Transfer from Erwinia herbicola to Escherichia coli of a plasmid associated with biocontrol of fire blight

Strains of Erwinia herbicola effective in the biocontrol of fire blight of hawthorn were used to investigate the possibility that the antagonistic activity is coded by plasmid-born genes. Agarose gel electrophoresis of isolated plasmids from four antagonistic Erw. herbicola strains showed a band of a supercoiled 12 kb plasmid in each strain, with a second band greater than 16.2 kb consistently seen in two strains. Erw. herbicola strains showed resistance to penicillin-G, which could be conferred on penicillin-G sensitive Escherichia coli TG1 by transformation with a pure Erw. herbicola plasmid preparation. Transformed strains of Esc. coli appeared to contain the Erw. herbicola 12 kb plasmid, but not the > 16.2 kb plasmid. In an agar plate assay, Esc. coli transformants produced an inhibition zone against Erw. amylovora similar to those produced by the original Erw. herbicola strains. In two biocontrol assays, the transformed Esc. coli strains had a suppressive effect on disease development on infected pear fruit slices and hawthorn blossoms.     

Biological control of fire blight of apple and pear with antagonistic Lactobacillus plantarum

Lactic acid bacteria (LAB) can be a source of biological control agents (BCA) of fire blight disease. Several species of LAB are inhabitants of plants and are currently used as biopreservatives of food because of their antagonistic properties against bacteria, and are considered as generally safe. Candidates to BCA were selected from a large collection of LAB strains obtained from plant environments. Strains were first chosen based on the consistency of the suppressive effect against E. amylovora infections in detached plant organs (flowers, fruits and leaves). Lactobacillus plantarum strains PC40, PM411, TC54 and TC92 were effective against E. amylovora in most of the experiments performed. Besides, strains PM411, TC54 and TC92 had strong antagonistic activity against E. amylovora and also other target bacteria, and presented genes involved in plantaricin biosynthesis (plnJ, plnK, plnL, plnR and plnEF). The strains efficiently colonized pear and apple flowers; they maintained stable populations for at least 1 week under high RH conditions, and survived at low RH conditions. They were effective in preventing fire blight on pear flowers, fruits and leaves, as well as in whole plants and in a semi-field blossom assay. The present study confirms the potential of certain strains of L. plantarum to be used as active ingredient of microbial biopesticides for fire blight control that could be eventually extended to other plant bacterial diseases.     

Biological control of peach twig blight (Monilinia laxa) with Penicillium frequentans

In 1986 and 1988 various preparations of spores and or mycelium of the antagonist Penicillium frequentans , alone or in alternation with captan were applied to field-grown peach trees inoculated with Monilinia laxa , the cause of twig blight. Some preparations of P. frequentans containing nutrients gave significant reductions in severity of disease (from 38 to 80%) over control, comparable to that given by captan. Combinations of P. frequentans and captan gave a level of control similar to or less than that given by the antagonist or chemical alone. Populations of P. frequentans on shoots were monitored after application in 1986. When applied with nutrients, levels of P. frequentans on shoots remained higher than when the fungus was applied in the absence of nutrients. In a glasshouse experiment the effect of P. frequentans treatments on twig blight was influenced by the type and amount of M. laxa inoculum applied.     

Biological control of peach twig blight (Monilinia laxa) with Epicoccum nigrum

Various preparations of spores and mycelium of the antagonist Epicoccum nigrum , alone or in combination with Captan, were applied in four different field trials to peach trees inoculated with Monilinia laxa , the cause of twig blight. Biocontrol obtained after application of E. nigrum was variable each year, depending on the releative disease severity in the first 2–3 weeks after infection and the climatic conditions. In three out of four trials, treatments with E. nigrum were comparable with Captan in reducing disease severity and combinations of E. nigrum and Captan gave a level of disease suppression similar to that given by the antagonist or chemical alone. In the remaining trial, combinations of E. nigrum and Captan were necessary to obtain successful disease suppression.     

The various means of dissemination of the fire blight bacterium Erwinia amylovora

A brief review is presented of the means of dissemination of Erwinia amylovora. The bacterium exists in the form of ooze, strands, epiphytic populations and endophytic populations. The status of endophytic bacteria in budwood and fruits is discussed. International dissemination of fire blight has taken place by wind, probably by birds, and by movement of budwood. It has been suggested that fruits may be involved, but there is international disagreement whether measures should be taken to protect against this pathway of dissemination.     

Effectiveness of preventing flowering of hawthorn in protecting pear orchards from fire blight infection

Since 1984 when a new Ministerial Regulation on fire blight came into force, there have been 20 protected regions in the Netherlands, where nurseries of rosaceous plants, and pear and apple orchards are extra protected against fire blight. This policy is also necessary to meet the requirements of the European Community (EC) on fire blight. Two of the measures in the protected regions are the prohibition of flowering of the native hawthorn (Crataegus monogyna andC. laevigata), and destruction of blighted plants. In the unprotected regions, flowering is allowed, and destruction of blighted plants is limited to a zone of 500 metres around orchards.For three years, the effectiveness of preventing flowering of hawthorn in protecting pear orchards against fire blight infection was studied in the field. Five test areas of about 3 km × 3 km were chosen with hawthorns and pear orchards. Two of these areas were in protected regions and three in unprotected regions. The more than 50 000 hawthorns in the areas were grouped into 1125 sites of hawthorn. The 126 orchards larger than 0.2 ha contained about 180 000 trees.During the three years light to moderate epidemics of fire blight were observed in the regions. Fire blight occurred in 2.3% of the non-flowering sites and 19.8% of the flowering (or fruiting) sites at least once in 1987, 1988 or 1989. The prohibition of flowering for hawthorn in protected areas was rather well implemented, so that in protected areas a smaller proportion of sites of hawthorns had fire blight (4.1%) than in unprotected areas (14%). Moreover, there were fewer sites per square kilometre in the protected areas (13) than in the unprotected areas (26).In protected areas, 53% and in unprotected areas 59% of the pear orchards had fire blight during 1987, 1988 or 1989. The difference was not significant. The first reason for the ineffectiveness of the preventing of flowering prevention in hawthorn to control fire blight in pear orchards was the inadequate hygiene of the pear orchards in both types of region. If it be assumed that a new focus is most probably initiated by the nearest existing focus, the second reason was that fire blight hardly spread from hawthorn to pear in the period of this study. Spread of fire blight within pear orchards and between pear orchards occurred frequently.     

Stem injection of prohexadione carboxylic acid to protect blossoms of apple trees from fire blight infection (Erwinia amylovora)

Journal of Plant Diseases and Protection - Stem injection of prohexadione carboxylic acid (pca) was tested to prevent blossom infection by the fire blight pathogen Erwinia amylovora on apple trees,...     

Real‐time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence

Fire blight is the most devastating bacterial disease of rosaceous plants. Forecasting fire blight infections is important to allow for countermeasures that reduce economic damage in pome fruit production. Current computerized forecasting models are solely based on physical factors such as temperature and moisture, but not on the actual presence of the pathogen Erwinia amylovora. Although the inoculum concentration is considered to be crucial for infection and disease outbreak, most current approaches used for identification of fire blight inoculum including morphological, biochemical, serological, and DNA‐based methods are nonquantitative. Based on a real‐time PCR approach previously published, an improved protocol to be used directly on whole bacteria in the field is described. The method allows for early detection and quantification of the pathogen prior to the occurrence of first symptoms. There is a clear correlation between bacterial abundance and subsequent disease development. However, in some cases, no disease symptoms could be observed despite a pathogen load of up to 3·4 × 10⁶ cells per blossom. Integration of the amount of pathogen detected into refined prediction algorithms may allow for the improvement of applied forecasting models, finally permitting a better abatement of fire blight.     

梨火疫病菌对噻唑锌及其他几种杀细菌剂的敏感性

噻唑锌是近期在我国登记为防治梨火疫病的新型杀菌剂。为明确梨火疫病菌Erwinia amylovora对噻唑锌及其他几种杀细菌剂的敏感性现状,本研究选用2016年-2020年从新疆不同地区分离的50个E. amylovora 代表菌株,通过药剂-病原菌共培养结合平板菌落法测定E. amylovora 对噻唑锌的敏感性,比较不同来源菌株的敏感性差异,并分析E. amylovora 对噻唑锌与农用链霉素、噻霉酮和春雷霉素的敏感性差异。结果表明,来自新疆不同地区、不同寄主和不同年份的E. amylovora 菌株对噻唑锌的敏感性差异明显,EC50分布在2.67~38.13 μg/mL, 平均（18.60±6.35） μg/mL。阿克苏地区阿瓦提县的杜梨分离菌株H10最敏感,EC50为2.67 μg/mL,巴州轮台县的山楂分离菌株Y126的敏感性最低,EC50为38.13 μg/mL,二者的EC50相差13.28倍。供试E. amylovora 菌株对噻唑锌的敏感性频率分布呈连续的单峰曲线,符合正态分布,将EC50平均值(18.60±6.35)μg/mL确定为新疆梨火疫病菌对噻唑锌的敏感基线,适用于新疆梨火疫病菌对噻唑锌的抗药性检测。供试E. amylovora 菌株对噻唑锌的抗性水平为0.13~2.05 (均值为1.00±0.35),均为敏感菌株,未发现抗药性菌株。相关性分析表明,E. amylovora 对噻唑锌的敏感性与农用链霉素（r=0.138,P=1.56）、噻霉酮(r=0.417,P=0.78)和春雷霉素(r=0.434,P=0.65)的敏感性之间不存在相关性(P>0.05)。本研究结果为后期评估梨火疫病菌对噻唑锌的抗药性风险奠定了基础,为制定梨火疫病的合理用药策略提供了依据。     

梨火疫病菌的RPA快速检测技术的研发

梨火疫病菌Erwinia amylovora是我国进境植物检疫性有害生物, 主要侵染梨、苹果、山楂、海棠等多种蔷薇科植物引起梨火疫病。该病自1780年在美国被发现以来, 已经传播到世界多个国家, 对我国的果树生产也构成一定的威胁。严格植物检疫是全球防控梨火疫病菌扩散危害的重要方法之一, 目前我国植物检疫部门主要从实验室分离鉴定、分子检测和田间症状识别及快速检测方面开展工作。本研究基于重组酶聚合酶扩增(RPA)技术和E.amylovora 16S-23S ITS区间的保守序列设计的引物和探针, 建立了实时荧光RPA和侧流层析试纸条RPA两种检测方法。经测试, 在39℃条件下, 这两种检测方法都具有良好的特异性, 分别能在20 min和10 min内完成扩增。实时荧光RPA对E. amylovora菌株DNA的检测阈值为1.38×10-2 ng/μL;侧流层析试纸条RPA对E. amylovora菌悬液和DNA的检测阈值分别为104 cfu/mL和1.38×10-3 ng/μL。侧流层析试纸条RPA体系因反应快速、读取结果方便、不需要复杂仪器操作可以满足现场快速检测的需求。     

Biological control of cucumber powdery mildew with Tilletiopsis minor under greenhouse conditions

In growth cabinet and greenhouse experiments the efficacy ofTilletiopsis minor in controlling cucumber powdery mildew decreased as humidity was lowered. This effect could be counteracted by formulation of the mycoparasite with lipophilic substances, like Hora Oleo 11E and lipids from milk. These formulations, without the mycoparasite, were also found to be deleterious to powdery mildew development. In the long run, however, formulations withT. minor gave better biological control of cucumber powdery mildew than formulations alone.     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Notes on the role of water potential in the pathogenesis of fire blight,caused by Erwinia amylovora

The rate of multiplication of fire blight causing bacteriumErwinia amylovora (Burrill) Winslow et al. depends on the availability of water. Water availability can be quantified by means of the parameter water potential. The relationship between water potential and relative multiplication rate ofE. amylovora was derived from experiments of L. Shaw (1935). This relationship appears to be applicable toE. amylovora in plant tissues and in nectar of flowers.Multiplication and expansion ofE. amylovora in a restricted space, e.g. an intercellular hole, creates a pressure, which may cause schizogenic cavities in soft tissue. Strong tissue, however, may be able to resist this multiplication pressure of the bacteria, so that symptom progression can be prevented. A hypothesis is formulated on how the multiplication pressure may be quantified by means of the parameter water potential. Expansion of bacterial ooze may alo be due to absorbtion of water without increase of dry weight (e.g. a daily cycle of shrinkage and expansion). This expansion may give rise to a swelling pressure, which again may be quantified by means of the parameter water potential.Samenvatting De vermenigvuldigingssnelheid van de bacterie die bacterievuur veroorzaakt (Erwinia amylovora (Burrill) Winslow et al.,), hangt af van de beschikbaarheid van water. De beschikbaarheid van water kan worden gekwantificeerd met de parameter waterpotentiaal. De relatie tussen waterpotentiaal en relatieve vermenigvuldigingssnelheid vanE. amylovora werd afgeleid uit experimenten van L. Shaw (1935, Cornell University Agricultural Experiment Station, Ithaca. Memoir 181). Deze relatie kan zowel worden toegepast op de pathogenese in planteweefsel als op de epifytische ontwikkeling in nektar.Vermenigvuldiging vanE. amylovora in een beperkte ruimte, bijvoorbeeld in een intercellulaire holte, creëert een druk, die tot scheuren van zacht weefsel kan leiden. Sterk weefsel kan de vermenigvuldigingsdruk van de bacteriën vermoedelijk wel weerstaan, zodat uitbreiding wordt verhinderd. In een hypothese wordt beschreven hoe de vermenigvuldigingsdruk zou kunnen worden gekwantificeerd met behulp van de parameter waterpotentiaal. Wateropname door bacterieslijm zonder toename van het drooggewicht (bijvoorbeeld een dagelijkse gang van krimpen en zwellen) kan ook leiden tot een druk. Deze druk kan eveneens worden berekend met de parameter waterpotentiaal.     

The undiscovered potential of dehydroproline as a fire blight control option

The non‐protein amino acid 3,4‐dehydro‐l ‐proline (DHP) significantly reduced the incidence of fire blight infection on immature pear fruits infected with wildtype Erwinia amylovora. DHP also inhibited biofilm formation in both streptomycin‐sensitive and ‐resistant strains of E. amylovora and induced dispersal of preformed biofilms in the streptomycin‐sensitive strain. The investigations shed light on the hitherto undiscovered ability of DHP to inhibit bacterial biofilms and its potential as a chemical control option for fire blight.     

内生芽孢杆菌TB2防治辣椒疫病效果及其机理初探

 内生芽孢杆菌(Bacillus sp.) TB2对辣椒疫霉(Phytophthora capsici)引起的辣椒苗和果疫病有良好的防治效果,菌液浸种处理的苗接种病菌后20 d防病效果可达70.0%;菌液浇灌后48 h的苗接种病菌后20 d防病效果可达88.1%;菌液喷雾处理的果接种病菌后14 d防病效果可达65.2%。喷雾或浇灌菌株培养液后间隔24 h以上接种病原菌的防病效果比二者同时接种时高。生防菌菌体及其胞外分泌物均有防病作用,其胞外分泌物的速效性较好,菌体的持效性较好。菌株胞外分泌物对病菌菌丝生长、孢子囊形成和游动孢子释放均有明显的抑制作用。菌液可诱导植物抗性相关酶的变化,但其与病菌共同接种于辣椒时诱导作用可相互影响。疫霉菌处理可使辣椒果的丙二醛(MDA)含量和过氧化物酶(POD)、超氧化歧化酶(SOD)、过氧化氢酶(CAT)活性出现明显的峰值;菌液处理可诱导辣椒SOD和POD活性增强;但菌液与病菌共同处理后,辣椒果的MDA含量和SOD、POD、CAT的活性均较低,与清水对照接近,表现出一种相对稳定的状态。     

Heat treatment of plant propagation material for the control of fire blight

The thermal sensitivity of Erwinia amylovora was determined at 45° C and 50°C. In vitro assays with eight differetit strains showed that thermal death times did not exceed 70min at 45° C and 50min at 50° C (95% confidence interval). Heat treatments of naturally infected apple and pear shoots were performed using a moist and a dry heat procedure. When shoots were wrapped in wet cotton cloths (wet heat) and maintained in an incubator, no bacterial growth could be detected after an incubation of 5 h at 45° C. When shoots were sealed in polyethylene bags and immersed in water (dry heat), no Erwinia amytovora could be isolated after an immersion of 3 h at 45° C. Incubation at 50°C for 1 –2h in either condition did not eradicate the pathogen entirely. The failure rate of grafts using budwoods treated at the effective time-temperature combinations did not exceed 25%.
The present investigations showed that the use of thermotherapy techniques for controlling Erwinia amylovora in propagation material of apple and pear might be possible.     

Biological control of fusarium seedling blight disease of wheat and barley

ABSTRACT Fusarium fungi, including F. culmorum, cause seedling blight, foot rot, and head blight diseases of cereals, resulting in yield loss. In a screen for potential disease control organisms and agents, Pseudomonas fluorescens strains MKB 100 and MKB 249, P. frederiksbergensis strain 202, Pseudomonas sp. strain MKB 158, and chitosan all significantly reduced the extent of both wheat coleoptile growth retardation and wheat and barley seedling blight caused by F. culmorum (by 53 to 91%). Trichodiene synthase is a Fusarium enzyme necessary for trichothecene mycotoxin biosynthesis; expression of the gene encoding this enzyme in wheat was 33% lower in stem base tissue coinoculated with Pseudomonas sp. strain MKB 158 and F. culmorum than in wheat treated with bacterial culture medium and F. culmorum. When wheat and barley were grown in soil amended with either chitosan, P. fluorescens strain MKB 249, Pseudomonas sp. strain MKB 158, or culture filtrates of these bacteria, the level of disease symptoms on F. culmorum-inoculated stem base tissue (at 12 days post- F. culmorum inoculation) was >/=31% less than the level on F. culmorum-inoculated plants grown in culture medium-amended soil. It seems likely that at least part of the biocontrol activity of these bacteria and chitosan may be due to the induction of systemic disease resistance in host plants. Also, in coinoculation studies, Pseudomonas sp. strain MKB 158 induced the expression of a wheat class III plant peroxidase gene (a pathogenesis-related gene).