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。     

拟康宁木霉Hailin产几丁质酶条件优化及其抑菌能力评价

采用透明圈法测定了拟康宁木霉(Trichoderma koningiopsis) Hailin菌株是否产几丁质酶,并通过单因素和正交试验对Hailin菌株产几丁质酶的发酵条件进行优化;用对峙培养和培养基中添加几丁质的方法检测Hailin菌株及其几丁质酶粗酶液对7种植物病原真菌的拮抗能力。结果表明,Hailin菌株在胶体几丁质的诱导下可以产生明显的透明圈,即可产生几丁质酶。Hailin菌株产几丁质酶的优化条件为葡萄糖5.0 g/L、硝酸钾10.0 g/L、Mn~(2+)0.08 mol/L、Mg~(2+)0.05 mol/L、初始pH为4、装液量为50 mL/150 mL。优化条件下,Hailin菌株产几丁质酶活性可达到0.784 U/mL,较优化前几丁质酶活性相比提高了2.57倍。Hailin菌株对7种植物病原真菌均有抑菌效果,抑菌率在44.44%～85.71%之间;Hailin菌株几丁质粗酶液对7种植物病原菌中的6种具有抑菌效果,抑菌率为5.88%～71.52%。Hailin菌株分泌的几丁质酶在Hailin菌株抑制不同病原菌过程中发挥的抑菌作用存在显著差异。     

蝇蛆甲壳素对深绿木霉T2产生几丁质酶的诱导作用

分别采用EDTA除钙和氢氧化钠-戊醇反应体系,并结合微波加热处理2次的优化工艺,从蝇蛆中提取甲壳素、壳聚糖,探讨二者对深绿木霉T2菌株产生几丁质酶的影响,并对深绿木霉T2几丁质酶活性及其对4种病原真菌的拮抗作用进行了研究。结果表明,提取的蝇蛆甲壳素的产出率为75.0%,灰分含量为17.3%;蝇蛆壳聚糖制备获得脱乙酰度为95.5%、含水量为8.36%的高质量壳聚糖产品,且碱用量明显下降,反应时间由4 h缩短为6 min;蝇蛆甲壳素培养基诱导培养的深绿木霉T2几丁质酶活性为0.9721 U/mL,高于PDB液体培养基、SCMS营养液和蝇蛆壳聚糖培养基;不同营养条件下,蝇蛆甲壳素培养基均能够诱导深绿木霉T2菌株产生几丁质酶,且最佳产酶培养时间为120 h,酶活性峰值为1.8340 U/mL;经蝇蛆甲壳素诱导后的深绿木霉T2菌株对4种病原菌的抑菌效果明显高于对照菌株T2。     

草酸对重寄生真菌盾壳霉分生孢子萌发和菌丝生长的影响

 本文研究了草酸对核盘菌的重寄生真菌盾壳霉(Coniothyrium minitans)分生孢子萌发和菌丝生长的影响。结果表明:草酸对盾壳霉分生孢子萌发没有明显促进作用,在酸碱性非缓冲基质(水琼脂)和酸碱性缓冲基质中,抑制盾壳霉分生孢子萌发的最低浓度分别为150和700μg/mL。在马铃薯葡萄糖琼脂培养基中,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且浓度为300~500μg/mL的草酸对盾壳霉的菌丝生长具有明显的促进作用。在以草酸为唯一碳源的合成培养基中,在酸碱性非缓冲的条件下,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为500μg/mL时对盾壳霉菌丝生长具有促进作用,而当草酸浓度为2500μg/mL时,盾壳霉菌丝则停止生长。在酸碱性缓冲的合成基质中,草酸浓度为100~4000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为1500~2500μg/mL时对盾壳霉菌丝生长具有促进作用。在含草酸钙的混浊培养基(以草酸为唯一碳源)上,盾壳霉菌落区域形成了透明圈。上述结果说明盾壳霉能忍耐一定浓度的草酸而进行分生孢子萌发及菌丝生长,且这种真菌可能对草酸分子具有分解作用。     

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%。     

产生几丁质酶的食线虫真菌绿粘帚霉Gliocladium virens CFCC80915对南方根结线虫卵孵化的影响

 根结线虫卵壳主要由几丁质和蛋白质构成。寄生根结线虫卵或产毒真菌产生几丁质酶特性是评价食线虫真菌生物防治潜力的重要生化指标之一。利用还原糖法和pNP法分别测定绿粘帚霉(Gliocladium virens)的几丁质酶的内切酶和外切酶活性。第14d内切酶活性达到最高值,其酶活为53.1μmol/h mL;外切酶活性第26d达到高峰,其酶活为0.432μmol/h mL。利用活性染色电泳测其几丁质酶的分子量分别为75.8kDa、42.8kDa和39.6kDa。表明筛选到的绿粘帚霉CFCC80915菌株具有较高产生几丁质酶的活性。绿粘帚霉12d的培养滤液对根结线虫卵孵化7d后的抑制率达到92.9%。显微观察线虫卵壳变形和破坏情况的结果表明,绿粘帚霉CFCC80915产生的几丁质酶可以引起根结线虫卵壳的裂解,抑制根结线虫卵的孵化。     

木霉几丁质酶对烟草赤星病菌的作用

以指形管培养法分别测定几丁质酶粗酶液和纯化的几丁质酶混合液(2种几丁质酶)对烟草赤星病菌孢子萌发的抑制作用。结果表明,较高浓度(25 2U)几丁质酶粗酶液在48h内强烈抑制孢子萌发和芽管伸长,或致芽管畸形和细胞壁破裂;几丁质酶混合液对赤星病菌的孢子萌发也表现出明显的抑制作用,但在相同或相近酶活性条件下,纯化的几丁质酶混合液(9 4U)和粗酶液(12 6U)对赤星病菌孢子萌发的抑制率(处理24h时)分别为46%和84 3%,前者明显低于后者。采用孢子液悬滴法接种烟苗(K 326)叶片测定木霉几丁质酶对赤星病菌致病性的影响。结果表明,几丁质酶粗酶液浓度越高,对孢子萌发抑制时间越长,抑制率越高;其浓度为4 9、9 8、19 5U/ml的抑制率7d时分别为36 8%、56 2%和57 6%。     

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。     

抗真菌物质HSAF发酵全合成培养基的筛选

热稳定抗真菌因子是产酶溶杆菌OH11产生的一种抗真菌活性物质,可开发成环境友好型生物杀菌剂。为了进一步提高HSAF发酵产量,本研究通过单因素试验筛选出最佳碳源、氮源、金属离子及磷酸缓冲盐并进行正交试验优化,确定了最适的合成培养基配方为葡萄糖8 g/L、（NH₄）₂SO₄ 1 g/L、FeCl₃ 8 mg/L、K₂HPO₄1 g/L、KH₂PO₄0.5 g/L。在此培养基下,HSAF产量达到（215.46±6.85）mg/L,比优化前提高了92.24%。该培养基优势在于成分已知、含量稳定,不仅能避免培养基中复杂成分造成发酵产量的波动,而且有利于对胞内代谢特征和HSAF合成关键因素进行深入研究,进而提高产量并降低生产成本。     

木霉几丁质酶及其对植物病原真菌的拮抗作用

 木霉(Trichoderma spp.)作为重要的植病生防因子(biocontrol agents)一直受到普遍关注。木霉菌株产生的包括几丁质酶在内的细胞壁降解酶,在木霉重寄生中起重要作用。本文论述木霉几丁质酶的种类、诱导产生、理化特性及其对植物病原真菌的拮抗作用,并对木霉几丁质酶及其基因的生防应用进行讨论。     

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.     

土壤中盾壳霉双标记平板计数法的建立及应用

盾壳霉是一种重要的生防菌,其在土壤中的存活数量直接关系到防治病害的效果。然而目前没有对土壤中盾壳霉直接计数的方法,构建一种简单易行的土壤中盾壳霉计数方法对研究盾壳霉在土壤中的存活动态具有重要意义。本研究利用农杆菌转化法构建了潮霉素基因和绿色荧光蛋白基因标记的双标记盾壳霉菌株,并测定转化子的生长速度、产孢量和菌核致腐能力,初步分析了该方法计数土壤盾壳霉的有效性和可行性。结果显示,潮霉素基因和绿色荧光蛋白基因可以稳定地遗传和表达,并且部分转化子生长速度、产孢量和菌核致腐能力与出发盾壳霉菌株JNCM没有显著差异。加入土壤中的盾壳霉转化子可以在含潮霉素(50μg/mL)、氯霉素(100μg/mL)和链霉素(100μg/mL)的PDA平板培养,杂菌得到充分抑制,呈现绿色荧光的盾壳霉转化子被有效检出,检出限达到2×103个/g土。本研究所构建的计数方法能有效检出施入土壤中的盾壳霉并进行活菌计数,可以用于盾壳霉JN-CM产品在土壤中的定殖、生长、繁殖和存活情况的研究。应用双标记平板计数法研究了不同温度、湿度、接种量和添加菌核等条件下盾壳霉JN-CM在土壤中的存活规律。结果显示,在含有核盘菌菌核的土壤中,盾壳霉JN-CM可以通过重寄生维持一段时间(12周)的数量增长,在长达半年左右(24周)的时间里其存活率仍然可以维持在65%左右。在不含菌核的土壤中,在一般土壤温度(10~20℃)范围内,无论土壤水分含量高低,其半年存活率也可以维持在50%左右。因此,可以预测,连续施用盾壳霉JN-CM生防制剂,可以使其数量在土壤中长期维持在一定的水平范围,达到长效防治效果。     

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.     

盾壳霉抗菌核净菌株的诱导及其特性初步研究

为筛选可与菌核净配合防治作物菌核病的盾壳霉菌株,进行了紫外线辐射和药剂驯化试验,分别得到4个和2个盾壳霉抗菌核净突变菌株,并研究了其生物学特性。结果表明:除菌株D-1外,其余突变菌株生长速率(菌落直径57.00 ~62.75 mm,生物量6.38 ~8.63 mg/mL)均大于亲本菌株(菌落直径54.00 mm,生物量5.91 mg/mL);大部分突变菌株产孢量(对数值7.33 ~8.16)小于亲本菌株(8.40);菌核净对突变菌株的EC50值是亲本菌株的2.23 ~191.84倍;突变菌株D-3、U-6有微弱的几丁质酶活性,且除U-4外的突变菌株葡聚糖酶活性增强(亲本菌株8.96 U,突变菌株9.18 ~17.61 U)。综合分析,U-6菌株既有较高的葡聚糖酶活性又有几丁质酶活性,并能保持较快的生长速率,是值得开发的优良菌株。     

Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape

In two field trials with oilseed rape, Coniothyrium minitans was applied to soil as a maizemeal-perlite preparation in order to determine its effect on sclerotial survival and apothecial production of Sclerotinia selerotiorum. The mycoparasite infected sclerotia and decreased sclerotial survival, carpogenic germination and production of apothecia. Effects were greatest when inoculum of C. minitans was applied in autumn, at the time of sowing, rather than when it was applied in spring. C. minitans survived in soil for 2 years and spread to adjacent control plots and infected sclerotia within those plots. However, despite the fact that the inoculum potential of S. selerotiorum was reduced by C. minitans treatment, no disease control was obtained either in trial 1, where disease levels were low (0-20% of plant stems affected), or in trial 2, where disease levels were high (up to 70% of plant stems affected). Possible reasons for this failure of C minitans to control sclerotinia disease in oilseed rape, and strategies to improve its efficacy in the field, are discussed.     

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。     

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.     

不同培养基继代培养蜡蚧轮枝菌对产孢量和烟粉虱毒力的影响

为了探究不同培养基继代培养的蜡蚧轮枝菌对烟粉虱的作用效果,通过继代培养方法测定JMC-01、JMC-02菌株的产孢量、对烟粉虱的毒力及其几丁质酶活性。结果表明,JMC-01、JMC-02菌株在添加蝉蜕和烟粉虱的PDA培养基中连续培养5代的产孢量、对烟粉虱的毒力、几丁质酶活性均高于无添加的PDA培养基,且JMC-01菌株的各项参数均高于JMC-02菌株。JMC-01菌株在添加蝉蜕的PDA培养基中T_1和T_3代的产孢量最大,均为1.279×10~7个/mL,T_3代对烟粉虱的毒力最大,为88.33%,T_5代的几丁质酶活最高,为0.268 3 U/mL;在添加烟粉虱的PDA培养基中,JMC-01菌株的最大产孢量、对烟粉虱的最高毒力和最高几丁质酶活分别出现在S_2、S_4和S_5代,分别为1.270×10~7个/mL、88.33%和0.325 4 U/mL。回归分析结果显示,JMC-01、JMC-02菌株几丁质酶活性与烟粉虱校正死亡率呈显著的正相关,且拟合优度较好。本试验表明JMC-01、JMC-02菌株均对烟粉虱具有较强的毒力,添加蝉蜕和烟粉虱的培养基可以维持菌株生长特性、增加产孢量、延缓菌株退化、增强菌株的几丁质酶活性,为烟粉虱的生物防治提供了新的菌种资源。     

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.