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。     

黑龙江省马铃薯早疫病菌生物学特性研究

[目的]明确黑龙江省马铃薯早疫病菌(Alternaria solani)生物学特性的多样性.[方法]通过平板培养研究分离自黑龙江省马铃薯早疫病菌菌株的生物学特性.[结果]各分离株在PDA培养基上菌落生长速度、颜色、形态、成带现象等差异较大.其中的5个代表性供试菌株在PDA、CMA、OA培养基上生长较好,均能在10～35℃生长,但最适温度有一定的差异;供试菌株均能在pH 4～10的条件下生长,最适pH为6～8;光照对各分离株菌落生长有促进作用,各菌株对pH、光的反应不完全一致.[结论]说明黑龙江省马铃薯早疫病菌的生物学特性具有明显的遗传多样性.     

10种杀菌剂对板栗疫病菌的抑制作用

为了筛选防治板栗疫病的高效、低毒、低残留杀菌剂,采用菌丝生长速率法测定了10种杀菌剂对栗疫病菌菌丝生长的抑制效果。结果表明,苯醚甲环唑+丙环唑、苯醚甲环唑、咯菌腈、咪鲜胺和多氧霉素对栗疫病菌具有很强的抑制作用,EC₅₀在0.014 8～8.752μg/mL之间,为生产上有效地防治板栗疫病提供了依据。     

梨火疫病菌噬菌体的初步研究

为找到梨火疫病菌适度专化性的噬菌体，用于该病的检测和鉴定，从英国病山楂枝条上分离到一株噬菌体，测定了其寄主范围、潜育期、繁殖量等，还观察了其形态。分离到的噬菌体为高度专化性噬菌体，不能单位用于梨火疫病菌的检测和鉴定，可与其他梨火疫病菌噬菌体株系配合使用。     

抑制番茄早疫病菌活性化合物的 高通量筛选方法研究

以OD 630 值作为评价指标,在高通量筛选设备上建立了番茄早疫病菌Alternaria solania的高通量定量筛选方法,并对嘧菌酯、百菌清、 NFDA1 醚唑、霜脲氰、甲霜灵、代森锰锌、福美双和咪鲜胺8种常用杀菌剂对番茄早疫病菌孢子萌发和菌丝生长的抑制效果进行了评价,结果与孢子萌发法和目测筛选法所得结论基本一致。     

棉铃疫病菌对甲霜灵的抗药性风险研究

在实验室条件下,对分离自安徽省芜湖、无为、宣城、望江、肥东、寿县、滁州等7个地区的棉铃疫病菌(Phytophthora boehmeriae Sawada)对甲霜灵的抗药性进行了测定。结果显示,甲霜灵对各供试菌株的EC50分布为0.008 3～0.079 1μg／mL,平均为0.026 1 μg／mL,远小于1 μg／mL,且供试菌株在含甲霜灵质量浓度为1μg／mL的LBA平板上菌丝生长几乎完全被抑制,表明各供试菌株对甲霜灵十分敏感,但其敏感程度地区间存在较大差异。通过室内药剂直接诱变,获得抗甲霜灵菌株,经抗性测定结果表明,抗性菌株的抗性水平较其敏感亲本菌株高13 000倍以上,提示安徽省棉铃疫病菌对甲霜灵具有潜在的抗药性风险。     

甲霜灵抑制棉疫病菌卵孢子的产生

在离体和活体条件下,测定了甲霜灵抑制棉疫病菌(Phytophthora boehmeriae Sawada)卵孢子产生的作用。结果表明,在含甲霜灵0.04μg/ml的利马豆培养基(LBA)平板上,棉疫病菌的卵孢子产生量仅为对照的50％,0.10μg/ml条件下,病菌完全不能产生卵孢子。在接种的棉苗叶片上,喷施10～20μg/ml的甲霜灵可以明显抑制病菌在病组织内产生卵孢子,卵孢子产生量比对照下降了13～160倍,喷施40μg/ml的甲霜灵,受侵染子叶不发病。     

大豆携带菜豆晕疫病菌PCR检测技术研究

晕疫病是豆科作物上重要的检疫性细菌病害。本研究在传统 PCR 检测技术的基础上,分析了细菌洗涤液、洗涤时间以及洗涤液容量对一粒带菌大豆检出率的影响,以及有效检出不同带菌率大豆的最佳检测时间。结果表明,洗涤液种类、洗涤时间以及洗涤液容量对检测结果影响不大,但对不同带菌率大豆洗涤时间的长短直接影响病菌的检出率,实验表明振荡洗涤时间8 h,可检测到的大豆种子最低带菌率为0.0625%。本研究所建立的检测方法简便易行,可有效应用于口岸检测进口大豆中的菜豆晕疫病菌。     

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。     

栗疫病菌不同毒力菌株生物学特性的研究

 对不同毒力、含或不含dsRNA的菌株的生物学特性进行了研究。含dsRNA的弱毒力菌株的分生孢子后代,可分化出白色和桔黄色两种菌落类型,菌落白色的单孢菌株,可继续分化出白色和桔黄色两种类型的后代,桔黄色的单孢菌株保持稳定,不再发生分化。不含dsRNA的菌株,未见培养性状发生分化。对含dsRNA的弱毒力菌株的第2代两种类型的单孢菌株进行了毒力测定和dsRNA检测,桔黄色的单孢菌株的毒力水平达到或接近强毒力菌株的水平,不含dsRNA;而白色的单孢菌株的毒力很弱,且都含有dsRNA。对不同毒力菌株分生孢子的萌发率进行了比较,结果表明,孵育12h后,即有部分菌株的分生孢子开始萌发,强毒力菌株孢子的萌发最早,含dsRNA的弱毒力菌株的孢子萌发较迟;孵育24h后,各菌株孢子的萌发率均达到90%以上,差异已不太明显,但芽管的分枝程度有较大差异,强毒力菌株的芽管(菌丝)大量分枝,初步形成菌丝丛,而弱毒力菌株,不论是否含有dsRNA,芽管的分枝程度均较低。     

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.     

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.     

栗疫病菌菌株的毒性联合与毒性分离现象的初步研究

 对来自3个中国板栗居群的共18个栗疫病菌株进行单株及混合菌株的毒力评价,结果表明各单株毒力差异显著,混合菌株存在毒性联合与毒性分离现象。同一种群的组合中有64.4%为毒性联合,不同种群的组合其毒性分离率高达88%,同一种群组合的毒性联合强度比不同种群组合的毒性联合强度大,而不同种群组合的毒性分离强度比同一种群组合的毒性分离强度大。大多数组合的毒性联合或毒性分离的性状表达稳定。     

Low diversity of vegetative compatibility types and mating type of Cryphonectria parasitica in the southern Balkans

The diversity of vegetative compatibility (vc) types and mating type was estimated in populations of the chestnut blight fungus, Cryphonectria parasitica , throughout Macedonia and from selected areas in Greece. Nearly all of the 786 isolates (94%) from Macedonia were in a single vc type, EU-12; all 379 isolates from Greece were EU-12. Only six of 20 populations in Macedonia had more than one vc type. The diversity of vc types in the most diverse populations of Macedonia was comparable with the least diverse populations found previously in Italy. All but six of the 313 isolates assayed had the same mating type, MAT-1 , and no perithecia of Cryphonectria parasitica were observed in any population. These results lead to the conclusion that sexual reproduction does not occur in these populations. The lack of vc type diversity may indicate a high potential for the spread of hypoviruses and successful biological control with transmissible hypovirulence. However, if sexual reproduction should occur in Macedonian populations, up to 32 vc types would be possible by recombination among vegetative incompatibility loci.     

Chestnut blight fungus in Croatia: diversity of vegetative compatibility types,mating types and genetic variability of associated Cryphonectria hypovirus 1

In order to improve understanding of its diversity, 338 isolates of Cryphonectria parasitica, the causal agent of chestnut blight, were sampled from 10 chestnut populations throughout chestnut‐growing coastal and continental areas of Croatia. Eighteen vegetative compatibility (VC) types were identified. The VC type EU‐1 was the most widespread, comprising 42·9% of the isolates, followed by EU‐2 (21%) and EU‐12 (14·2%). In respect to the occurrence of the main VC types, the C. parasitica populations in Croatia combined features of both northwestern and southeastern European populations. Perithecia and mating‐type ratios of approximately 1 : 1 were found in all populations, suggesting that sexual reproduction of the fungus is common in Croatia. Natural hypovirulence was also evident in all populations, with incidence of hypovirus‐infected isolates ranging from 12·7% in Istria‐Buje to 66·6% in the continental part of the country. A total of 36 hypovirus‐infected isolates sampled throughout Croatia were analysed in ORF‐A and ORF‐B by RT‐PCR/RFLP analysis. All viral isolates belonged to the Italian subtype of Cryphonectria hypovirus 1 (CHV‐1) and were closely related to the isolates found in other European countries. The RFLP patterns found were also identical or similar to the patterns of three isolates collected in Croatia 22 years ago, suggesting a slow evolution of the hypovirus.     

Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis‐mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co‐culturing of the same isolates, revealing the specificity and accuracy of the PCR‐based molecular vic genotyping assay. Twenty‐six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.