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1.
单克隆抗体检测栗疫菌dsRNA的研究初报 总被引：4，自引：0，他引：4
本文利用dsRNA特异性单克隆抗体,采用双抗体夹芯ELISA法,检测了栗疫菌中的dsRNA.结果表明,该法不仅能从栗疫菌低毒力菌株的总核酸粗提液中检测到dsRNA而不受其它核酸的干扰,而且比电泳法简便迅速,在大量筛选含dsRNA的栗疫菌低毒力菌株中具有很大的实用价值。 相似文献
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辣椒疫霉生防菌的双重筛选 总被引：16，自引：2，他引：16
利用抑菌圈-定殖力双重测定法进行辣椒疫霉拮抗菌筛选。结果表明:有6株拮抗菌(28G14、03GY02②、F1、B2、95W06和31-4G02)在辣椒幼苗根茎部的定殖能力较强(定殖密度大于2.6×104cfu/g根),其中B2和F1的定殖密度为176.0和134.0,分别是致病菌辣椒疫霉(27.0×104cfu/g根)的6.5倍和5.0倍;这6株拮抗菌均具有较高的相对防效(81.2%~100%),不能定殖的菌株相对防效低(0~37.9%),说明抑菌圈-定殖力双重测定法是筛选具有双重功能生防菌的一种有效而简便的方法;6株有效菌在灭菌和未灭菌土壤中的相对平均防效分别为93.3%和87.0%;拮抗菌的相对防效与接种时间有关,有效菌株数按十叶期(44株)>开花期(38株)>结椒期(36株)的顺序排列。 相似文献
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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。 相似文献
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抑菌圈-定殖力双重测定法筛选青枯病生防细菌 总被引：10，自引：0，他引：10
本研究首先用平皿抑菌圈法筛选出55个拮抗青枯菌的细菌菌株,将番茄幼苗在各菌菌悬液中浸根12h后栽种于温室未灭菌的土壤中,结果发现,有22个菌株在幼苗根部的定殖能力较强(终定殖密度大于104 cfu/g根),其中革兰氏阴性土壤细菌占同类菌的86.3%,革兰氏阳性土壤细菌占同类菌的13.0%,10个无致病力产细菌素的青枯菌菌株在根部的终定殖密度均低于104 cfu/g根,其定殖能力弱于致病青枯菌。有17个拮抗菌菌株在番茄幼苗根部的定殖密度超过所有致病菌。温室生防结果证明,抑菌圈-定殖力双重测定法对于筛选生防菌株是一种行之有效的方法。 相似文献
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抑菌圈－定殖力双重测定法筛选青枯病生防细菌 总被引：16，自引：0，他引：16
本研究首先用平皿抑菌圈法筛选出５５个拮抗青枯菌的细菌菌株，将番茄幼苗在各菌菌悬液中浸根１２ｈ后栽种于温室未灭菌的土壤中，结果发现，有２２个菌株在幼苗根部的定殖能力较强（终定殖密度大于１０４ｃｆｕ／ｇ根），其中革兰氏阴性土壤细菌占同类菌的８６．３％，革兰氏阳性土壤细菌占同类菌的１３．０％，１０个无致病力产细菌素的青枯菌菌株在根部的终定殖密度均低于１０４ｃｆｕ／ｇ根，其定殖能力弱于致病青枯菌。有１７个拮抗菌菌株在番茄幼苗根部的定殖密度超过所有致病菌。温室生防结果证明，抑菌圈－定殖力双重测定法对于筛选生防菌株是一种行之有效的方法 相似文献
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荧光假单胞菌CZ菌株定殖及抗病毒活性研究 总被引：2，自引：0，他引：2
荧光假单胞菌Pseudomonas fluorescens CZ能分泌包括碱性磷酸酶在内的抗病毒蛋白抑制烟草花叶病毒Tobacco mosaic virus(TMV)侵染?本文利用抗生素利福平逐级诱导获得抗药性标记菌株CZ-rif, 生测试验显示:野生型和抗药型菌株的发酵上清和粗蛋白液对TMV的体外钝化效果均大于90%, 无显著差异?灭菌和非灭菌的根际土拌CZ-rif菌液后盆栽烟苗, 取根际土在含抗生素的平板上检测定殖菌量, 结果显示, CZ-rif能够在烟株根际土壤中有效定殖, 第31天在灭菌土中的定殖菌量为4.3×104 cfu/g, 显著大于在非灭菌土中的定殖菌量6×103 cfu/g, 在叶面上的定殖菌量显著低于根际土壤?叶面喷施CZ-rif菌液后24 h接种病毒, 对TMV-GFP的预防效果为45.85%?田间试验中喷淋菌株发酵稀释液, 对烟草花叶病毒病的防效为42.02%?此外, 水培试验显示生防菌发酵液对烟草幼苗具有促生作用?总之, 荧光假单胞菌CZ能在烟草根际定殖和促进植株生长, 叶面喷施能钝化TMV并抑制其初侵染, 可以研发防治病毒病害的生物制剂? 相似文献
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枯草芽孢杆菌B_3菌株对小麦根系和茎基部的定殖作用研究 总被引：1，自引：0，他引：1
本文研究了枯草芽孢杆菌Ｂ＿３菌株经种子处理后在小麦根系及茎基部的定殖能力和影响其定殖的可能因子。结果表明，Ｂ＿３在田间自然土壤中定殖小麦根系及茎基部的时间分别为６０天和４２天以上。培养不同时间的Ｂ＿３菌悬液处理种子表明，播后２２天内，培养２４小时的菌在小麦上的定殖量高于培养４８和７２小时。用ＮＢ培养的Ｂ＿３菌悬液经离心弃上清，并用新鲜ＮＢ液悬浮菌体后处理种子，在播后１９天，Ｂ＿３定殖量比不处理菌悬液的定殖量增加。Ｂ＿３与化学杀菌剂混合处理种子，以及土壤经灭菌处理均可增加Ｂ＿３在小麦上的定殖量。 相似文献
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非致病性尖孢镰刀菌FJAT-9290的定殖特性及对番茄枯萎病的防治效果 总被引：1，自引：0，他引：1
为评价非致病性尖孢镰刀菌FJAT-9290对不同植物的致病性和定殖能力,利用该菌株所含的无毒基因SIX1特异性检测引物P12-R1/P12-F2跟踪其在不同植物中的侵入与定殖情况,并研究其对番茄植株生长特性的影响及对番茄枯萎病的防治效果。结果显示,接种120 d内,菌株FJAT-9290对所供试的11种植物均未造成危害,但在侵入时间与定殖方面存在差异。该菌株最易侵入番茄植株,接种第5天即可在茎基部检测到;其次为甜椒、甜瓜、西瓜和香蕉等植株,接种10 d时可在茎基部检测到;但在韭菜、香葱和马唐草上均未检测到。该菌株在番茄与茄子植株的定殖时间最长,达90 d;其次为甜椒、香蕉和粉蕉,至少60 d;在甜瓜、西瓜和黄瓜上为40~50 d。该菌株能促进番茄植株生长,显著提高其株高和叶片数量,对番茄枯萎病的盆栽与田间防治效果分别达76.70%和69.56%。表明菌株FJAT-9290具有良好的定殖能力且对番茄枯萎病具有较好的防治效果。 相似文献
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Md. Iqbal Faruk Masatoshi Izumimoto Nobuhiro Suzuki 《Journal of General Plant Pathology》2008,74(6):425-433
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. 相似文献
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ABSTRACT Strains of the chestnut blight fungus, Cryphonectria parasitica, have been genetically engineered to contain an integrated full-length cDNA copy of the prototypic virulence-attenuating hypovirus CHV1-EP713. Unlike natural hypovirulent C. parasitica strains, these transgenic hypovirulent strains are able to transmit virus to ascospore progeny under laboratory conditions. This ability provides the potential to circumvent barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system. During July 1994, transgenic hypovirulent strains were introduced into a Connecticut forest site (Biotechnology Permit 94-010-01). Subsequent analysis of the release site confirmed hypovirus transmission from transgenic hypovirulent strains to ascospore progeny under field conditions. Additionally, it was possible to recover transgenic hypovirulent strains from the test site as long as 2 years after the limited, single-season release. Evidence also was obtained for cytoplasmic transmission of transgenic cDNA-derived hypovirus RNA, including transmission to mycelia of a virulent C. parasitica canker after treatment with conidia of a transgenic strain. Finally, a transgenic hypovirulent strain was recovered from a superficial canker formed on an untreated chestnut tree. Genetic characteristics of the recovered strain suggested that the canker was initiated by an ascospore progeny derived from a cross involving an input transgenic hypovirulent strain. The durability of a molecular marker for field-released cDNA-derived hypovirus RNA is discussed. 相似文献
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P. Zamora A. González Casas M. Dueñas R. San Martin J. J. Diez 《European journal of plant pathology / European Foundation for Plant Pathology》2017,148(1):65-73
Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and León (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well. 相似文献
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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. 相似文献
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Seçil Akıllı Çiğdem Ulubaş Serçe Yakup Zekai Katırcıoğlu Salih Maden Daniel Rigling 《European journal of plant pathology / European Foundation for Plant Pathology》2013,135(2):323-334
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. 相似文献
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L. Krstin S. Novak‐Agbaba D. Rigling M. Krajačić M. Ćurković Perica 《Plant pathology》2008,57(6):1086-1096
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. 相似文献
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ABSTRACT In France, chestnut blight, caused by Cryphonectria parasitica, has been controlled since 1974 in orchards, but never in coppice forests, by releasing hypovirulent strains infected with CHV1 hypovirus. We tested the hypothesis that this biological control (BC) has lead to a decrease in blight severity, spread of hypovirulence, and change in C. parasitica populations. The low severity of chestnut blight was confirmed in the six regions studied (subdivided into zones). The remission of cankers was associated with the presence of white isolates presumed to be hypovirulent. These two parameters were also correlated, at the zonal level, to the frequency of sites where BC was used. However, the estimates of the natural background level of hypovirulence, independent of BC, ranged from 4% in forests in Dordogne to 60% in orchards in Lozère. Differences in the rate of hypovirulent isolates among regions were consistent with the diversity of vegetative compatibility (VC) types in populations of C. parasitica. The highest VC-type diversity and mean allelic diversity for known vegetative incompatibility (vic) genes were observed in Dordogne. We showed that the current diversity of VC types in populations of C. parasitica was lower than in 1981. We found 30 VC types among 1,113 isolates of C. parasitica. Ten VC types were incompatible with known EU testers, suggesting that one additional vic gene or allele at one of the six vic loci known should be present in Europe. 相似文献
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ABSTRACT Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia. 相似文献
20.
C. Kubo E. Nakazono-Nagaoka K. Hagiwara H. Kajihara S. Takeuchi K. Matsuo T. U. Ichiki T. Omura † 《Plant pathology》2005,54(5):615-620
New strains of Melon necrotic spot virus (MNSV), designated MNSV-YS and MNSV-KS, caused much more severe growth retardation on melon plants than MNSV-NH, which was previously reported as the most severe strain of MNSV in Japan. MNSV-YS spread much more quickly than MNSV-NH in infected plants, and induced more severe growth retardation, even though the appearance of necrotic lesions on inoculated cotyledons was much slower. MNSV-KS had properties intermediate between those of the other two strains. The results suggest that faster-spreading strains can multiply more rapidly as a result of lower levels of activity in inducing necrotic lesions in melon plants. The complete sequences of MNSV-YS and MNSV-KS were determined, and an RT–PCR–RFLP method based on these sequences was successfully developed to detect and discriminate between the three strains. 相似文献