除草剂精禾草克和乙草胺对油菜菌核病生防菌盾壳霉生长和寄生的影响

本文研究了油菜田间常用除草剂精禾草克和乙草胺对油菜菌核病生防菌盾壳霉Conio-thyrium minitans的影响。结果表明它们对盾壳霉菌丝生长和分生孢子萌发均有显著的抑制作用,其中精禾草克对盾壳霉菌丝生长和孢子萌发的抑制中浓度分别为2.95mg/L和7.80mg/L;乙草胺对菌丝生长和孢子萌发的抑制中浓度分别为137.45mg/L和120.90mg/L。精禾草克可以抑制盾壳霉寄生核盘菌Sclerotinia sclerotiorum菌核,当精禾草克的使用量达田间使用浓度时,盾壳霉不能寄生核盘菌菌核;而乙草胺对盾壳霉寄生菌核的影响较小,在田间使用浓度1250mg/L时,盾壳霉仍可寄生菌核;乙草胺和盾壳霉在田间使用浓度条件下混合使用,60d后菌核腐烂指数与单独使用盾壳霉没有显著差异,均大于75。上述结果表明在田间使用量条件下,乙草胺可以和盾壳霉生防制剂混用,而精禾草克不宜和盾壳霉混用。     

核盘菌菌核围微生物群落分析及其对盾壳霉重寄生的影响

重寄生真菌盾壳霉(Coniothyrium minitans)是核盘菌的一种生防菌,它通过寄生核盘菌菌核,减少初侵染来源,从而达到防病效果.但在田间自然土壤中,核盘菌菌核围微生物对盾壳霉寄生菌核的影响还不清楚.本研究对核盘菌菌核围微生物进行了分离鉴定,并评估了菌核围细菌对盾壳霉重寄生的影响.结果 表明,不同取样时间和不...     

利用油菜秸秆培养重寄生菌盾壳霉分生孢子

研究了油菜秸秆作为基质培养植物病原菌核盘菌的重寄生菌盾壳霉分生孢子,并从盾壳霉分生孢子萌发及其抑制核盘菌菌核子囊盘萌发等方面评价了所获得的盾壳霉分生孢子的质量。结果表明:盾壳霉野生菌株Chy-1和Zs-1,以及Chy-1的突变菌株SV-5-2(抗杀菌剂vin-clozolin)可以利用油菜秸秆为基质进行培养,有利于3个菌株的菌丝生长、分生孢子器及分生孢子的产生,分生孢子产量可达2·4×109~3·4×109个孢子/g干秸秆。水分含量和发酵时间影响盾壳霉分生孢子产量。在接种量为5×105个孢子/g干秸秆的条件下,以干秸秆中含水量为3~6ml/g,在20℃下发酵12d较为适宜。水琼脂平板试验表明:在20℃下培养48h,盾壳霉分生孢子的萌发率达到90%以上。将油菜秸秆基质培养的盾壳霉分生孢子接种于土壤中,无论是夏季试验,还是秋季试验,对核盘菌菌核萌发及存活具有显著的抑制作用。2003年夏季,4·0×106个孢子/m处理其核盘菌菌核萌发数比对照减少26·3%;秋季该处理比对照减少57·1%~88·0%。     

盾壳霉控制油菜菌核病菌再侵染及其叶面存活动态的研究

 本文评估了施于油菜(Brassica napus)叶片上的盾壳霉(Coniothyrium minitans)控制油菜菌核病菌再侵染能力,探讨了其作用机理,并测定了盾壳霉分生孢子在油菜叶面上的存活动态。结果如下:叶面上的盾壳霉对油菜菌核病菌的初侵染影响较小,但在高剂量(> 10⁶孢子/ml)时可以控制病斑的扩展。所有供试剂量的盾壳霉均可不同程度地控制再侵染。盾壳霉分生孢子可在叶面病部迅速萌发,48 h和72 h时孢子萌发率分别为51%和95%,而在健康叶面上6 d未能检测到萌发的孢子。自携带盾壳霉的叶面病部不能分离到核盘菌,表明叶面上的盾壳霉已寄生并破坏了核盘菌再侵染菌丝。自油菜叶面上分离到的盾壳霉菌落数随时间延长而降低,但其分生孢子至少可以在叶面上存活28 d。这即表明,在叶面上适时适量地添加盾壳霉可以控制油菜菌核病的为害。     

影响盾壳霉寄生核盘菌菌核的几个生态因子的分析

 在室内测定了盾壳霉(Coniothyrium minitans)对不同寄主上的核盘菌(Sclerotinia sclerotiorum)菌核的寄生致腐作用,研究了温度、含水量和土壤类型等生态因子对核盘菌菌核的寄生致腐作用的影响,通过检测土壤的呼吸速率探讨了它在土壤中定殖与核盘菌菌核的关系。结果表明:盾壳霉能寄生致腐核盘菌属所有供试菌株的菌核;寄生致腐菌核的最适温度是20℃,最适相对含水量为50%~60%;盾壳霉在供试的8种土壤中均能寄生致腐菌核,对它们的pH值要求不严格,但土壤类型影响其寄生致腐速度;在土壤中添加菌核和菌核提取液都可不同程度地刺激它的生长。     

莴苣上发现一种新的核盘菌菌核病

 从湖北省神农架莴苣上分离到一种产菌核病原真菌。这种真菌在培养特性上同核盘菌、三叶草核盘菌和小核盘菌既存在着明显差异,又存在着相似之处。其菌核易萌发成子囊柄,但在一般散射光下柄顶端难以发育成子囊盘。对偶得的1枚子囊盘观察表明这种真菌符合核盘菌属真菌的特征,并且同核盘菌属3个近缘种的菌株不同。可溶性蛋白质和多种酶同功酶电泳分析结果表明这种真菌不同于核盘菌属的3个常见种,但同它们亲缘关系较近。这种新菌核病的初侵染来源是菌核萌发产生的菌丝,通过病健接触构成再侵染,在病组织上形成菌核越冬越夏。离体和活体致病性测定结果表明这种真菌只能侵染莴苣,不能侵染油菜、小白菜、萝卜和胡萝卜。     

盾壳霉对油菜长效专用配方肥料的敏感性评价

油菜菌核病是油菜生产中的重要病害,盾壳霉是核盘菌的重寄生真菌,在菌核病防治方面具有重要的生防潜力。为了明确盾壳霉与油菜长效专用配方肥料混合施用的可行性,本文研究了盾壳霉对油菜长效专用配方肥的敏感性。结果发现,低浓度的油菜长效专用配方肥（1.5和7.5 mg/mL）对盾壳霉菌丝生长、菌落及菌丝尖端形态和分生孢子萌发等无明显影响,高浓度的油菜长效专用配方肥对盾壳霉生长和孢子萌发有一定影响,在饱和浓度（560 mg/mL）条件下的油菜长效专用配方肥,24 h盾壳霉孢子的萌发率为0.83%,然而在96 h时萌发率可达到95%;油菜长效专用配方肥对盾壳霉产孢和寄生致腐菌核的能力无明显影响,寄生菌核30 d后,致腐指数均在50以上。研究结果表明,在田间生产中,盾壳霉可以与油菜长效专用配方肥料混合施用,达到轻简化栽培的目的。     

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。     

核盘菌菌核萌发多样性的研究

在15、20、25和28℃条件下培养核盘菌菌核,再在20℃下诱导菌核萌发,结果将不同来源的50个菌株分成5类:①菌核易进行菌丝型萌发;②4种温度下形成的菌核均易进行子囊盘型萌发;③高温(25和28℃)下形成的菌核进行子囊盘萌发,而低温(15或20℃)下形成的菌核则不易萌发;④15℃下形成的菌核易产生子囊盘,而其它温度下形成的菌核都不能萌发;⑤4种温度下形成的菌核都不能萌发。5类菌株各占2％、6％、22％、6％和64％。对第2、3类菌株而言,形成菌核时温度越高,菌核越易萌发。进一步分析说明菌核萌发多样性和菌株来源有一定的关系。对其中3个菌株的21个单子囊孢子后代菌核萌发特性的研究结果表明,菌核萌发特性具有遗传稳定性。这说明核盘菌菌株间菌核萌发习性存在着明显的多样性,且具有遗传稳定性,可用于研究这一病菌的群体结构。     

人参核盘菌菌核的菌丝型萌发特性研究

人参核盘菌菌核在自然环境及人工培养条件下很难产生子囊盘,主要通过菌核进行菌丝型萌发侵染寄主。为明确人参核盘菌菌核的菌丝型萌发特性,对人参核盘菌菌核的萌发历程和影响因素进行了研究。结果表明,人参核盘菌菌核吸水性强,前期吸水速度较快,菌核吸水饱和后,是吸水前菌核平均质量的1.99倍。菌核吸水后3 d开始菌丝型萌发,8 d时菌核的菌丝型萌发率到达97.8%,而未吸水的菌核则不能萌发产生菌丝。菌核在干燥的土壤环境中的菌丝型萌发率极低,土壤湿度越大越利于菌丝型萌发。菌核龄越大所需的菌丝型萌发时间越长。15.0～22.5℃为菌核菌丝型萌发的适宜温度。pH为6.0时,其菌核的菌丝型萌发率最高。     

Long-Term Biosanitation by Application of Coniothyrium minitans on Sclerotinia sclerotiorum-Infected Crops

ABSTRACT The effect of the fungal mycoparasite Coniothyrium minitans applied as a spray to crops infected with Sclerotinia sclerotiorum (causal agent of white mold) on contamination of soil with S. sclerotiorum sclerotia was studied in a 5-year field experiment. Sclerotial survival also was monitored during two subsequent years, when the field was returned to commercial agriculture. In a randomized block design, factorial combinations of four crops and three treatments were repeated 10 times. Potato (Solanum tuberosum), bean (Phaseolus vulgaris), carrot (Daucus carota), and chicory (Cichorium intybus), which are all susceptible to S. sclerotiorum, were grown in rotation. Plots were treated with C. minitans or Trichoderma spp. or were nontreated (control). Crops were rotated in each plot, but treatments were applied to the same plot every year. After 3 years during which it showed no effect on sclerotial survival, the Trichoderma spp. treatment was replaced by a single spray with C. minitans during the fourth and fifth years of the trial. The effect of treatments was monitored in subsequent seasons by counting apothecia as a measure of surviving S. sclerotiorum sclerotia and scoring disease incidence. Trichoderma spp. did not suppress S. sclerotiorum, but C. minitans infected at least 90% of S. sclerotiorum sclerotia on treated crops by the end of the each season. C. minitans lowered the number of apothecia compared with the other treatments during the second year after the bean crop. C. minitans reduced the number of apothecia by approximately 90% when compared with the control and Trichoderma spp. treatments and reduced disease incidence in the bean crop by 50% during the fifth year of the trial, resulting in a slightly higher yield. In 1993, but not 1994, a single spray with C. minitans was nearly as effective at reducing apothecia as three sprays (monitored in 1995). The final population size of sclerotia in soil at the end of the 7-year period was lower in all C. minitans plots than at the beginning of the trial, even in plots where two highly susceptible bean crops were grown during the period. The results indicate that the mycoparasite C. minitans has the potential to keep contamination of soil with sclerotia low in crop rotations with a high number of crops susceptible to S. sclerotiorum.     

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.     

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.     

Potential for Integrated Control of Sclerotinia sclerotiorum in Glasshouse Lettuce Using Coniothyrium minitans and Reduced Fungicide Application

ABSTRACT All pesticides used in United Kingdom glasshouse lettuce production (six fungicides, four insecticides, and one herbicide) were evaluated for their effects on Coniothyrium minitans mycelial growth and spore germination in vitro agar plate tests. Only the fungicides had a significant effect with all three strains of C. minitans tested, being highly sensitive to iprodione (50% effective concentration [EC(50)] 7 to 18 mug a.i. ml(-1)), moderately sensitive to thiram (EC(50) 52 to 106 mug a.i. ml(-1)), but less sensitive to the remaining fungicides (EC(50) over 200 mug a.i. ml(-1)). Subsequently, all pesticides were assessed for their effect on the ability of C. minitans applied as a solid substrate inoculum to infect sclerotia of Sclerotinia sclerotiorum in soil tray tests. Despite weekly applications of pesticides at twice their recommended concentrations, C. minitans survived in the soil and infected sclerotia equally in all pesticide-treated and untreated control soil trays. This demonstrated the importance of assessing pesticide compatibility in environmentally relevant tests. Based on these results, solid substrate inoculum of a standard and an iprodione-tolerant strain of C. minitans were applied individually to S. sclerotiorum-infested soil in a glasshouse before planting lettuce crops. The effect of a single spray application of iprodione on disease control in the C. minitans treatments was assessed. Disease caused by S. sclerotiorum was significantly reduced by C. minitans and was enhanced by a single application of iprodione, regardless of whether the biocontrol agent was iprodione-tolerant. In a second experiment, disease control achieved by a combination of C. minitans and a single application of iprodione was shown to be equivalent to that of prophylactic sprays with iprodione every 2 weeks. The fungicide did not affect the ability of C. minitans to spread into plots where only the fungicide was applied and to infect sclerotia. These results indicate that integrated control of S. sclerotiorum with soil applications of C. minitans and reduced foliar iprodione applications was feasible, did not require a fungicide tolerant isolate, and that suppression of Sclerotinia disease by C. minitans under existing chemical control regimes has credence.     

Interactions between Coniothyrium minitans and Sclerotinia minor affect biocontrol efficacy of C. minitans

Coniothyrium minitans, marketed as Contans, has become a standard management tool against Sclerotinia sclerotiorum in a variety of crops, including winter lettuce. However, it has been ineffective against lettuce drop caused by S. minor. The interactions between C. minitans and S minor were investigated to determine the most susceptible stage in culture to attack by C. minitans, and to determine its consistency on S minor isolates belonging to four major mycelial compatibility groups (MCGs). Four isolates of S. minor MCG 1 and 5 each from MCGs 2 and 3 and one from MCG 4 were treated in culture at purely mycelial, a few immature sclerotial, and fully mature sclerotial phases with a conidial suspension of C. minitans. Sclerotia from all treatments were harvested after 4 weeks, air dried, weighed, and plated on potato dextrose agar for recovery of C. minitans. S. minor formed the fewest sclerotia in plates that received C. minitans at the mycelial stage; C. minitans was recovered from nearly all sclerotia from this treatment and sclerotial mortality was total. However, the response of MCGs was inconsistent and variable. Field experiments to determine the efficacy of C. minitans relative to the registered fungicide, Endura, on lettuce drop incidence and soil inoculum dynamics were conducted from 2006 to 2009. All Contans treatments had significantly lower numbers of sclerotia than Endura and unsprayed control treatments, and drop incidence was as low as in Endura-treated plots (P > 0.05). Although the lower levels of lettuce drop in Contans treatments were correlated with significantly lower levels of sclerotia, the lower levels of lettuce drop, despite the presence of higher inoculum in the Endura treatment, was attributable to the prevention of infection by S. minor. A useful approach to sustained lettuce drop management is to employ Contans to lower the number of sclerotia in soil and to apply Endura to prevent S. minor infection within a cropping season.     

Germination of Sclerotinia minor and S. sclerotiorum Sclerotia Under Various Soil Moisture and Temperature Combinations

ABSTRACT Sclerotial germination of three isolates each of Sclerotinia minor and S. sclerotiorum was compared under various soil moisture and temperature combinations in soils from Huron and Salinas, CA. Sclerotia from each isolate in soil disks equilibrated at 0, -0.03, -0.07, -0.1, -0.15, and -0.3 MPa were transferred into petri plates and incubated at 5, 10, 15, 20, 25, and 30 degrees C. Types and levels of germination in the two species were recorded. Petri plates in which apothecia were observed were transferred into a growth chamber at 15 degrees C with a 12-h light-dark regime. All retrievable sclerotia were recovered 3 months later and tested for viability. Soil type did not affect either the type or level of germination of sclerotia. Mycelial germination was the predominant mode in sclerotia of S. minor, and it occurred between -0.03 and -0.3 MPa and 5 and 25 degrees C, with an optimum at -0.1 MPa and 15 degrees C. No germination occurred at 30 degrees C or 0 MPa. Soil temperature, moisture, or soil type did not affect the viability of sclerotia of either species. Carpogenic germination of S. sclerotiorum sclerotia, measured as the number of sclerotia producing stipes and apothecia, was the predominant mode that was affected significantly by soil moisture and temperature. Myceliogenic germination in this species under the experimental conditions was infrequent. The optimum conditions for carpogenic germination were 15 degrees C and -0.03 or -0.07 MPa. To study the effect of sclerotial size on carpogenic germination in both S. minor and S. sclerotiorum, sclerotia of three distinct size classes for each species were placed in soil disks equilibrated at -0.03 MPa and incubated at 15 degrees C. After 6 weeks, number of stipes and apothecia produced by sclerotia were counted. Solitary S. minor sclerotia did not form apothecia, but aggregates of attached sclerotia readily formed apothecia. The number of stipes produced by both S. minor and S. sclerotiorum was highly correlated with sclerotial size. These results suggest there is a threshold of sclerotial size below which apothecia are not produced, and explains, in part, why production of apothecia in S. minor seldom occurs in nature.     

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.     

A method for inducing apothecia from sclerotia of Sclerotinia sclerotiorum

An isolate of Sclerotinia sclerotiorum from oilseed rape was grown on sterilized wheat grain for 3 weeks at 20 C, followed by 4 weeks at 4^oC. Harvested sclerotia were buried 1 cm deep in compost in plastic containers and kept at 10^oC until apothecial stipes appeared (c. 6 weeks). When these dishes were placed under near-UV light (14 h/day) at 22^oC, apothecia matured in 5 days. The method also induced apothecia from sclerotia of 35 other isolates of S. sclerotiorum obtained from 17 different hosts.