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.     

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。     

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。     

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。     

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。     

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...     

Effect of formulation on the rhizosphere competence and biocontrol ability of Trichoderma atroviride C52

The rhizosphere competence of the biological control agent Trichoderma atroviride isolate C52 was studied on onion roots both in the glasshouse and in the field when introduced into soil in a range of formulations. Proliferation of T. atroviride in the rhizosphere was formulation-dependent. A pellet formulation maintained the fungal concentration at 10⁵ cfu per g soil, whereas solid-substrate and seed-coating formulations gave concentrations of 10⁴ and 10¹ cfu per g soil, respectively. To facilitate rhizosphere-competence studies, a UP-PCR band profile generated with primer L45 for isolate C52 was used to enable conclusive identification of T. atroviride C52 when recovered from soil. When isolate C52 was introduced into Sclerotium cepivorum -infested soil as both pellet and solid-substrate formulations, there was no statistically significant difference in the disease control between these treatments, but the pellet treatment doubled the percentage of healthy plants compared with the control treatment.