Evaluation of biological control agents for managing cucurbit powdery mildew on greenhouse-grown melon

An evaluation was made of the ability of two mycoparasite-based products AQ10® ( Ampelomyces quisqualis ) and Mycotal® ( Lecanicillium lecanii ), as well as three strains of Bacillus subtilis , to manage powdery mildew disease, caused by Podosphaera fusca on melon seedlings maintained under different regimes of relative humidity and on plants grown under greenhouse conditions in Spain. In every case fungal and bacterial biocontrol agents (BCAs) performed better under conditions of high relative humidity (90–95% RH). In greenhouse experiments, the effectiveness of the mycoparasites to manage powdery mildew was absolutely dependent on mineral oil. The strains of B. subtilis provided disease control similar to that achieved with the mycoparasites or the fungicide azoxystrobin. Microscopic analysis showed the ability of these bacterial strains to efficiently colonize leaf surfaces and revealed the occurrence of antagonistic interactions between biological agents and P. fusca structures. These results confirmed the usefulness of these BCAs for managing powdery mildew on greenhouse-grown cucurbits either as single products or as a component of integrated control programmes.     

Existence of different physiological forms within genetically diverse strains of <Emphasis Type="Italic">Ampelomyces quisqualis</Emphasis>

Powdery mildew fungi are parasitized by strains of the genetically distinct Ampelomyces quisqualis. To investigate whether differences in the phylogeny and other cultural, morphological and physiological characteristics of these different strains are related to differences in their geographic origins or the host species from which they were isolated, several A. quisqualis strains isolated from different species of Erysiphaceae collected in different countries and possessing different ITS rDNA sequences were selected and characterized. The results revealed some significant variation among the selected strains, which provides evidence for the existence of different physiological forms within the A. quisqualis species. Two groups that display differential growth on artificial media were identified. These groups also differ in the morphology of their mycelium, but not in the morphology of their pycnidia and conidia. Temperature greatly affected the in vitro growth of the A. quisqualis strains and growth rate was closely correlated to colony color. Differences in the conidial germination of distinct strains were observed during the recognition phase of the parasitic relationship. The germination of each of the investigated strains was greatly stimulated by all of the examined powdery mildew species and not only by the conidia of their original hosts. An Italian strain isolated from grapevine in the Trentino Alto-Adige region was identified as the strain that germinates the most quickly in the presence of powdery mildew conidia. Phylogenetic analysis revealed that these A. quisqualis strains can be classified into five different genetic groups, which generally correlate with the fungal host of origin and morphological and growth characteristics.     

Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

ABSTRACT A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.     

Overwintering of Ampelomyces mycoparasites on apple trees and other plants infected with powdery mildews

Apple shoots and aerial parts of 13 other plant species infected with powdery mildews during the previous season were collected in late winter and early spring between 1998 and 2003 at a total of 34 sample sites in Hungary. Samples were examined for the presence of overwintering structures of Ampelomyces, common mycoparasites of powdery mildews. Pycnidia and resting hyphae resembling those of Ampelomyces were found on six plant species, including apple. Their viability and subsequent mycoparasitic activity of the hyphae emerging from the overwintered fungal structures were studied in vitro to determine whether they can serve as sources of primary inocula of Ampelomyces in the spring. Overwintered pycnidia of Ampelomyces collected in the spring, and produced in both the ascomata and the conidiophores of powdery mildews during the previous season, initiated the life cycle of these mycoparasites when placed close to fresh powdery mildew colonies in vitro. Similarly, thick-walled resting hyphae, found in the dried powdery mildew mycelia which covered the overwintered aerial parts of the host plants, also germinated and gave rise to new intracellular pycnidia of Ampelomyces when powdery mildew colonies were inoculated with them in vitro. On apple trees, Ampelomyces mycoparasites overwintered as resting hyphae in the dried powdery mildew mycelia covering the shoots and in the parasitized ascomata of Podosphaera leucotricha on the bark and the scales of the buds. Approximately 31% of the field samples collected from apple trees in spring between 1998 and 2003 contained overwintered structures of Ampelomyces. Artificial bursting of apple buds in the laboratory showed that both P. leucotricha and Ampelomyces start their life cycle during or soon after bud burst, but Ampelomyces can only slowly follow the spread of its mycohost on infected leaves. Most probably, the mycoparasites did not overwinter in the dormant hyphae of P. leucotricha in the buds, but only on the bark and the bud scales, as their hyphae were not found in the young hyphae of apple powdery mildew that appeared on the leaf tissues during bud burst. This study demonstrated that Ampelomyces mycoparasites can survive the winter in the field as pycnidia and as resting hyphae in the dried mycelia of their mycohosts.     

A single nucleotide polymorphism in the β-tubulin gene distinguishing two genotypes of Erysiphe necator expressing different symptoms on grapevine

The biotrophic fungus Erysiphe necator (formerly Uncinula necator ), for which two genetic groups have been described in European vineyards, is the causal agent of grapevine powdery mildew. By analysing the pathogen population with respect to polymorphism in the sequence of the β-tubulin gene, which distinguishes two groups of isolates, a new tool was developed for epidemiological and population studies and tested in the vineyard. As in many ascomycetes, the β-tubulin gene of E. necator ( Entub ) includes six introns and seven exons and encodes a 447-amino-acid protein. A single nucleotide polymorphism (SNP) in the intron-3 region of the Entub gene distinguished two genetic groups (A and B). This method was used to examine differences in the ratio of the two groups from a total of 289 grape powdery mildew samples collected at the beginning of the growing season from either flag shoots or leaves with sparse-spot symptoms in four different vineyards. The SNP in the intron-3 region of the β-tubulin gene, similar to SNPs in the CYP51 gene, was associated with genotypes A and B of E. necator and confirmed the existence of two sympatric populations of the pathogen in the French vineyards. Differences in the relative proportions of each group varied with the presence or absence of flag-shoot symptoms and with the region in which isolates had been collected.     

Persistence and spatial autocorrelation of clones of Erysiphe necator overwintering as mycelium in dormant buds in an isolated vineyard in northern Italy

The population structure of the grape powdery mildew fungus, Erysiphe necator (formerly Uncinula necator), has been hypothesized to vary from being clonal to highly diverse and recombining. We report here on the structure of an E. necator population sampled during a 4-year period from an isolated vineyard in northern Italy (Voghera, Pavia Province). We obtained 54 isolates of E. necator that overwintered asexually as mycelium in grapevine buds and caused severe symptoms on the emerging shoots, known as flag shoots. All isolates were genotyped for mating type, four multilocus polymerase chain reaction (PCR)-based markers (a total of 64 loci were scored), and two single-copy loci designed to identify genetic subgroups in E. necator. All isolates had the same mating type and single-locus alleles that correlate to isolates from flag shoots in other areas. Only 2 of the 64 loci scored from multilocus markers were polymorphic; 46 of the 54 isolates had the same multilocus haplotype. Seven isolates had a second haplotype that was recovered over 3 years, and only a single isolate was found with a third haplotype. Both variant haplotypes differed from the main clonal haplotype by single loci. Spatial autocorrelation analyses showed that vines with flag shoots were not aggregated within years, but they were aggregated between consecutive years. These results demonstrate that this subpopulation of E. necator on flag shoots is composed of a single clonal lineage that has persisted for at least 4 years. We speculate that the lack of diversity in the flag shoot subpopulation in this vineyard is the result of restricted immigration from surrounding areas and genetic drift operating through founder effects and periodic bottlenecks. We propose a model that integrates epidemiology and population genetics to explain the variation observed in genetic structure of E. necator flag shoot subpopulations from different vineyards or viticultural regions.     

Morphological,molecular phylogenetic and pathogenic analyses of Erysiphe spp. causing powdery mildew on cashew plants in Brazil

Cashew powdery mildew is presently the most important disease of cashew trees in all Brazilian growing regions. Although it was described over a century ago, it had never threatened the Brazilian cashew industry until the first decade of the 21st century. Morphological and pathogenic evidence indicated the possibility of different pathogen species being involved in early and late types of cashew powdery mildew. This study was designed to elucidate this issue by comparing two different powdery mildew fungi occurring on cashew plants in Brazil according to the morphological characteristics, phylogenetic relationships with closely related powdery mildew fungi and pathogenic relationships. Based on morphology, molecular phylogenetics and pathogenicity on cashew, it was shown that two species of powdery mildew specimens are without question associated with cashew trees. One species, which infects young immature tissues such as shiny leaves, flowers and young fruits, is Erysiphe quercicola, while Erysiphe necator is associated exclusively with mature leaves. This is the first report of both E. quercicola and E. necator causing cashew powdery mildew, and the first detection of E. necator on cashew.     

Effects of Diffuse Colonization of Grape Berries by Uncinula necator on Bunch Rots, Berry Microflora, and Juice and Wine Quality

ABSTRACT Production of grape (principally cultivars of Vitis vinifera) for high-quality wines requires a high level of suppression of powdery mildew (Uncinula necator syn. Erysiphe necator). Severe infection of either fruit or foliage has well-documented and deleterious effects upon crop and wine quality. We found that berries nearly immune to infection by U. necator due to the development of ontogenic resistance may still support diffuse and inconspicuous mildew colonies when inoculated approximately 3 weeks post-bloom. Fruit with diffuse mildew colonies appear to be healthy and free of powdery mildew in late-season vineyard assessments with the naked eye. Nonetheless, presence of these colonies on berries was associated with (i) elevated populations of spoilage microorganisms; (ii) increased evolution of volatile ethyl acetate, acetic acid, and ethanol; (iii) increased infestation by insects known to be attracted to the aforementioned volatiles; (iv) increased rotting by Botrytis cinerea; and (v) increased frequency of perceived defects in wines prepared from fruit supporting diffuse powdery mildew colonies. Prevention of diffuse infection requires extending fungicidal protection until fruit are fully resistant to infection. Despite a perceived lack of improvement in disease control due to the insidious nature of diffuse powdery mildew, potential deleterious effects upon crop and wine quality thereby would be avoided.     

Glycolytic activities in some fungicolous fungi

Some fungal species isolated from other fungi were tested for their capacity to degrade chitin, cellulose and laminarin. Only three entomophagous fungi degraded native chitin. However colloidal chitin was degraded by all fungi tested except four. Cellulolytic activity was mainly found in fungi with a poor performance against cucumber powdery mildew. This suggests that the capacity to produce cellulase is not important for successful mycoparasitic activity against cucumber powdery mildew. A good correlation was found between the capacity to degrade laminarin and the mycoparasitic activity of the fungi that were investigated.     

河南省西部山区小麦白粉菌群体遗传多样性分析

为揭示河南省小麦白粉菌群体遗传结构、起源及进化关系,采用简单重复序列区间(inter-simple sequence repeats,ISSR)和扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记技术对河南省西部山区4个小麦产区的35个小麦白粉菌单孢分离菌株进行了群体遗传多样性分析。结果显示:ISSR和AFLP分析均将35个菌株分为3个组,组Ⅰ包括来自卢氏和灵宝的大部分菌株;组Ⅱ包括来自栾川、卢氏和巩义的菌株;组Ⅲ由4个地区的个别菌株组成,同时包含1个闭囊壳释放子囊孢子获得的菌株。ISSR分析出菌株遗传距离分布在0.0139～0.6592之间,扩增多态性比率为64.83%,各菌株间的Shannon指数为0.2749;而AFLP分析所得的各菌株遗传距离变化幅度在0.1257～0.9322之间,扩增多态性比率为82.68%,各菌株间的Shannon指数为0.5100。可见,河南省小麦白粉菌具有丰富的遗传多样性,研究所用的2种方法均可用于遗传多样性分析,其中AFLP分析小麦白粉菌群体表现出更为丰富的遗传多样性。     

白粉寄生孢寄生黄瓜白粉菌的特性研究

采用白粉寄生孢（Ampelomyces quisqualis Ces.AQ）接种离体条件下赛璐酚上黄瓜白粉菌［Sphaerotheca fuliginea (Schlecht)Poll.］和活体条件下黄瓜白粉菌后,通过棉兰染色和显微观察分析,初步明确了白粉寄生孢的侵染寄生过程。白粉寄生孢分生孢子产生芽管可入侵黄瓜白粉菌的分生孢子、菌丝、分生孢子梗;有时黄瓜白粉菌串生的分生孢子、分生孢子梗可被2~3条白粉寄生孢的菌丝寄生,随着寄生过程的进一步发展,黄瓜白粉菌的分生孢子梗基部膨大成无色的椭圆形或球形,其上逐渐产生由黄色至褐色的白粉寄生孢的分生孢子器,器内含有大量的分生孢子。     

Analysis of population structure of Erysiphe necator using AFLP markers

An analysis of the population structure of Erysiphe necator from Spain was carried out to determine if population structure of grapevine powdery mildew is partitioned by origin of primary inoculum, i.e. overwintering ascomata on grapevine bark and mycelia in dormant buds. Ten of the 31 isolates collected were from flag shoots, i.e. shoots covered with sporulating mycelia that arise from dormant infected buds. Genetic variation was assessed using 61 AFLP markers. Cluster analysis revealed two distinct groups with 63% genetic similarity. Principal coordinates analysis ( pccorda ) produced a similar distribution of isolates in two groups. Isolates were not well identified within their groups by the origin of primary inoculum. Analysis of molecular variance ( amova ) determined that the origin of primary inoculum was not a significant component in the genetic variation in the population of E. necator . It was concluded that these two subpopulations are not separated by the type of primary source of inoculum.     

小麦白粉病菌基因组DNA的微量提取及ISSR-PCR反应体系的优化

为了寻找适合小麦白粉菌基因组DNA微量提取的方法,本试验利用改进的CTAB法、电钻微量研磨一管法、FastPrep DNA试剂盒法和Chelex-100法分别提取小麦白粉菌基因组DNA。比较得出,在微量提取时,CTAB法的提取率较高;在分生孢子量较多时,FastPrep DNA试剂盒方法提取的DNA质量较高,这两种方法提取的DNA均适用于ISSR-PCR。采用正交设计L16(45)法优化了适合于小麦白粉病菌群体的ISSR体系,确定了25μL时优化的反应体系:1×buffer、模板DNA0.5ng、dNTP0.14mmol/L、TaqDNA聚合酶1U、引物1.4μmol/L、Mg2+1.8mmol/L。利用该体系筛选出了一批多态性较好的ISSR引物,为小麦白粉病菌遗传多样性研究奠定了基础。     

Microcyclic conidiogenesis in powdery mildews and its association with intracellular parasitism by <Emphasis Type="Italic">Ampelomyces</Emphasis>

Microcyclic conidiogenesis (MC), a process defined as the production of conidia on a spore without any, or only a minimal, involvement of hyphal growth, has recently been reported in a little known powdery mildew species, Oidium longipes. To investigate whether this was an isolated case or it is a more general phenomenon in powdery mildew fungi, germinating conidia of eight species of the Erysiphales were examined using light microscopy. The following species were included in this work: Erysiphe necator on grapevine, Blumeria graminis f. sp. hordei on barley, Podosphaera xanthii on cucumber, Erysiphe sp. on Ligustrum vulgare, O. longipes on Petunia x grandiflora, O. neolycopersici on tomato, Golovinomyces cichoracearum on Rudbeckia laciniata and Sawadaea sp. on Acer negundo. In all these species, up to 4% of the germinated conidia exhibited MC. Moreover, when colonies of E. necator and O. neolycopersici, on detached grapevine and tomato leaves, respectively, were treated with a conidial suspension of Ampelomyces, the intracellular pycnidia of these mycoparasites appeared in microcyclic conidiophores. This represents a yet undescribed method of accelerating asexual reproduction in this mycoparasite. In the life cycle of powdery mildews, the importance of MC is still not clear but it should be taken into consideration when conidial germination is studied on the host surface for purposes such as epidemiology or species identification.     

What types of powdery mildew can infect wheat-barley introgression lines?

This work is a detailed study of the infection of fungal biotrophic pathogens causing powdery mildew diseases on introgression lines originating from the intergeneric hybridisation between wheat and barley (Triticum aestivum L. × Hordeum vulgare L.). Powdery mildew fungi are among the most widespread biotrophic pathogens of plants also and infect dicot and monocot species. Most powdery mildew species are strictly host specific. They colonize only a narrow range of species or one particular host species. The intergeneric hybridisation between wheat and barley could result in expansions of host ranges of the barley powdery mildew. Our experiments covered natural infections in the field and artificial infections under greenhouse conditions. Formae speciales of powdery mildew were identified on the basis of the sequencing results of ribosomal internal transcribed spacer sequences (rDNA-ITS). We identified Blumeria graminis f.sp. tritici isolate 14 (HM484334) on the wheat parent and all wheat-barley introgression lines and B. g. f. sp. hordei isolate MUMH1723 (AB 273556) on the barley parent, respectively. The wheat-barley introgression lines were inoculated with barley powdery mildew under greenhouse conditions. According to our results the added barley chromosomes (or segments) do not cause host range expansion of barley powdery mildew.     

Paecilomyces farinosus destroys powdery mildew colonies in detached leaf cultures but not on whole plants

Since 2001, several isolates of Blumeria graminis, the causal agent of cereal powdery mildew, maintained on detached leaves at the John Innes Centre, Norwich, UK, have spontaneously become infected with an unknown filamentous fungus whose mycelia have quickly overgrown the powdery mildew colonies and destroyed them completely. A total of five isolates of the contaminant were obtained and identified as Paecilomyces farinosus based on morphological characteristics and rDNA ITS sequence data. To determine whether these P. farinosus isolates can be considered as biocontrol agents (BCAs) of powdery mildews, we studied the interactions between P. farinosus and the following four powdery mildew species: B. graminis f.sp. hordei infecting barley, Oidium neolycopersici infecting tomato, Golovinomyces orontii infecting tobacco and Podosphaera fusca infecting cucumber. The powdery mildew colonies of all these four powdery mildew species were quickly destroyed by P. farinosus in leaf cultures but neither conidial suspensions nor cell-free culture filtrates of P. farinosus isolates could suppress the spread of powdery mildew infections on diseased barley, tomato, tobacco or cucumber plants in the greenhouse. It is concluded that P. farinosus cannot be considered as a promising BCA of powdery mildew infections although it can destroy powdery mildew colonies in detached leaf cultures and can be a menace during the maintenance of such cultures of cereal, apple, cucurbit and tomato powdery mildew isolates.     

Effect of seventeen fungicolous fungi on sporulation of cucumber powdery mildew

Nineteen isolates of 17 different fungal species thriving upon other fungi were tested for their ability to control sporulation of cucumber powdery mildew,Sphaerotheca fuliginea. More than half of the fungi reduced the number of healthy conidiophores to less than 10%.Tilletiopsis albescens was superior toAmpelomyces quisqualis. Three species, viz.A. quisqualis, Aphanocladium album andT. albescens, were selected for further greenhouse experiments.Samenvatting Negentien isolaten behorend tot 17 verschillende schimmelsoorten werden getoetst op hun bruikbaarheid voor de biologische bestrijding van komkommermeeldauw aan de hand van effecten op sporulatie. Meer dan de helft van de getoetete isolaten reduceerde het aantal gezonde conidioforen tot minder dan 10%. De werking vanTilletiopsis albescens was enigszins beter dan die van de meestal gebruikte hyperparasietAmpelomyces quisqualis. Van de getoetste schimmels werden er drie, n.l.A. quisqualis, Aphanocladium album enT. albescens, geselecteerd voor kasproeven.     

解淀粉芽孢杆菌LJ1诱导黄瓜抗白粉病的研究

解淀粉芽孢杆菌LJ1是从土壤中分离得到的一株对黄瓜白粉病具有较好防效的生防细菌。田间试验发现,用LJ1发酵上清100倍稀释液喷施黄瓜幼苗,在施药后14 d时其对黄瓜白粉病的防效可达83.45%。为研究LJ1防治病害的作用机制,用LJ1发酵上清100倍稀释液喷施黄瓜幼苗,测定黄瓜叶片中的超氧化物歧化酶(SOD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)等与诱导抗病性相关的酶活性和信号分子水杨酸含量的变化,并检测了苗期根围土壤中真菌的动态。结果显示,经过LJ1发酵液处理后3种酶的活性和水杨酸的含量在不同时间点均有一个骤增的过程,其活性显著高于对照,并且7 d后土壤中的可培养真菌数量急剧减少。说明LJ1发酵液中有诱导黄瓜产生抗病性的物质,并且诱导后分泌的抗性物质对真菌具有广谱性。