大黄酚对黄瓜白粉病菌的抑制作用研究

 采用子叶喷雾法分别测定了大黄酚(chrysophanol)对黄瓜白粉病菌(Sphaerotheca fuliginea)的保护和治疗生物活性。结果表明,大黄酚能有效降低黄瓜白粉病的病情指数,其保护和治疗作用的EC₅₀分别为0.23μg/mL和25.88μg/mL,对照药剂50%硫磺悬浮剂保护作用的EC₅₀为59.75μg/mL,10%苯咪甲环唑可湿性水分散粒剂治疗作用的EC₅₀为13.55μg/mL。在此基础上,利用2.4μg/mL大黄酚溶液处理黄瓜子叶24h后接种白粉病菌,在不同时段对病组织进行透明染色观察白粉病菌的变化情况。结果发现,大黄酚可有效降低黄瓜白粉病菌分生孢子的萌发率和萌发芽管个数,并能抑制菌丝的生长以及降低新生分生孢子的个数。接菌后24h,清水对照萌发率为66.6%,而大黄酚处理后的孢子不萌发;接菌后48h,对照菌丝的平均长度为414.7μm,大黄酚处理的菌丝平均长度为40.1μm;接菌后144h,对照的新生分生孢子串最多为8个孢子,大部分为6-7个,而大黄酚处理后的新生分生孢子串孢子个数最多为2个,大部分为1个。接菌7d后对照的病情指数为72.2,大黄酚处理组的病情指数仅为2.1,其保护作用防治效果达到97.1%,远高于50%硫磺悬浮剂65.4%的防效。     

采用孢子萌发法测定化合物对黄瓜白粉病菌的生物活性

接种黄瓜白粉菌后16 h的黄瓜叶片用透明胶带在子叶表面粘孢子取样的平均发芽率与用台盼蓝染色后在光学显微镜下调查的发芽率分别为83.7%和85.0%,t测验无显著差异,表明透明胶带取样的发芽率可以代表黄瓜叶片上白粉病菌孢子的发芽率。采用孢子萌发法和叶碟法分别测定了醚菌酯、植物源活性组分大黄素甲醚(P3D)对8个黄瓜白粉菌菌株的EC₅₀。统计结果表明:采用两种方法测定醚菌酯P、3D对黄瓜白粉菌的EC₅₀的相关系数的平方值分别为0.880、.99,表明两种方法的测定结果有很强的相关性;采用该孢子萌发法可评价化合物对植物专性寄生病菌如白粉菌孢子萌发的生物活性。     

黄瓜白粉病菌对己唑醇的敏感基线及实验室抗性获得

为了评价黄瓜白粉病菌对己唑醇的抗性风险,采用子叶法测定了山东地区50个黄瓜白粉病菌菌株对己唑醇的敏感性。结果表明,各菌株敏感性存在较大差异,EC50值最低为0.0017μg/mL,最高达0.16μg/mL,平均值为0.028μg/mL,由于所有菌株均采自未使用过脱甲基抑制剂类杀菌剂的黄瓜日光温室,敏感性频率呈近似正态分布,可作为黄瓜白粉病菌对己唑醇的相对敏感基线。以野生菌株为材料,以紫外线诱导和药剂驯化相结合获得了2个黄瓜白粉病菌对己唑醇的抗性菌株,比较了抗性菌株和敏感菌株的适合度和分生孢子的竞争能力。结果表明,无药情况下2个抗性菌株与野生菌株适合度无显著差异。抗性菌株和敏感菌株分生孢子以80∶20混合培养7代后,抗性菌株的频率变化不明显;50∶50混合培养7代,中抗菌株的抗性频率明显降低,而高抗菌株的抗性频率降低不明显;20∶80混合培养7代,抗性菌株的频率变低甚至消失。     

枯草芽孢杆菌CAB-1抑菌蛋白对黄瓜白粉病的防治作用

采用子叶喷雾法分别测定了枯草芽孢杆菌Bacillus subtilis CAB-1所产抑菌蛋白粗提物对黄瓜白粉病的保护和治疗生物活性。结果表明该抑菌蛋白粗提物能显著降低黄瓜白粉病的病情指数,其保护和治疗作用的效果分别为73.33%和76.85%;EC50分别为175μg.mL-1和125μg.mL-1。利用曲利苯兰染色法检测抑菌蛋白粗提物对黄瓜白粉菌Sphacrotheca fuliginea生长的影响,结果表明该抑菌蛋白粗提物能有效降低白粉菌分生孢子的萌发率及萌发的芽管个数,造成菌丝顶端畸形膨大呈球状并减少新生分生孢子的个数。接菌3 d后,对照的孢子萌发率为36.98%,而经抑菌蛋白粗提物处理后的孢子萌发率仅为4.8%;接菌7 d后对照每个分生孢子梗上串生孢子个数最多为7个,多数5～6个;而抑菌蛋白粗提物处理后每个分生孢子梗上串生孢子个数最多为2个,大部分为1个。     

橡胶树白粉病杀菌剂生物测定接种方法研究

对橡胶树白粉病菌的3种接种方法－抖粉法、涂抹法和喷雾法进行比较,并测定了喷雾法不同接种量对橡胶白粉病病情指数和10 mg/L三唑酮相对防效的影响。结果表明,喷雾法较抖粉法和涂抹法重复性更好,结果更稳定;在进行杀菌剂对橡胶树白粉病菌的室内盆栽毒力测定时,应采用喷雾法,接种量以5×10 4~10×104个/mL为宜。     

新型杀菌剂氯苯肟唑对蔬菜主要病原菌的抑制作用及田间防病效果

对新化合物氯苯肟唑作为农用杀菌剂的效果进行了评价。分别采用孢子萌发法、含毒介质法、离体叶片法和盆栽试验法,测试了氯苯肟唑对不同蔬菜病原真菌的抑制作用;此外,还进行了20%氯苯肟唑水乳剂防治黄瓜白粉病和黑星病的田间药效试验。室内毒力测定结果显示,氯苯肟唑对供试的几种病原菌均具有较高的杀菌活性,其中500 mg/L的处理对番茄枯萎病菌、辣椒疫病菌、黄瓜黑星病菌的活性与对照药剂相当,对菜豆菌核病菌、黄瓜灰霉病菌和黄瓜白粉病菌的抑制率虽然低于对照药剂,但抑菌效果均在80%以上。田间试验中20%氯苯肟唑水乳剂40 mg/L药液对黄瓜白粉病和黄瓜黑星病的防效可分别达80％和84％。室内生物活性测定结果与田间试验结果一致。     

黄瓜炭疽病菌对杀菌剂的敏感性的优化测定方法研究

本文以黄瓜炭疽病菌(GloeosporiumorbiculareArs)为研究对象,采用随机试验设计,比较了不同培养基、孢子萌发方法对病菌孢子萌发的影响,建立了一套测定炭疽病菌分生孢子对杀菌剂敏感性的简易方法。结果表明:不同培养基培养的分生孢子,均以使用玻片水琼胶表面萌发法的萌发效果最好,且以PDA培养基培养的分生孢子在玻片水琼胶表面萌发率最高、最稳定。此外使用该方法测定了黄瓜炭疽病菌对福美双、百菌清的敏感性。     

黄瓜炭疽病菌对杀菌剂敏感性的优化测定方法研究

本文以黄瓜炭疽病菌(Gloeosporium orbiculare Ars)为研究对象，采用随机试验设计，比较了不同培养基、孢子萌发方法对病菌孢子萌发的影响。建立了一套测定炭疽病菌分生孢子对杀菌剂敏感性的简易方法。结果表明：不同培养基培养的分生孢子。均以使用玻片水琼胶表面萌发法的萌发效果最好．且以PDA培养基培养的分生孢子在玻片水琼胶表面萌发率最高、最稳定。此外使用该方法测定了黄瓜炭疽病菌对福美双、百菌清的敏感性。     

大黄酚对黄瓜白粉病的生物活性及内吸传导性研究

采用喷雾法研究了大黄酚对黄瓜白粉病的生物活性及其在黄瓜组织中的内吸传导性。结果表明,大黄酚对黄瓜白粉病菌具有较高毒力,感染白粉病的叶片经200 mg/L的大黄酚处理2 d后,霉状物变褐色,病斑枯死。扫描电子显微镜观察显示,黄瓜白粉病菌经大黄酚处理后,菌丝和分生孢子梗扭曲、变形,分生孢子塌陷。盆栽试验表明,利用大黄酚防治黄瓜白粉病具有较长的持效期,黄瓜子叶上喷施100 mg/L的大黄酚,20 d后接种病原菌,其防效仍达84.83%,与有效成分500 mg/L的硫磺悬浮剂防效相近。此外,大黄酚在黄瓜组织中具有一定的跨层传导性,其横向传导性较弱,几乎无向顶及向基的传导性。     

8种杀菌剂对黄瓜枯萎病菌的室内毒力测定

采用菌落直径法,分别测定了7大类8种常用杀菌剂对黄瓜枯萎病菌的室内毒力。结果表明,8种供试杀菌剂对黄瓜枯萎病菌的作用效果均较好,其中三唑类丙环唑的毒力最高,EC50值为0.023mg/L。咪鲜胺、苯醚甲环唑、咯菌腈、百菌清和嘧菌酯的毒力也较高,EC50均低于1.0mg/L;多菌灵和异菌脲对黄瓜枯萎病的毒力分别为1.105和1.181mg/L,仍有较好的作用效果。     

四氟醚唑对黄瓜的安全性及其对黄瓜白粉病的防治效果

为明确四氟醚唑对黄瓜植株的安全性,采用浸种及茎叶喷雾处理的方法测定其对不同生长时期黄瓜植株的影响,采用子叶保湿培养法测定白粉病菌对其敏感性,并在田间进行防治白粉病的药效试验,综合评价了四氟醚唑对黄瓜白粉病的效果。浸种处理后,黄瓜子叶上白粉病发病率明显降低,对黄瓜株高和根长的抑制率、茎周增长率及叶绿素含量增加率与己唑醇相比均较低;发芽期及幼苗期的黄瓜植株使用四氟醚唑处理,新生节间均出现轻微的抑制伸长现象;4％四氟醚唑水乳剂对黄瓜白粉病菌的EC50为0．8146mg／L,敏感毒力高于50％醚菌酯水分散粒剂;田间间隔期10天喷雾,在末次药后7天对黄瓜白粉病的防治效果为76．02％～85．77％,与5％己唑醇水乳剂防效相当,明显高于50％醚菌酯水分散粒剂的防效。表明四氟醚唑对黄瓜生长安全且用药间隔期长,是防治白粉病的高效轮换药剂。     

A foliar spray of micronutrient solutions induces local and systemic protection against powdery mildew (Sphaerotheca fuliginia) in cucumber plants

A single spray of solutions of 0.005M H₃BO₃, 0.0025M CuSO₄, and 0.0025 MnCl₂, on the upper surface of the first true leaf of cucumber plants 2 h before inoculation with a conidial suspension of Sphaerotheca fuliginea, induced systemic protection against powdery mildew in leaves 2 and 3 without causing any damage on the induced leaf (first leaf). A similar level of systemic protection was observed when plants were induced by micronutrients, 2, 24 and 72 h before challenge with S. fuliginea. The level of protection induced by various concentrations varied from solution to solution. In general, the systemic protection induced by K₂HPO₄ was similar to that by the microelements. Spraying of a 1:1 mixture of phosphate and micronutrient solutions did not improve the systemic protection over that obtained with each of the solutions alone. Increasing the inoculum concentration of S. fuliginea increased the number of powdery mildew colonies produced on both induced and non-induced plants and has relatively affected the systemic protection on induced plants. A single foliar spray of micronutrient solutions, as a prophylactic treatment, on the upper surface of all the leaves of 3-leaf stage cucumber plants significantly inhibited powdery mildew development. A single spray of MnCl₂ on leaf 1 elevated peroxidase activity in the soluble fraction and caused an enhancement of -1,3-glucanase content in the ionically bound fractions of leaf 2 of non-inoculated plants. Forty-eight hours after inoculation, the level of both fractions of the enzymes increased in non-treated plants and decreased (-1,3-glucanase) or remained unchanged (peroxidase) in treated (induced) plants as compared to non-treated plants. The possible mechanism for this protection, and the use of microelements and phosphate solutions as inducers for systemic protection and as agents for disease control are discussed.     

Phytogeny and Cross-infectivity of Powdery Mildew Isolates (Podosphaera fuliginea s. lat.) on Cosmos and Cucumber

To clarify the relationship between the phylogeny and infectivity of isolates of Podosphaera fuliginea s. lat. (= Sphaerotheca fuliginea s. lat.) from cosmos and cucumber, more than 50 powdery mildew isolates from these two plants were subjected to nucleotide sequencing or PCR-RFLP analysis of the rDNA internal transcribed spacer (ITS) region and cross-inoculation tests. The isolates from both cosmos and cucumber are genetically monotypic, and there are six nucleotide substitutions in the rDNA ITS region between isolates from cosmos and cucumber. Cross-inoculation tests of these isolates revealed that isolates from cosmos are not pathogenic on cucumber. Although isolates from cucumber produced conidia on leaves of cosmos in the laboratory, the conidial density was much lower than that from isolates from cosmos. This result, as well as the fact that the cucumber strain was not isolated from cosmos in fields, suggests that isolates from cucumber do not infect cosmos in the field. Therefore, powdery mildews on cosmos and cucumber can be regarded to have become specialized for their hosts both genetically and pathogenically. The present study reconfirms the close relationship between phylogeny and infectivity of powdery mildew fungi. Host specialization may be a trigger that causes genetic divergence of powdery mildew fungi. Received 28 June 2000/ Accepted in revised form 4 September 2000     

The Effect of Film-Forming Polymers on Powdery Mildew of Cucumber

In pot experiments, powdery mildew of cucumber, caused bySphaerotheca fuliginea, was reduced by 64-82% by the film-forming polymer Vapor Gard and by up to 55% by Wilt Pruf. In experiments in polyethylene-covered greenhouses, no harmful effect to the crop was caused by these materials. In one experiment significant disease reductions of 19-67% and 60-96% were obtained by 3% Safe Pack and by the fungicide fenarimol (Rubigan), respectively. The combined treatment of Safe Pack with the fungicide was significantly more effective than either treatment alone, 72-78 days after planting. In another experiment, disease that appeared late in the season was reduced by up to 80, 100, 30 and 96% by Vapor Gard, Folicote, Wilt Pruf and fenarimol, respectively, and was statistically different from the nontreated control in some of the treatments and dates. Most tested substances inhibited germination ofS. fuliginea conidia and growth of its hyphae on leaves of cucumber.     

黄瓜白粉病菌在不同抗性黄瓜材料上的侵染过程

白粉病是黄瓜生产中发生普遍,危害严重的主要病害之一。pm5.1和PM5.2是黄瓜上的2个白粉病抗性位点,本文对7份不同抗病基因型的黄瓜自交系进行了黄瓜白粉病抗性鉴定,并开展了黄瓜白粉病菌侵染过程的研究,对侵染后12、24、72 h的萌发率、菌丝形成率及菌落形成率等进行了分析。结果表明,当基因型为PM5.1PM5.1 pm5.2pm5.2时,黄瓜病情指数最高,表现为高感白粉病;当基因型为PM5.1PM5.1PM5.2 PM5.2和pm5.1pm5.1pm5.2pm5.2时,表现为中感白粉病;当基因型为pm5.1pm5.1PM5.2PM5.2时黄瓜自交系病情指数最低,表现为抗白粉病。分生孢子在抗、感黄瓜自交系植株叶片上均能萌发,但只能在感病黄瓜材料上完成整个无性生长周期,产生分生孢子。此外,黄瓜白粉病菌分生孢子在感病材料上的萌发率、菌丝形成率及菌落形成率均高于抗病材料。     

Prior Inoculation with Non-pathogenic Fungi Induces Systemic Resistance to Powdery Mildew On Cucumber Plants

A spray inoculation of the first leaf of 2-leaf stage cucumber plants with a non-pathogenic isolate of Alternaria cucumarina or Cladosporium fulvum before a challenge inoculation with the pathogen Sphaerotheca fuliginea induced systemic resistance to powdery mildew on leaves 2–5. Systemic resistance was expressed by a significant (p < 0.05) reduction in the number of powdery mildew colonies produced on each leaf of the induced plants, as compared with water-sprayed plants. Systemic resistance was evident when a prior inoculation with each of the inducing fungi was administered 1, 3 or 6 days before the challenge inoculation with S. fuliginea. Increasing the inoculum concentration of A. cucumarina or C. fulvum enhanced the systemic protection and provided up to 71.6% or 80.0% reduction, respectively, in the number of colonies produced on upper leaves, relative to controls. Increasing the inoculum concentration of S. fuliginea used for challenge inoculation, increased the number of powdery mildew colonies produced on both induced and non-induced plants. Pre-treated plants, however, were still better protected than controls, indicating that the level of systemic protection was related to the S. fuliginea inoculum concentration. The induction of systemic resistance against powdery mildew by biotic agents, facilitates the development of a wide range of disease management tools.     

Persistence of resistance to fungicides in Sphaerotheca fuliginea

Isolates ofSphaerotheca fuliginea collected in 1981–1983 in cucumber glasshouses in the Netherlands were tested for their sensitivity to benzimidazole fungicides, dimethirimol, dinocap and pyrazophos.Resistance to dinocap was not observed, although this fungicide has been used for over 30 years. Resistance to benzimidazole fungicides and dimethirimol has been persistent since these fungicides were withdrawn for control of cucumber powdery mildew more than 10 years ago. Although pyrazophos has only been used incidentally after 1977, the level of resistance has not decreased.Factors possibly involved in the persistence of resistance and implications for disease control in practice are discussed.Samenvatting De gevoeligheid voor twee benzimidazool-fungiciden en dimethirimol, dinocap en pyrazofos werd getoetst vanSphaerotheca fuliginea isolaten die in 1981–1983 verzameld waren in komkommerkassen in Nederland. Resistentie tegen dinocap werd niet waargenomen, ofschoon dit fungicide al meer dan 30 jaar wordt gebruikt.De resistentie tegen benzimidazool-fungiciden en dimethirimol, die meer dan 10 jaar geleden werden teruggetrokken voor de bestrijding vanS. fuliginea, was persistent. Hoewel pyrazofos slechts incidenteel gebruikt is sinds 1977, is het resistentieniveau niet teruggelopen. Factoren die mogelijk betrokken zijn bij de persistentie van resistentie en de gevolgen voor de ziektebestrijding worden besproken.     

Seed treatment for the control of Sphaerotheca fuliginea in muskmelon seedlings

The effects of powdery mildew (caused by Sphaerotheca fuliginea )on muskmelon seedlings and the efficacy of seed treatment with systemic fungicides were examined under controlled environmental conditions. The fresh weight of seedlings infected with powdery mildew was substantially less than that of uninoculated seedlings 5 weeks after inoculation, and the inoculated seedlings shrivelled 1 week later. The fungicides fenarimol and triadimenol applied to the seeds did not affect germination and suppressed powdery mildew effectively when applied at a rate equivalent to 8 g fungicide product per kg seed. Under conditions moderately conducive to the pathogen, fenarimol markedly reduced disease severity and its effect when applied as a seed treatment was as effective as foliar treatment. When conditions were highly conducive to the pathogen, seed treatment with fenarimol was not effective. Development of powdery mildew and efficacy of the seed treatments were related to soil type: disease developed earlier and more intensely on seedlings grown in sandy soil than those in organic medium, and seed treatment had a more pronounced effect on seedlings grown in sandy soil. The results indicate that fungicidal treatment of muskmelon seeds may offer an adequate means of reducing powdery mildew infections on seedlings.     

Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants (Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.