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.     

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.     

重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

重寄生真菌盾壳霉Coniothyrium minitans是核盘菌Sclerotinia sclerotiorum的重要生防菌。为了探讨盾壳霉胞外蛋白酶在寄生核盘菌过程中的作用,采用明胶平板法对盾壳霉寄生核盘菌菌核产生的蛋白酶活性进行了检测,并进一步采用福林酚法定量测定蛋白酶活性,研究盾壳霉产生胞外蛋白酶的培养条件及影响蛋白酶活性的因子。试验结果表明,在被盾壳霉寄生的核盘菌菌核中检测到蛋白酶活性,表明蛋白酶可能参与盾壳霉重寄生作用。发现核盘菌菌核浸出液培养基适合盾壳霉产生胞外蛋白酶,摇培(20℃、200r/min)5d时蛋白酶活性最高,达到0.22U/mL。盾壳霉胞外蛋白酶酶促反应的最适温度为60℃,最适pH7.0。当温度不高于40℃时,蛋白酶酶活较稳定。5mmol/L的金属离子Mg²⁺、Zn²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Li⁺和K⁺等对蛋白酶酶活没有显著影响(P>0.05),而Fe²⁺(5mmol/L)显著(P<0.05)提高了蛋白酶活性。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)敏感,说明盾壳霉产生的胞外蛋白酶可能主要是丝氨酸蛋白酶。这些结果为盾壳霉胞外蛋白酶的分离纯化和功能研究奠定了基础。     

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.     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: control of sclerotinia disease in glasshouse lettuce

The effects of Coniothyrium minitans inoculum quality and an 8-week interval between inoculum application and crop planting on sclerotinia ( Sclerotinia sclerotiorum ) disease in three successive lettuce crops were investigated in a glasshouse trial. Spore suspensions of three isolates of C. minitans (Conio, IVT1 and Contans) applied at 10⁸ CFU m⁻² and a standard Conio maizemeal–perlite application (06 L m⁻², 10¹¹ CFU m⁻²) were assessed for their ability to control S. sclerotiorum . Only the maizemeal–perlite inoculum (isolate Conio) consistently reduced sclerotinia disease. In the third lettuce crop only, isolates IVT1 and Contans formulated by Prophyta and isolate IVT as an oil–water formulation, all applied as spore suspensions, reduced disease at harvest compared with the untreated control. Recovery, viability and C. minitans infection of sclerotia buried during the 8-week period prior to each of the three lettuce crops, and of sclerotia formed on the crop, were tested. Only the maizemeal–perlite inoculum (isolate Conio) reduced the recovery of sclerotia buried in soil for weeks between inoculum application and crop planting, reducing their viability and increasing infection by C. minitans . Eight weeks was sufficient to enable C. minitans to infect sclerotia of S. sclerotiorum , and may account for disease control. After harvest of the second and third crops, maizemeal–perlite treatment (isolate Conio) reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore-suspension treatments. The effect of inoculum concentration and the influence of soil temperature (varying with time of year) on infection of sclerotia by C. minitans are discussed.     

Biological Control of Sclerotinia pseudotuberosa and Other Fungi During Moist Storage of Quercus robur Seeds

The fungal pathogen Sclerotinia pseudotuberosa is a major cause of deterioration during storage of Quercus robur seeds (acorns) and along with other, mainly saprotrophic fungi, contributes to the decline of viability and vigour in the acorn population. Hot-water thermotherapy (HWT; 41 °C for 2.5 h) killed the fungal pathogen S. pseudotuberosa and prolonged the storage life of acorns. The addition of the systemic fungicide benomyl to the HWT and/or the broad-spectrum fungicide thiram as a seed dressing further enhanced the storage life of acorns. Three fungal antagonists, Coniothyrium minitans, Trichoderma sp. (KW3) and Trichoderma virens (G20), were also applied as a film-coating to acorns using a polyvinylacetate sticker achieving ca. 10⁷–10⁸ viable conidia per acorn. The biological treatments provided protection against infection and the spread of infection of S. pseudotuberosa and other fungi on the acorns during storage over several months. All treated and stored acorns grew normally following sowing in seedbeds. The Trichoderma species were more effective than C. minitans, with T. virens being the most effective. T. virens reduced pathogen spread from acorns infected with S. pseudotuberosa to `clean' acorns when T. virens coated infected and 'clean' acorns were mixed together. However, T. virens was less effective than HWT at preventing the proliferation of this pathogen within individual acorns that were infected before coating. A combination of HWT and subsequent coating with T. virens provided a more effective control against both S. pseudotuberosa and saprotrophic fungi than when either treatment was applied alone.     

油菜菌核病抗(耐)病性筛选方法的研究

本文评价了不同接种鉴定方法在油菜菌核病抗(耐)病性筛选上的应用。结果表明:花期离体或活体叶片菌丝体接种和限期接种法能显著区分不同品种(系)的抗感水平;苗期菌丝体悬浮液接种和试管苗接种法适于早期育种世代进行大规模筛选。对83份品种(系)鉴定结果,821、083等呈轻度感病,大部分属中感至高感范围。     

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.     

Identification of Greenhouse Characteristics which Affect the Incidence of Sclerotinia sclerotiorum (Lib.) de Bary in Pepper Crops in a Mediterranean Climate

The effect of the main construction factors and design features of greenhouses on the incidence of white rot disease, caused by Sclerotinia sclerotiorum (Lib.) de Bary, was studied in peppers grown under plastic-roofed greenhouses typical of Almería (SE Spain). At the height of the pepper-growing season in the province, fifty greenhouses were repeatedly sampled. Incidence of the disease was measured and the different construction features of the greenhouses were evaluated: types of roof cover, colour and age of the plastic cover, dimensions (ground plan, surface area, average height), estimated ventilation capacity and the presence of netting on the sides of the greenhouses (laterals). Statistical analyses were undertaken to see if the choice of various design options for each factor had any significant influence on the incidence of the disease. To achieve this single factor ANOVA was undertaken. The incidence of this disease was significantly affected (p<0.05) by the following greenhouse characteristics: ground plan, surface area, mean height, colour of the plastic cover, estimated ventilation and use of lateral netting.     

陕西省核盘菌不同分离株对油菜的致病性

采用带菌牙签活体茎秆穿刺接种法和离体叶片菌饼接种法,以3个品种的油菜为供试寄主,对陕西省5个地区10个县的油菜和其它5种寄主共495个核盘菌分离株进行了致病性测定。结果显示,两种接种方法接种3个不同油菜品种所反映的不同分离株的致病趋势一致。离体叶片菌饼接种蓉油12号除来自勉县和城固县的2个分离株不能致病外,其余493个分离株均能致病,但各分离株所致病斑直径差异很大,为0.5～42.6mm,其中病斑直径在10～40mm的分离株占94.7%,属于优势群体。同一地区甚至同一地块均存在不同致病力的分离株,不同寄主来源的分离株在油菜离体叶片上均能致病。说明核盘菌种内存在明显的致病性分化,这种分化与地理来源和寄主来源均无明显的相关性。     

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).     

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.     

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.     

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.     

花生-玉米间作种植模式对蓟马类害虫的控制效果

为明确花生-玉米间作种植模式对蓟马类害虫的控制效果,通过3年田间试验系统调查花生单作、玉米单作和花生-玉米间作3种种植模式下蓟马及其主要捕食性天敌东亚小花蝽Orius sauteri的种群数量动态特征及不同种植模式下东亚小花蝽和蓟马的益害比。结果显示,花生-玉米间作种植模式下东亚小花蝽种群密度显著高于花生单作种植模式,花生-玉米间作种植模式下82.94%以上的东亚小花蝽来源于玉米斑块;与2种单作种植模式相比,花生-玉米间作种植模式未显著减少蓟马的种群密度;东亚小花蝽种群密度与蓟马种群密度呈显著线性关系;花生-玉米间作种植模式下东亚小花蝽与蓟马的益害比均显著低于其他2种单作种植模式。表明花生-玉米间作种植模式在局域农田小范围空间尺度下能显著提高捕食性天敌的种群密度,但并不能显著减少蓟马类害虫种群数量。     

<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.     

山东省不同地区蔬菜菌核病菌对四霉素的敏感性

采用菌丝生长速率法测定了四霉素对采自山东省不同地区不同蔬菜作物的151株菌核病菌的毒力作用,同时比较了其对蔬菜菌核病菌不同生育阶段的抑制活性,并通过离体叶片法评价了四霉素对蔬菜菌核病的防治效果。结果表明:菌核病菌对四霉素比较敏感,敏感性频率呈单峰正态分布,151株病菌菌丝生长的平均EC₅₀值为 (0.29 ± 0.01) μg/mL,该值可作为蔬菜菌核病菌对四霉素的敏感基线。此外,经四霉素处理后,该病菌的菌核数量以及干重明显降低,菌核明显变小;2 μg/mL的处理对菌核萌发的抑制率达到100.00%。 离体黄瓜叶片接种试验表明,四霉素对菌核病具有较好的保护和治疗效果,且保护作用较为显著。在质量浓度为20 μg/mL时,四霉素对该病的防效显著高于对照药剂多菌灵和异菌脲。因此,四霉素具有防治蔬菜菌核病的潜在价值,可进一步通过田间试验验证其应用效果。     

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.     

噻虫嗪对丽蚜小蜂寄生烟粉虱的影响

为了明确噻虫嗪对烟粉虱天敌昆虫丽蚜小蜂Encarsia formosa的影响,在室内模拟田间噻虫嗪灌根和叶面喷雾2种施药方式,研究了施药后丽蚜小蜂的直接死亡率及其寄生Q型烟粉虱的生物学参数。结果表明,经噻虫嗪叶面喷雾后,丽蚜小蜂的死亡率高达98.10%,显著高于其它处理,丽蚜小蜂对烟粉虱若虫的寄生率为5.26%,显著低于其它处理;与清水对照相比,经噻虫嗪处理后丽蚜小蜂对烟粉虱的致死率降低,且叶面喷雾处理大于灌根处理;噻虫嗪灌根和叶面喷雾处理对丽蚜小蜂的羽化率和发育历期均无显著影响,但均能显著缩短丽蚜小蜂成虫的寿命。研究表明,噻虫嗪灌根施药方式对丽蚜小蜂的死亡率及寄生率影响较小,可将噻虫嗪灌根施药方式与释放丽蚜小蜂相结合以防治烟粉虱。