitrogen (as TN 34, Thames Nitrogen Co.) was applied to one set of pots in autumn 1982 and spring 1983. Production of apothecia was recorded in this set in 1983 only. In pots without added N apothecia were counted in 1983 and 1984 and sclerotia were recovered from these pots at the end of the experiment.
Overall, production of apothecia was not affected by soil type. More apothecia formed from cultured sclerotia and they appeared earlier than from natural sclerotia. More apothecia were produced from sclerotia placed at 0–2 cm than from those buried deeper. Fewer apothecia were produced in pots with added N and their production was delayed. Fewer sclerotia were recovered from the upper layers of soil with a higher clay content, and when sclerotia were obtained from oilseed rape rather than from cultures.     

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.