Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml^–1 for the homologous antigen and 50 ng ml^–1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.     

青稞种子带菌检测及杀菌剂消毒处理效果

为了研究青稞种子外部和内部携带真菌情况,比较不同杀菌剂对青稞种子的带菌消毒效果和对幼苗生长的影响,为青稞种子播前包衣处理和种传真菌病害防控提供依据,采用离体平皿法对云南迪庆‘云青1号’、‘云青2号’和‘短白青稞’3个主栽品种进行带菌检测,并对种子进行拌种或浸种处理测定6种杀菌剂对种子消毒效果,分析杀菌剂对种子发芽和幼苗生长的影响。结果表明:供试青稞种子表面携带的优势菌群为青霉(Penicilliumspp.)、镰刀菌(Fusariumspp.);种子内部寄藏的真菌主要为镰刀菌、核腔菌(Pyrenophoraspp.)、附球菌(Epicoccumspp.)、丝核菌(Rhizoctoniaspp.)、链格孢(Alternariaspp.)和木霉(Trichoderma spp.)。青稞不同品种的种子表面及内部携带的真菌种类差异较大。致病性测定表明,镰刀菌对种子萌发和幼苗生长影响最大,后期出现幼苗坏死现象。45%咪鲜胺EW、75%百菌清WP、50%福美双WP对青稞种子携带真菌均有显著抑制作用和消毒效果,50%福美双WP消毒效果最优,达100%;45%咪鲜胺EW、75%百菌清WP、50%福美双WP处理对青稞种子发芽和幼苗生长均无显著影响。     

西瓜种子带菌检测及杀菌剂消毒处理效果

采用离体平皿法对来自新疆等4个省区的14个西瓜品种进行种子带菌检测、分离纯化和鉴定,并测定了7种杀菌剂和1种种衣剂对种子带菌的消毒处理效果。结果表明,种子表面携带的优势菌群主要为青霉属Penicillium spp.、根霉属Rhizopus spp.、曲霉属Aspergillus spp.、交链孢属Alternaria spp.和镰孢属Fusarium spp.;种子内部寄藏真菌主要为青霉属、根霉属和曲霉属;不同品种之间种子表面携带真菌种类差异较大,种子内部寄藏真菌种类差异不明显;种壳带菌率一般高于种仁带菌率。15% FDDF ·霜·福悬浮种衣剂、福美双和代森锰锌对种子带菌消毒效果优于多菌灵、敌磺钠、 FDDF 霉灵、拌种灵和甲霜灵。     

Application of fungicides to foliage through overhead sprinkler irrigation – a review

Articles on chemigation with fungicides targeting foliage have been reviewed. They included 23 fungicides tested on 10 crops. Many studies compared chemigation to a check treatment, while others also included conventional methods. Chlorothalonil, followed by mancozeb, fentin hydroxide and captafol were the most studied fungicides, while peanut (Arachis hypogaea), potato (Solanum tuberosum), tomato (Lycopersicon esculentum ), and dry beans (Phaseolus vulgaris) were the most studied crops. Center pivot, followed by solid set, were the irrigation systems most frequently used. The minimum volume of water applied by some center pivots (25 000 litre ha⁻¹ ) is 25 times the maximum volume of water used by conventional ground sprayers. The reduction of fungicide residue on foliage caused by the very large volume of water used by chemigation might be offset by the following factors: (1) fungicide application at the time of maximum leaf wetness when fungi are most active, (2) complete coverage of plants, (3) reducing greatly the inoculum on plant and soil surface, (4) better control of some soil pathogens, and (5) more uniform distribution of fungicides by center pivot. Furthermore, chemigation avoids mechanical damage and soil compaction. Additionally, some systemic fungicides seem to be absorbed rapidly by the leaves, by root uptake from the soil, or by both. In general, all fungicides applied through irrigation water can lessen disease severity. However, when compared to conventional methods, chemigation with fungicides can be less, equally or more effective depending on crop, pathogen, disease severity, fungicide and volume of water. For Cercosporidium personatum control on peanuts, application of protectant fungicides through irrigation water is less effective than conventional methods, but the results with some systemic fungicides mixed with non-emulsified oil and applied through a relatively low volume of water (2.5 mm) are encouraging. Important diseases of potato and tomato can be controlled nearly as well by chemigation as by conventional methods without impairing yield. The main advantage of chemigation for these crops is avoiding a large number of tractor trips through the field and reduced costs of fungicide application. Chemigation has also been shown to be a good option for control of white mold [ Sclerotinia sclerotiorum] on dry beans. © 1999 Society of Chemical Industry     

新疆食葵种子健康检测和杀菌剂消毒处理初步研究

采用PDA平板法检测了来自新疆自治区的2个品种的食葵种子带菌情况,并测定了9种不同杀菌剂对食葵种子带菌消毒处理效果和对种子发芽及苗期生长的影响。结果表明,食葵种子表面携带的优势菌群主要为镰孢霉属(Fusarium)、链格孢属(Alternaria)和青霉属(Penicillium),种子内部寄藏真菌主要有链格孢属和曲霉属(As-pergillus),其他分离频率较高的真菌还有根霉属(Rhizopus)和枝孢属(Cladosporium)等;2个食葵品种种子表面携带真菌种类有较大差异,种子内部寄藏真菌种类差异不大。杀菌剂苯醚甲环唑、咯菌腈和咪鲜胺具有良好的种子带菌消毒处理效果。     

赤豆种内寄藏真菌的分离与鉴定

采用吸水纸法和琼脂平板法,从河北省6个赤豆品种中分离和鉴定出种内寄藏真菌6属,包括5个种和1个未定名种。对常见致病菌的侵入机理进行了初步分析探讨。     

Effect of two systemic fungicides on rice blast control in a rainforest zone of Nigeria

Abstract

Two systemic fungicides, benomyl (methyl 1‐((butylamino)‐carbonyl)‐1H‐benzimidazol‐2‐yl carbamate) (Benlate 50WP, E.I. du Pont de Nemours & Co.) and tricyclazole (5‐methyl‐1,2,4‐triazole (3,4‐b) benzothiazole) (Beam 75 WP, Eli Lilly & Co.), were sprayed on Faro 29, a popular shallow swamp rice, at full tillering stage for the control of natural infection of rice blast caused by Pyricularia oryzae Cav. in the rainforest zone of eastern Nigeria. The rates evaluated for each fungicide were a split application of 150 + 150, 300, and 400ga.i./ha of each fungicide. Both fungicides suppressed foliar and neck blast development, but tricyclazole was superior to benomyl. One application of tricyclazole at 400g a.i./ha at full tillering stage of rice successfully suppressed blast development and resulted in a significantly (P = 005) higher grain yield than the untreated control plants by an average of 42.17% during the 2 years of this study. Similarly, benomyl at 400g a.i./ha produced 18.94% more rice grain than the control. A fungicide such as tricyclazole may therefore be recommended for control of blast in areas where resistant varieties are not available or where popular resistant varieties become susceptible to one or the other phases of the disease.     

大豆种子携带病原菌对8种杀菌剂的敏感性

为筛选对大豆种子携带病原菌具有良好抑制效果的杀菌剂,采用菌丝生长速率法测定了从内蒙古大豆种子内部分离的4个属和种子表面分离的8个属的病原菌对不同作用机制杀菌剂的敏感性。结果表明,3种甾醇14α脱甲基酶抑制剂咪鲜胺、戊唑醇、氯氟醚菌唑对供试病原菌均有良好的抑制作用,其中氯氟醚菌唑抑菌效果最强,对所有供试病原菌的EC₅₀值均小于3.73 μg/mL。琥珀酸脱氢酶抑制剂氟吡菌酰胺对部分供试病原菌有较好的抑菌活性;线粒体呼吸链复合物III抑制剂嘧菌酯对除链格孢属Alternaria、Boeremia属外的多数供试病原菌具有良好的抑菌活性,EC₅₀值介于0.01~5.53 μg/mL之间;解偶联抑制剂氟啶胺仅对种子内部分离的织球壳属Plectosphaella无抑菌活性,对于其他供试病原菌的EC₅₀值均小于3.39 μg/mL。多菌灵和咯菌腈对多数供试病原菌抑菌活性良好,但对于链格孢属和织球壳属病原菌无抑菌作用。综上,可推荐使用咪鲜胺、戊唑醇、氯氟醚菌唑等甾醇14α脱甲基酶抑制剂与其他药剂科学复配来进行种子处理以防治大豆种子携带病原菌引起的主要病害。     

Haplotype diversity of Tilletiopsis spp. causing white haze in apple orchards in Northern Italy

White haze (WH), a manifestation of extensive colonization of anamorphic smut fungi of the genus Tilletiopsis on the surface of apple fruit, has recently been described as a new postharvest disorder of the cultivar Elstar. This study shows that these fungi can also affect apple fruit of different cultivars prior to harvest, and therefore seriously compromise the quality of the produce in Northern Italy. In order to identify the Tilletiopsis taxa involved in the induction of WH in this region, 48 isolates, mainly derived from fruit samples, were analysed by DNA sequencing of the internal transcribed spacer region. Six haplotypes were identified that could be assigned to three different phylogenetic lineages, the T. washingtonensis group, the T. pallescens group and the Entylomataceae. More than 40% of the isolates were classified as T. washingtonensis which had not previously been associated with WH. Furthermore, this is the first study that reports the occurrence of this species on field‐grown fruit samples. The extensive overgrowth of apple fruit by Tilletiopsis spp., appearing erratically in the last decade, may be a consequence of moist weather conditions and novel cultural practices, such as the use of hail nets and the intensive application of foliar nutrients.     

鳄梨蒂腐病毛色二孢属真菌对6种杀菌剂的敏感性

由毛色二孢属 (Lasiodiplodia spp.) 真菌引起的蒂腐病是对采后鳄梨最具破坏性的病害。为明确鳄梨蒂腐病原群体对杀菌剂的敏感性及6种不同类型杀菌剂在鳄梨蒂腐病防治中的应用潜力,采用菌丝生长速率法测定了来自海南和云南省鳄梨种植区的101个蒂腐病菌菌株对6种内吸性杀菌剂的敏感性,以优势种L. pseudotheobromae建立该种群的敏感性基线。结果表明：多菌灵、甲基硫菌灵、咪鲜胺和苯醚甲环唑对蒂腐病菌菌丝生长均表现出强烈的抑制活性,其平均EC₅₀值分别为 (0.06 ± 0.04)、 (0.72 ± 0.49)、 (0.86 ± 0.98) 和 (1.25 ± 1.38) μg/mL。其中优势种L. pseudotheobromae对上述4种杀菌剂的敏感性均呈连续单峰曲线,符合正态分布,可将相应的平均EC₅₀值作为鳄梨蒂腐病菌对上述4种杀菌剂的敏感性基线。分别有4%和8%的菌株其咪鲜胺和苯醚甲环唑的平均EC₅₀ 值 大于5 μg/mL;91% 和100% 的菌株对吡唑醚菌酯和嘧菌酯敏感性很低,平均EC₅₀值分别高达 (371.03 ± 353.38) 和 (622.86 ± 771.28) μg/mL,且为非正态分布。同时,针对上述可能已产生抗药性的菌株的靶标基因进行了测序和表达量分析,qRT-PCR结果表明：供试菌株中咪鲜胺和苯醚甲环唑的靶标基因CYP51未发生任何点突变,但经药剂处理12 h后,抗性菌株CYP51基因表达量较敏感菌株显著上调,推测该基因的过表达与病原菌对咪鲜胺和苯醚甲环唑的抗药性形成有关;然而,抗性菌株吡唑醚菌酯和嘧菌酯的靶标基因Cyt b却既没有发生点突变也没有呈现过表达,因此其抗药性形成机制还有待进一步探究。研究表明,我国鳄梨蒂腐病菌对多菌灵和甲基硫菌灵非常敏感,2种杀菌剂可考虑作为鳄梨蒂腐病防治的优先候选药剂,但该病原菌对另外2种供试杀菌剂的潜在抗性也不容忽视。     

植物病原真菌肌球蛋白及其抑制剂研究进展

肌球蛋白是一类在真核生物中广泛存在的功能性蛋白,在生物体细胞内的各项生命活动过程中扮演着重要的角色。此外,来源于植物病原真菌的肌球蛋白对真菌的生长发育和致病性也有着重要影响。基于这些特性,越来越多研究者把目光投入到了以肌球蛋白为靶标的农药创制研究当中。基于植物病原真菌来源的肌球蛋白进行抑制剂设计,从而开发具有新型作用机制的杀菌剂,将是防控植物病害、保障粮食安全的重要手段。本文重点介绍了植物病原真菌来源肌球蛋白及其抑制剂的研究进展,主要综述了植物病原真菌肌球蛋白的结构特征和生化功能,以及近年来已报道的肌球蛋白抑制剂类杀菌剂,可为植物病原真菌肌球蛋白靶标导向先导化合物的发现和新农药活性分子的合理设计提供参考。     

黄瓜和葡萄霜霉病菌对不同内吸杀菌剂的交互抗药性

为了确定甲霜胺、乙磷铝与恶唑烷酮、霜脲氰、安克和硫代磷酰胺酯之间的交抗型,在叶盘上共测试了黄瓜霜霉(PC)和葡萄霜霉(PV)的27个分离系对6种内吸杀菌剂的抗药水平,结果证实了在两种目标真菌上甲霜胺和乙磷铝之间的非交抗关系,甲霜胺和恶唑烷酮之间的正交互抗药性,在PC上甲霜胺和霜脲氰之间的非交抗关系。试验还表明在PC上甲霜胺和安克之间以及乙磷铝和霜脲氰之间的非交抗关系,在PV上甲霜胺和乙磷铝与硫代磷酰胺酯之间的非交抗关系。此外,试验发现了霜脲氰和安克在叶盘上对抗甲霜胺分离系具有很高的杀菌活性。     

Enzyme-linked immunosorbent assay (ELISA) and dispersedye immuno assay (DIA): Comparison of simultaneous and separate incubation of sample and conjugate for the routine detection of lettuce mosaic virus and pea early-browning virus in seeds

Two modifications of ELISA and DIA were compared in relation to sensitivity of detection of two plant viruses and suitability for large-scale routine testing. DIA is a solid phase immuno assay like ELISA, in which the enzyme conjugate is replaced by a dye sol conjugate and substrate incubation is replaced by immediate dissolving of the dye molecules from the conjugate with an organic solvent. Sample and conjugate were incubated separately (ELISA 1, DIA 1) or simultaneously (ELISA 2, DIA 2). The seed-borne viruses viz. lettuce mosaic virus (LMV) and pea early-browning virus (PEBV) were subjected to the assays. DIA detected LMV in a purified extract ofNicotiana benthamiana. However, compounds present in crude virus-free and virus-containing plant extracts strongly interfered with DIA, necessitating adaptation of DIA to plant viruses in crude extracts.With the extracts of lettuce and pea seeds ELISA 2, in comparison with ELISA 1, resulted in equal (LMV) or slightly higher (PEBV) extinction values and in a higher ratio between extinction values of virus-containing and virus-free samples. The higher sensitivity of ELISA 2 in combination with its higher efficiency as a result of simultaneous sample and conjugate incubation, indicates the potential of this method for large-scale indexing.Samenvatting Twee modificaties van ELISA en DIA werden vergeleken met betrekking tot hun gevoeligheid voor het aantonen van twee plantevirussen en hun geschiktheid voor routinematige toepassing. DIA is een serologische toetsmethode die veel overeenkomst vertoont met ELISA, maar waarin het enzymconjugaat is vervangen door een conjugaat met gedispergeerde kleurstofdeeltjes en de incubatie met enzym-substraat door het direct oplossen van de kleurstofmoleculen van het conjugaat met een organisch oplosmiddel. Incubatie van monster en conjugaat vond zowel gescheiden (ELISA 1, DIA 1) als gelijktijdig (ELISA 2, DIA 2) plaats. Twee met zaad overgaande virussen, te weten slamozaïekvirus (LMV) en vroege-verbruiningsvirus van erwt (PEBV) werden bij het onderzoek betrokken. Met DIA kon LMV worden aangetoond in een gezuiverd extract vanNicotiana benthamiana. In ruwe planteëxtracten bleken echter stoffen voor te komen die in DIA sterke niet-specifieke reacties tot gevolg hadden. Verder onderzoek is dan ook noodzakelijk om DIA geschikt te maken voor het aantonen van plantevirussen in ruwe extracten van planten. Betere resultaten werden verkregen met de beide ELISA-modificaties. Met de extracten uit slazaad en erwtezaad gaf ELISA 2 vergelijkbare (LMV) of iets hogere (PEBV) extinctiewaarden dan ELISA 1. Bovendien was de verhouding tussen de extinctiewaarden van virusziek materiaal en die van virusvrij materiaal, bij ELISA 2 hoger dan bij ELISA 1. De grotere gevoeligheid van ELISA 2 en de grotere doelmatigheid ten gevolge van de gelijktijdige incubatie van monster en conjugaat duiden op de bijzondere geschiktheid van deze methode voor routinematige toepassing op grote schaal.     

Performance of the Pseudomonas chlororaphis biocontrol agent MA 342 against cereal seed-borne diseases in field experiments

The bacterial biocontrol agent Pseudomonas chlororaphis, strain MA 342, was tested for activity against a number of cereal seed-borne diseases in a total of 105 field experiments carried out at different locations in Sweden during the years 1991-1996. Bacterial liquid culture was directly applied to pathogen-infested seeds of barley, oats, wheat and rye without additives. The seeds were then dried and sown in field together with fungicide-treated and untreated seeds used as controls. The bacterization controlled seed-borne diseases caused by Drechslera (Pyrenophora) graminea, D. teres, D. avenae, Ustilago avenae, U. hordei, and Tilletia caries, as effectively as guazatine + imazalil, and these effects were consistent over the years and over varying climatic zones. Diseases caused by pathogens like U. nuda, soil-borne T. caries and T. contraversa were not controlled and the bacterization gave less than full effect against diseases caused by Microdochium (Fusarium) nivale, and Bipolaris sorokiniana (Cochliobolus sativus). Bacterized seeds could be stored dry for at least two years without losing the disease suppressing effect of the bacterial treatment, when tested in the field.