黑龙江省部分地区马铃薯疮痂病菌种类及致病性鉴定

为明确黑龙江省马铃薯疮痂病病原菌的种类及其特征,2012-2013年从黑龙江省克山县、绥化市、哈尔滨市、杜尔伯特蒙古族自治县采集具有疮痂病病斑的马铃薯块茎,从中分离纯化病原菌,根据16SrRNA基因的差异采用分子手段对所分离的菌株进行种类和致病性鉴定,并对txtAB阳性菌株采用萝卜幼苗法或马铃薯致病性试验测定其致病性。共分离出74株菌株,鉴定出致病性菌株26株,其中Streptomyces scabies或S.europaeiscabiei 21株,S.turgidiscabies 3株和S.acidiscabies 2株。所有的致病性菌株中共有4种致病岛基因型,即nec1+/tomA+、nec1-/tomA+、nec1+/tomA-和nec1-/tomA。     

Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: quick diagnosis based on a PCR-based assay

A time-saving and cost-effective polymerase chain reaction (PCR)-based method was developed for species-specific detection of the scab pathogens ( Streptomyces scabies and S. turgidiscabies ) prevalent in potato ( Solanum tuberosum ) in northern Scandinavia. Species specificity of primers was verified using a collection of previously characterized Streptomyces strains isolated from potato scab lesions in Finland and Sweden. A total of 1245 scab lesions was tested from potato cvs Matilda and Sabina grown in the field in two geographic regions of Finland in 2000 and 2001. Freshly harvested or stored potato tubers were incubated at room temperature (18–21°C) under humid conditions for a few days. Bacterial growth was collected from scab lesions for DNA isolation and PCR. The two scab pathogens were detected in the same potato fields, tubers and scab lesions. The relative incidence of S. scabies was high in freshly harvested tubers but was much lower than that of S. turgidiscabies following storage. Both pathogens were seed-transmitted in Matilda and Sabina after 24 weeks of storage at 4°C.     

A survey of genetic variation in streptomyces isolates causing potato common scab in the United States

ABSTRACT Common scab is a serious disease of potatoes and other root and tuber crops, affecting crop quality and market value. The disease is caused by gram positive soil bacteria in the genus Streptomyces. Disease incidence and severity vary in different locations and years; this is due in part to variation in the environment (weather) and genetic variation in potato cultivars. Little information is available on the contribution of genetic variation by the pathogen. To examine genetic diversity in different locations within the United States, streptomycetes were isolated from lesions on field-grown potatoes from six states. Isolates were classified into species based on sequence of variable regions in the 16s rRNA gene. The presence of genes associated with the recently described S. turgidiscabies pathogenicity island (PAI) was also determined. About half of the isolates belonged to S. scabies or S. europaeiscabiei based on 16s rDNA sequence, and had characteristic features of the PAI. They were found in all six states, and were pathogenic on potato and radish. The remaining isolates included pathogens and nonpathogens. They were varied in appearance, and represent several species, including one pathogenic species not previously reported. Some pathogenic isolates lacked one or more genes characteristic of the PAI, although all had genes for biosynthesis of the pathogenicity determinant thaxtomin. In this relatively small survey, regional differences in scab-causing streptomycetes were seen. This report furnishes tools and baseline data for population genetic study of scab-causing streptomycetes in the United States.     

Genotypic and phenotypic characterization of Streptomyces species associated with potato crops in the central part of Colombia

In Colombia, Streptomyces scabiei (syn. S. scabies) is commonly believed to be the causal organism of scab disease in local potato crops. However, very little is known about this organism and about the diversity and pathogenicity of the Streptomyces species associated with potato crops in Colombia. This study, therefore, aimed to elucidate aspects regarding the diversity of these bacteria associated with potato crops in a particular region of Colombia and evaluate their pathogenicity. We obtained 33 isolates of Streptomyces from netted, superficial and deep-pitted potato scab lesions from two main potato-producing regions in Colombia. Of these, 17 were pathogenic based on in vitro and in planta assays. None of these isolates carried the txtA, txtB, or nec1 genes, commonly associated with pathogenicity in Streptomyces, and characteristic of the pathogenicity island (PAI). We also characterized all isolates based on phenotypic characteristics and analysed their phylogenetic relationships using the 16S rRNA, atpD, recA, rpoB, and trpB genes. The isolates were highly diverse, placed in nine clades with 15 different phenotypes. The 17 pathogenic isolates were placed into three clades, namely S. pratensis, S. xiamenensis, and unknown species. This study is a preliminary investigation towards understanding scab disease in Colombia through the study of both pathogenic and nonpathogenic species present in scab disease lesions in potatoes. Also, this is the first report of Streptomyces species associated with potato tubers in Colombia.     

Detection and characterization of Streptomyces causing potato common scab in Western Europe

A PCR-based diagnostic method was developed for direct detection from tuber lesions of pathogenic Streptomyces causing common scab of potato. Primers were designed to amplify a fragment of the txtAB ( txtA and txtB ) genes, which are pathogenicity determinants in the main pathogenic Streptomyces species. The method was evaluated on 84 naturally infected potato samples, comprising 19 potato cultivars, harvested in the years from 2000 to 2004 in the Netherlands, the UK, France, Germany and Spain. Pathogenic Streptomyces in tuber lesions were detected by PCR in 70 samples and were also successfully isolated from these 70 samples. All pathogenic isolates showed the basic general phenotypic traits of the S. scabiei phenetic cluster. RFLP analysis of amplified rRNA sequences, together with carbon source utilization and repetitive BOX profiles, allowed most isolates to be assigned to S. europaeiscabiei , which emerged as the main cause of potato common scab in Western Europe.     

Differences in host range, pathogenicity to potato cultivars and response to soil temperature among Streptomyces species causing common and netted scab in France

The pathogenicity and ecology of some isolates representative of the four main Streptomyces species ( S. scabies , S. europaeiscabiei , S. stelliscabiei and S. reticuliscabiei ) identified as pathogenic to potato tubers were investigated. Three pathogenicity groups could be distinguished. Group 1 included all isolates of S. scabies , S. europaeiscabiei and S. stelliscabiei from common scab lesions of potato and other susceptible root crops. All these produced similar symptoms and were pathogenic to potato, carrot and radish. Group 2 included all isolates from S. reticuliscabiei netted scab lesions; they were pathogenic to both tubers and roots of only a few potato cultivars, and did not infect carrot or radish. Group 3 included three isolates of S. europaeiscabiei from netted scab lesions on cv. Bintje, which produced either common or netted scab symptoms depending on the potato cultivar or plant species. In an experiment on a few isolates from each of the three groups, held at various soil temperature regimes, the three from group 1 were most pathogenic at higher temperatures (20°C or 20/30°C), the two from group 2 were most pathogenic at a lower temperature (17°C). The group 3 isolate caused netted scab symptoms on susceptible cultivars at low temperatures (≤ 20°C) and deep-pitted lesions at higher temperatures. Since the groups identified differ in ecological requirements, it is important to adapt the control methods to the pathogenic species present in the soil.     

Isolation and characterization of Streptomyces species from potato common scab lesions in Norway

The present survey was conducted to isolate and characterize Streptomyces species from common scab lesions of potato in Norway. Bacteria were isolated from scab lesions on tubers sampled in two consecutive years at different locations in Norway spanning ～1400 km from south to north. In total, 957 independent isolations from individual tubers were performed, with 223 putative pathogenic isolates obtained from 29 different potato cultivars and 130 different fields. Streptomyces europaeiscabiei was the most abundant species isolated from common scab lesions (69%), while 31% of the isolates obtained were S. turgidiscabies. Streptomyces scabies was not found. Pathogenicity of selected Streptomyces isolates was tested on potato. The ability of the bacterial isolates to infect potato was consistent with the presence of the txtAB operon. The results revealed no pattern in geographical distribution of S. europaeiscabiei and S. turgidiscabies; both could be found in the same field and even the same lesion. Four different pathogenicity island (PAI) genotypes were detected amongst the txtAB positive isolates: nec1+/tomA+, nec1–/tomA+, nec1+/tomA? and nec1?/tomA?. The current findings demonstrate that there is genetic variability within species and that the species are not spread solely by clonal expansion. This is thought to be the most comprehensive survey of Streptomyces species that cause common scab of potato in a European country.     

马铃薯疮痂病菌毒素及其致病性的研究

 本文对分离自我国不同地区的马铃薯疮痂病菌产生的毒素进行了研究。通过对不同菌株进行毒素提取、纯化和产毒分析发现各致病菌株均能产生同一类毒素,而非致病菌株均未见毒素产生。毒素的活性检测结果表明,毒素能使马铃薯薯片和萝卜幼苗产生与病菌作用相同的症状,说明毒素是疮痂病菌侵染过程中的主要致病因子。     

Pathogenicity of Streptomyces scabies Mutants Altered in Thaxtomin A Production

ABSTRACT To investigate the role of thaxtomin A in the pathogenicity of Streptomyces scabies, mutants altered in thaxtomin A production were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutants of S. scabies EF-35 could be differentiated according to levels of thaxtomin production. Mutants M1, M8, and M19 produced 2 to 20 times less thaxtomin A in oat bran medium than did EF-35. M1 and M19 were deficient in tryptophan catabolism. Thaxtomin production was reduced by about 300 times in mutant M16, which was a glutamic acid auxotroph. No thaxtomin A was detected in M13 culture supernatant. This mutant had a normal growth rate, was prototrophic, and catabolized tryptophan. Pathogenicity of mutants was tested on radish and potato. Mutants M1, M8, and M19 were pathogenic but, in most cases, less virulent than EF-35. M13 and M16 were nonpathogenic. These results suggest that thaxtomin A is an important pathogenicity determinant in S. scabies.     

Edaphic Soil Levels of Mineral Nutrients, pH, Organic Matter, and Cationic Exchange Capacity in the Geocaulosphere Associated with Potato Common Scab

ABSTRACT In order to determine possible relationships between geocaulosphere soil properties and severity of common scab of potato caused by Streptomyces scabies, soils were collected from representative commercial potato fields in Canada: in Simcoe and Dufferin Counties, Ontario and across Prince Edward Island (PEI) in August 2004. Soils immediately adjacent to tubers were sampled and analyzed for select edaphic factors and for pathogen presence using polymerase chain reaction (PCR) tests with primers that amplify a region of the TxtA gene involved in regulating the biosynthesis of the thaxtomin toxin family. Individual tubers were assessed visually for scab severity. The relationships between soil chemical factors and disease severity were investigated for each region to detect the strongest relationships. Principal component analysis revealed a distinctive clustering of samples with respect to disease severity in PEI but not in Ontario soils. Total and percent saturation of K (%K) were the only factors found associated with high disease severity in soils from both provinces. In PEI soils, pH, Mg, Ca, Cu, and %K, %Mg, %Ca, and %Na were associated with high disease severity, whereas cation exchange capacity (CEC) and Al were correlated with low disease severity soils. In Ontario, high Mn content was strongly correlated with low disease severity soils, whereas %K and organic matter content were correlated with disease severity. Partitioning samples into presence or absence of the TxtA PCR product with corresponding high or low severity showed further significant relationships in the data. There was an excellent correlation between Streptomyces spp. presence as detected by PCR and disease severity in PEI soils; however, the relationship was not as clear in Ontario soils, where many PCR-positive soils had low disease incidence. Principal component and partial least square analysis indicated that disease severity was predicted by soil factors such as organic matter, CEC, pH, Al, %Ca, %Mg, and %K for PEI but not for Ontario soils. The data reveal that the relationship between scab severity and soil chemical components is complex and potentially soil specific.     

Assessment of common scab-inducing pathogen effects on potato underground organs via computed tomography scanning

Common scab caused by Streptomyces scabies is a major bacterial disease of potato (Solanum tuberosum). Its best known symptom is superficial lesions on the surface of progeny potato tubers, observed at harvesting. In this study, effects of S. scabies on space occupancy by underground organs and on structural complexity of root systems are investigated during growth via computed tomography (CT) scanning. Two groups of potato plants were grown in a greenhouse in middle-sized plastic pots. Using a high-resolution X-ray CT scanner formerly used for medical applications, their underground organs and surrounding medium (sieved and autoclaved homogeneous sand) were submitted to CT scanning 4, 6, and 8 weeks after planting. For one group, sand was inoculated with the common scab-inducing pathogen (S. scabies EF-35) at potting. Space occupancy by underground organs was estimated via curve fitting applied to histograms of CT scan data, while three-dimensional skeletal images were used for fractal analysis. Root systems of diseased plants were found to be less complex than those of healthy plants 4 weeks after planting, and the relative growth rates derived from space occupancy measures were of different sign between the two groups from week 4 to week 8.     

拮抗链霉菌防治马铃薯疮痂病的大田试验研究

 本文报道了利用拮抗链霉菌防治马铃薯疮痂病的大田试验研究。拮抗链霉菌接种在0.8公顷的病田中。不同接种量和接种方法均显著地影响防治效果。3年的试验表明:拮抗菌系以蛭石接种形式防效优于种薯浸泡。一般情况下,连续2年接种或高接种量的防效优于1年或低接种量的防效。不同菌系的防效差异明显,单个拮抗菌的防效优于2个拮抗菌混合使用的效果。甜玉米和大豆轮作结合拮抗菌显著降低了马铃薯疮痂病的危害。     

西北地区马铃薯疮痂病病原菌鉴定及其生物学特性

为明确西北地区马铃薯疮痂病病原菌的种类和生物学特性,分别采用常规组织分离法和土壤混悬液分离法从宁夏、陕西和甘肃3个省区采集的29份疮痂病发病薯块和8份发病地块土壤中进行病原菌分离,并利用形态特征、生理生化特性和16S rDNA序列分析对病原菌进行鉴定。结果表明,从发病薯块和发病土壤中共分离到50株链霉菌Streptomyces spp.,通过回接法验证获得6株马铃薯疮痂病致病菌株。6株致病菌株的培养特性和形态特征差别较大;其中菌株G4-1、G9和SYN13不能以果糖和木糖为单一碳源,菌株SYNT3不能以棉子糖为单一碳源;除菌株NLG4-1外,其余5株菌株均能在络氨酸琼脂培养基上产生黑色素。经16S rDNA序列分析,菌株G4-1、G9与疮痂病链霉菌S. scabiei的相似率分别达99.47%和99.34%,菌株NLG4-1、SYNT3与S. enissocaesilis的相似率分别达97.90%和98.18%,菌株GBH2与加利利链霉菌S. galilaeus的相似率达99.93%,菌株SYN13与S. turgidiscabies的相似率达97.56%,表明西北地区马铃薯疮痂病病原菌至少存在4个种。     

Distribution and characterization of Streptomyces species causing potato common scab in Germany

Field‐grown potatoes showing scab infections were sampled in two successive years and analysed for prevailing Streptomyces strains. In 2008 and 2009, 293 Streptomyces isolates were collected in Germany and analysed for morphology, pathogenicity and strain type. Isolates varied in mycelium colour, sporulation and pigmentation. Based on their morphology, no clear differentiation of species was possible. At the genetic level, sampled isolates, as well as a number of type strains from culture collections, were characterized by PCR using 16S rRNA‐specific primers and PCR‐RFLP of the 16S–23S internal transcribed spacer (ITS) region with Hpy99I. Using this fingerprinting approach, Streptomyces species could be differentiated genotypically. The data from this study show that diversity among scab‐causing species in Germany is much higher than previously thought. Isolates belonged to various Streptomyces spp. previously associated with common scab. This is apparently the first report of pathogenic strains of S. europaeiscabiei, S. stelliscabiei, S. acidiscabiei, S. turgidiscabiei and S. bottropensis within Germany. Streptomyces europaeiscabiei was the predominant species found. Other scab‐causing species were identified, but their local distribution was uneven. For most of the isolates, the presence of the txtAB gene was demonstrated, indicating pathogenicity. This analysis is one of the first reports to examine the distribution of common scab‐causing species in Germany.     

马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

Phytotoxin produced by the netted scab pathogen, <Emphasis Type="Italic">Streptomyces turgidiscabies</Emphasis> strain 65, isolated in Sweden

Streptomyces spp. are a highly diverse group of bacteria most of which are soil-inhabiting saprophytes. A few are plant pathogens that produce a family of phytotoxins called thaxtomins and cause significant economic losses, e.g., by reducing the marketability of potato tubers (Solanum tuberosum). In northern Europe, S. scabies, S. turgidiscabies and S. europaeiscabiei are the most common plant pathogenic species. In this study, a Streptomyces strain isolated from a netted scab lesion on a tuber of potato cv. Bintje in northern Sweden was identified as S. turgidiscabies but was found to differ in the genomic region carrying genes required for thaxtomin biosynthesis. Our results showed that the strain did not produce thaxtomin but rather phytotoxin fridamycin E, which is an anthraquinone novel to plant pathogenic Streptomyces spp. Fridamycin E was shown to reduce or inhibit sprouting of potato microtubers in vitro. While fridamycin E is known to have antibiotic activity against Gram-positive bacteria, the inhibitory activity of fridamycin E on plant growth is a novel finding.     

Novel in vivo use of a polyvalent Streptomyces phage to disinfest Streptomyces scabies-infected seed potatoes

A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 10⁹ plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly ( P  < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced ( P  < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.     

Resistance of potato genotypes to common and netted scab-causing species of Streptomyces

Common and netted scabs are two disfiguring bacterial diseases of potato tubers, caused by various groups of Streptomyces species. Common scab, caused primarily by Streptomyces scabies and Streptomyces europaeiscabiei , is characterized by more or less deep pustules on the tuber surface, while symptoms of netted scab, caused mainly by Streptomyces reticuliscabiei , are superficial, corky alterations of the tuber periderm. Some isolates of S. europaeiscabiei are able to induce both common and netted scab symptoms, and therefore constitute a third pathogenicity group. Like most bacterial diseases, potato scabs would be best controlled by using resistant cultivars. Repeated experiments with soil artificially infested with isolates of three species representative of the three pathogenicity groups showed the level and stability of cultivar resistance, as well as the existence of a range of aggressiveness among different isolates. The distribution of scab severity indexes recorded on a collection of 16 potato cultivars and 27 breeding clones grown in soil infested with common scab-inducing isolates was continuous, suggesting isolate nonspecific quantitative resistance. Least susceptible cultivars were Nicola, BF15, Sirtéma, and Charlotte, while Urgenta, Désirée, Ondine and Bintje were very susceptible. The same genotypes proved either highly susceptible (e.g. cvs Bintje, Désirée or Carmine) or highly resistant (e.g. cvs Charlotte, Sirtéma, Monalisa, BF15 or Belle de Fontenay) to isolates forming netted scab symptoms, suggesting isolate-specific qualitative resistance. The ability was confirmed of some isolates of S. europaeiscabiei to induce one or the other type of symptoms depending on cultivar and soil temperature.     

云南省马铃薯疮痂病致病链霉菌种类组成研究

 为明确云南省马铃薯疮痂病病原菌(Streptomyces spp.)的种类及其生物学特征,自2013年从云南省13个马铃薯主产区采集疮痂病病样,共分离到200株链霉菌,通过温室盆栽致病性试验筛选出67株致病菌。通过形态学、生理生化指标、致病性测定及16S rDNA序列分析对获得的菌株进行鉴定。结果显示,引起云南地区马铃薯疮痂病的病原为10种链霉菌,分别为S. caviscabies、S. anulatus、S. scabies、S. turgidiscabies、S. acidiscabies、S. europaeiscabiei、S. luridiscabiei、S. enissocaesilis、S. griseus和S. aureofaciens。其中S. enissocaesilis和 S. anulatus为优势种群,S. caviscabies、S. anulatus和S. luridiscabiei为国内首次报道的病原菌。因此,认为云南省马铃薯疮痂病菌种类复杂多样。     

Using the TxtAB operon to quantify pathogenic Streptomyces in potato tubers and soil

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.