栗疫病菌侵染板栗枝条的显微观察

栗疫病是一种严重危害栗属植物的病害。为了明确栗疫病菌侵染板栗枝条的过程及侵染的关键时间点,本研究利用病理组织切片技术、显微镜和扫描电镜技术对栗疫病菌侵染板栗枝条的过程进行了观察。结果表明:接种栗疫病菌后0～5 h,菌丝先降解枝条表皮,进行横向营养生长的同时沿着伤口纵向侵染,为进入皮层做准备;接种后6 h病菌开始在表皮定殖,并侵入皮层;接种后9 h在皮层可观察到侵染性菌丝沿着细胞间隙向相邻细胞延伸;接种后12 h栗疫病菌侵入韧皮部,在皮层的侵染面积扩大。随着侵染程度加深,皮层、韧皮部等处细胞被菌丝降解,最终在形成层附近聚集。接菌后9 h为栗疫病菌侵染板栗枝条的关键时间点。     

Invasion genetics of the chestnut blight fungus Cryphonectria parasitica in Switzerland

Cryphonectria parasitica is the best-known example of an invasive forest pathogen in Europe. In southern Switzerland, chestnut blight was first reported in 1948 whereas, north of the Alps, it did not appear until the 1980s. Between 1995 and 2008, we sampled 640 C. parasitica isolates from nine populations south of the Alps and nine north of the Alps. Twelve historical isolates, collected between 1950 and 1972 in the south, were obtained from our collection. All 652 isolates were screened at 10 microsatellite loci to test for the existence of divergent genetic pools and to infer possible origins of haplotypes. In total, 52 haplotypes were identified. Structure software analysis indicated that 43 haplotypes (including all historical haplotypes) belonged to a main cluster, 6 haplotypes belonged to a different cluster, and 3 haplotypes had an intermediate allele pattern. All newly founded populations in northern Switzerland were initiated by one or just a few haplotypes from the main cluster, which probably came directly from the populations south of the Alps. Subsequently, genetic diversity increased through mutations, sexual reproduction, or new migrations. The highest increase in diversity was observed in populations where haplotypes from different genetic pools were encountered.     

板栗疫病菌脱毒方法的比较研究

 病毒与宿主相互作用中,存在宿主主动或被动摆脱病毒的机制。本研究用光照诱导-单孢分离、原生质体再生和菌丝尖端分离3种方法对携带低毒病毒的板栗疫病菌(Cryphonectria parasitica)的EP721、EP713和Euro7三个菌株的脱毒效率进行了比较。结果表明:经光照诱导-单孢分离后所有菌株均可获得脱毒菌株,而原生质体再生对EP721的脱毒效率最高,是继光照诱导-单孢分离脱毒法之后另一种新的有效的脱毒方法,特别适用于不产孢的真菌脱毒。     

Seasonal patterns of dispersal of ascospores of Cryphonectria parasitica (chestnut blight)

Infected barks of chestnut blight cankers, caused by Cryphonectria parasitica , were collected from a naturally infected orchard and incubated at different temperatures. Cankers started to discharge ascospores about a week after incubation at 15–25°C; most ascospores were collected at 20 and 25°C. When incubated at 5, 10 or 30°C, only a few cankers released a small number of ascospores and only during the later stages of incubation. However, the rate of formation of perithecia was not affected by the incubation temperature. The number of airborne ascospores was monitored using a volumetric spore trap in a chestnut orchard during 1996 and 1997. In both years, the number of ascospores trapped daily varied greatly, but in general it increased sharply from March onwards, reached a peak in May, and then declined steeply. There was a significant correlation between daily counts of ascospores and air temperature. Time-series transfer function (TF) analysis showed a positive association of the daily number of ascospores with increasing temperature, rain events and wet/humid conditions. In general, values predicted by the TF model agreed well with the observed pattern. However, a multiple regression equation based on TF analysis failed to provide a satisfactory prediction of the daily number of ascospores.     

Genetic diversity of Cryphonectria parasitica causing chestnut blight in South Tyrol (northern Italy)

European Journal of Plant Pathology - European chestnut (Castanea sativa) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus...     

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real-time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV-1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV-1 was found in seven isolates from three different locations and was always subtype-I, which has limited hypovirulence. Therefore, although CHV-1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV-1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade.     

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.     

Characterization of mutants of the chestnut blight fungus (<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>) with unusual hypovirus symptoms

The type virus of the family Hypoviridae, Cryphonectria hypovirus 1 strain EP713 (CHV1-EP713), infects Cryphonectria parasitica, the filamentous causal fungus of chestnut blight, and reduces its virulence. This pathosystem serves as a model to study fungus-mycovirus interactions. We previously developed a genetic screening protocol for host factors associated with symptom induction by CHV1-EP713 and its mutants. In the procedure the standard field fungal isolate EP155 was transformed by cDNA from a mild hypovirus mutant Cys(72), launching virus infection, and mutagenized by random plasmid insertion with pHygR conferring hygromycin resistance. We now report an extension of the study to characterize different mutant strains, with different phenotypes than their parental strain TCys(72)-1. TCys(72)-1 is moderately reduced in pigmentation and sporulation compared to the uninfected wild-type strain EP155. Mutants sfb1, sfb2 and k202 were characterized biologically and molecularly in comparison to the previously isolated mutant (namA) and the parental strain. These mutants harbored one (sfb1) or more copies (sfb2 and k202) of the mutagenic plasmid, pHygR. The three mutants had similar biological attributes; that is, vegetative growth rate, conidiation and virulence (assay on apples) was reduced on potato dextrose agar media, relative to the parental strain and pigmentation was the same or slightly increased. Interestingly, viral dsRNA accumulation levels were apparently unaltered in these mutants. The screening method was efficient for mining fungal mutants with unusual hypovirus symptoms. Further, characterization of the mutants provides interesting insights into symptom induction by the hypovirus.     

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double-stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis . These dsRNA-containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA-free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence-associated traits as those of the original dsRNA-containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis .     

Clonal population structure and introductions of the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, in Asturias,northern Spain

To understand the history of introductions of the chestnut blight fungus, Cryphonectria parasitica, in the Principality of Asturias in northern Spain, we conducted an extensive survey of chestnut blight and collected C. parasitica from 216 sites. All 778 isolates were assayed for vegetative compatibility (vc) type, whereas a subsample of 301 isolates was assayed for mating type, and 189 isolates were genotyped at 16 microsatellite markers. We found low diversity for all markers. Nearly all isolates (95%) were compatible with vc type EU-1 and had the same microsatellite multilocus haplotype, or differed from the most common type by mutation at one locus. Approximately 5% of the isolates were vegetatively compatible with EU-13 and only two isolates (< 1%) were compatible with EU-3; five different microsatellite haplotypes were found among isolates in these latter two vc types. The overall mating-type ratio was 218 MAT-1: 81 MAT-2, with both mating types represented in each of the three vc types. Microsatellite haplotypes based on ten markers used in France showed that most isolates in Asturias were either identical to or only one marker different from one of the seven most common genotypes in France, RE103. Based on these ten markers alone, the population of C. parasitica in Asturias, would appear to have been founded by a single genotype from the C1 lineage (to which RE103 belongs) found in eastern France and northern Italy. However, additional genotyping by vc types suggests the introduction of multiple genotypes, with different vc types. The exact source for introduction into Asturias cannot be determined without additional genotyping of isolates from other locations. Regardless of their origin, the low diversity of vc types makes this population ideal for deploying hypovirulence because there will be few barriers for virus transmission between individuals.     

Diversity of vegetative compatibility types and mating types of Cryphonectria parasitica in Slovenia and occurrence of associated Cryphonectria hypovirus 1

Cryphonectria parasitica, the causal agent of chestnut blight, has been present in Slovenia since at least 1950. To improve understanding of its diversity, 254 isolates of the fungus from 11 Slovenian populations were sampled. Fifteen vegetative compatibility (vc) types were identified. The dominant vc type was EU‐13, comprising 40·1% of all isolates tested, followed by EU‐1 (19·7%), EU‐2 (12·2%) and EU‐12 (9%). The vc type diversity in the most diverse population sampled in Slovenia was higher than in the populations found previously in northern Italy and Croatia. Both mating types and perithecia were observed in surveyed populations. Natural hypovirulence was found in six out of seven populations tested, with frequencies ranging from 72·2% in the population sampled near the Croatian border to 11·1% in the population sampled near the Austrian border. All identified hypoviral isolates (21) belonged to the Italian subtype of Cryphonectria hypovirus 1 and were closely related to the hypoviruses found in other European countries. Despite the high vc type diversity, incidence of hypovirulence was also high, indicating widespread natural biological control of the disease.     

板栗炭疽病田间综合防治技术研究

板栗（CastaneamollissimaBI．）是我国重要的干果之一，栗实病害种类较多，发生规律不同。自１９９８年来，作者在对山东省板栗病害种类进行系统研究基础上，认为板栗炭疽犤Colletotrichumgloeosporioides（Penz．）Sacc．犦病是栗实中的最严重种类，病原菌在田间侵染叶片和果实，并发病，于贮藏期在栗实上严重发生。对较多种类的栗实病害，其防治应分为两种情况，采用不同对策。生长至贮藏期病害的防治，如板栗炭疽病，应重点抓防治病害在生长期的侵染和发展；贮藏期病害的防治主要在贮藏前预防，贮藏期加强贮藏环境管理。目前尚未见有…     

行间覆盖方式对板栗园土壤及板栗产量品质的影响

试验于2019年在河北迁西地区展开,以8 a生板栗品种‘燕山早丰’(Castanea mollissima‘Yanshanzaofeng’)为研究对象,采用随机区组设计,在种植行间利用杂草、黑膜进行覆盖,共设置(Ⅰ)清耕(CK);(Ⅱ)覆膜;(Ⅲ)覆草;(Ⅳ)覆草+覆膜4个处理,研究行间覆盖对板栗园土壤理化性质、光合特性以及叶片生长发育和果实产量品质的影响,并从经济效益方面进行了分析比较。结果表明:行间覆草处理可以明显使土壤温度日较差变小,在0～20cm土层,夏季(6月30日)不同处理的温差日变化范围为Ⅱ(3.65℃)Ⅰ(2.82℃)Ⅲ(1.73℃)Ⅳ(1.62℃),秋季(10月30日)不同处理的温差日变化范围为Ⅱ(2.14℃)Ⅰ(2.01℃)Ⅲ(1.78℃)Ⅳ(1.69℃);对于20～40 cm土层的土壤温度日变化与0～20 cm土温变化趋势一致,但是没有明显的峰值,波动范围在0.5℃左右。在生长期内(4—10月),覆膜、覆草、覆草+覆膜处理的平均土壤含水量分别比CK高1.78%、3.34%和5.11%,可见覆草+覆膜处理的蓄水保湿效果最为显著,其贮水量在生育期末较生育期初增加39.56 mm,且土壤水分利用效率可达2.25 kg·hm~(-2)·mm~(-1)。不同覆盖处理均能显著降低0～20 cm土层的土壤容重,增加土壤总孔隙度,变化幅度为2%～8%,提高板栗园土壤有机质和速效养分含量,均以覆草和覆草+覆膜处理效果较好。而且覆草+覆膜处理可显著增加百叶干质量、叶面积和比叶质量,分别比CK高9.61%、2.39%、7.05%。覆草和覆草+覆膜处理下板栗叶片的水分利用效率(2.67μmol·mmol~(-1)和3.01μmol·mmol~(-1))显著高于CK(2.10μmol·mmol~(-1)),SPAD值分别比CK高4.62%和6.95%。杂草覆盖可以提高板栗单粒重、坐果率和出实率,降低空苞率,进而增加产量,其中覆草和覆草+覆膜处理的坐果率较大,分别可达75.68%、79.48%,其产量分别比CK高10.07%、15.00%,而且杂草覆盖还能有效改善板栗果实品质。从经济学角度分析,覆草处理的净收益(13 052.80元·hm~(-2))和产投比(5.08)最高,经济效益最大,可以在干旱半干旱山区板栗园应用。     

一种简便易行的栗疫病菌单孢分离方法

介绍了一种分离栗疫病菌单分生孢子的简便易行的方法。此方法可以排除菌丝段的干扰,也适用于分生孢子器中产生微小分生孢子的其他真菌。     

Diversity of Hypoviruses and Other Double-Stranded RNAs in Cryphonectria parasitica in North America

ABSTRACT Double-stranded (ds) RNAs in Cryphonectria parasitica were randomly sampled from nine subpopulations in North America using an antibody-based detection system for dsRNA. dsRNA was detected in 166 (28%) of a total of 595 C. parasitica isolates sampled by immunoblotting. Incidence of dsRNA infection within subpopulations ranged from 0% in samples from New Hampshire and Ontario to 100% in County Line, MI. Most of the dsRNAs sampled were approximately 9 to 13 kb in size. dsRNAs from 72 isolates analyzed by probing Northern blots with (32)P-labeled dsRNAs were in one of three hybridization groups. One hybridization group was widespread throughout eastern North America, being found in New York, New Jersey, Maryland, West Virginia, Kentucky, and Michigan. These dsRNAs hybridized to dsRNA from the previously described C. parasitica isolate SR2 from Maryland and are referred to as SR2-type dsRNAs. The second hybridization group was found almost exclusively in Michigan. The Michigan dsRNAs cross-hybridized to Cryphonectria hypovirus 3-GH2 (CHV3-GH2) and are referred to as CHV3-type dsRNAs.One dsRNA sampled from Kentucky hybridized to CHV3-type dsRNAs from Michigan. This dsRNA was probably derived from a fungal isolate that had been intentionally released for biological control at this same site 10 years previously and had become established in Kentucky. The third hybridization group was found only in New Jersey. These dsRNAs were much smaller than all other dsRNAs (3 and 5 kb) and were found in all 11 isolates that were probed; two of these isolates also had SR2-type dsRNA in mixed infections. None of the North American dsRNAs hybridized to CHV1 from Europe, even though CHV1 has been released in numerous locations in eastern North America for biological control of chestnut blight. Similarly, no dsRNAs hybridized to CHV2-NB58, a hypovirus found previously in New Jersey. Mixed infections of SR2-type and CHV3-type dsRNAs were found in 13 of 15 isolates from Frankfort, MI, while another nearby subpopulation (County Line) was infected with only CHV3-type dsRNAs. The distribution of dsRNA hybridization groups in C. parasitica thus presents a mixed picture, since one hybridization group is widespread, whereas two others are primarily restricted to smaller geographic areas.     

Residues of diflubenzuron on horse chestnut (Aesculus hippocastanum) leaves and their efficacy against the horse chestnut leafminer, Cameraria ohridella

Residues of the insect growth regulator diflubenzuron were quantified on horse chestnut (Aesculus hippocastanum L.) leaves treated with a diflubenzuron 480 g litre^?1 SC, Dimilin. To analyse the samples, an analytical procedure was developed involving a simple extraction step followed by high‐performance liquid chromatography on an octadecyl‐modified silica column with methanol + 0.01 M ammonium acetate mobile phase. The results showed diflubenzuron to be highly stable on horse chestnut leaves; more than 4 months (127 days) after application, 38% (on average) of the insecticide still remained on/in the leaves. The data confirmed biological observations showing diflubenzuron's long‐term efficacy against the horse chestnut leafminer, Cameraria ohridella Deschka & Dimi?, which is the most important pest of the horse chestnut in Europe. The hypothesis of possible penetration of diflubenzuron into the leaf mass is explored and discussed. Copyright © 2006 Society of Chemical Industry