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。     

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.     

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.     

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.     

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.     

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.     

Effect of temperature on the transmission of Potato mop-top virus from seed tuber and by its vector, Spongospora subterranea

Potato mop-top virus (PMTV) causes disease in both the growing plant and tubers (spraing) of potato and is transmitted by the plasmodiophorid Spongospora subterranea , the cause of powdery scab. The effect of temperature during plant growth on the transmission of PMTV from infected seed tubers and from infested growing media was investigated in a series of glasshouse experiments. Symptoms developed on foliage of plants derived from infected seed tubers but none developed when PMTV was transmitted by S. subterranea in soil. The incidence of foliar symptoms was greatest on plants grown at 12°C, less at 16°C, few at 20°C and absent at 24°C. The transmission of PMTV from infected seed tubers was not significantly affected by temperatures between 12 and 24°C, but when the virus was transmitted by S. subterranea , minimal tuber infection occurred at 24°C and no differences were recorded at temperatures between 12 and 20°C. The incidence of powdery scab on tubers was greatest at 12 and 16°C and very low at 20 and 24°C. However, the incidence and severity of root galling caused by S. subterranea , was greatest at 20 and very low at 24°C. The incidence of powdery scab was greater on tubers of plants derived from infected seed tubers grown in a fluctuating temperature regime of 12 h at 20°C followed by 24 h at 12°C than on those grown at a constant 20°C, whereas the incidence of tuber infection by PMTV and spraing was similar for both regimes. This demonstrates that infection of roots can occur at a higher temperature than that for powdery scab on tubers and that this root infection can enable the transmission of PMTV into the potato plant.     

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.     

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.     

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.     

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。     

Effects of concanamycins produced by <Emphasis Type="Italic">Streptomyces scabies</Emphasis> on lesion type of common scab of potato

Streptomyces scabies, causative agent of common scab of potato, produces the phytotoxins concanamycin and thaxtomin. In a potato tuber slice assay to study the contribution of concanamycins to lesion development, concanamycin A had weak necrosis-inducing activities; >10× the amount of thaxtomin A was needed to produce equivalent lesion severity. Concanamycins were detected in tubers inoculated with S. scabies, which caused deep-pitted lesions but not in those inoculated with Streptomyces acidiscabies, which caused corky, raised lesions. In field-grown, diseased potatoes, concanamycin content tended to be higher in tubers with deep-pitted lesions than in those with corky, raised lesions.     

Reaction of potato cultivars to primary and secondary infection by potato mop-top furovirus and strategies for virus detection1

Potato cultivars in elite seed production in Finland were tested for susceptibility to primary and secondary infection by potato mop-top furovirus (PMTV) in field and pot experiments during 1987 and 1988. The tubers of all 22 cultivars tested became infected under favourable conditions in naturally infested soil, and most of them showed external and internal spraing symptoms when stored first at 18°C for 2 weeks and then 2 weeks at 8°C. The second incubation cycle increased the percentage of tubers manifesting symptoms and the halving of tubers also favoured symptom development. The cultivars most tolerant to primary infection were Pito, Hertha and Record, and the most susceptible were Sabina, Olympia, Saturna and Matilda. The same cultivars, excluding Saturna, were also the most susceptible to secondary infection, which decreased tuber yield up to 37%. The virus could be detected fairly reliably in soils by the bait-plant method, and in the tubers after a repeated cycle of incubation at 18 and 8°C. Electron microscopy and serological methods are useful in both virus detection and identification but they still have severe limitations.     

Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea

The effects of soil inoculum level and three environmental factors (soil type, soil moisture regime and temperature) on the incidence and severity of powdery scab caused by Spongospora subterranea were investigated in potato plants grown under controlled environmental conditions. Symptoms of powdery scab on tubers were assessed visually, after which DNA was extracted from tuber peelings and quantified in a real-time polymerase chain reaction assay using primers and a TaqMan® probe specific to S. subterranea to establish tuber infection levels. Soil inoculum concentration of S. subterranea did not significantly affect the incidence and severity of either tuber infection or powdery scab symptoms at maturity. No significant differences in disease incidence and severity were found between sandy, loamy and clay soils, although the two lighter soils yielded more powdery scab than clay soil. Constant dampness of the soil resulted in significantly more disease than a fluctuating moisture regime. Infection and disease levels were high at all three temperatures tested (9, 12 and 17°C), but symptoms were most severe at 12°C. The percentage of plants with infected tubers did not increase after tuber initiation, although the amount of S. subterranea DNA detected in tubers and the severity of powdery scab symptoms increased in mature plants. Latent tuber infections were found to be common, especially under conditions suboptimal for disease development. This new information may be important for the prevention of powdery scab in potato-growing areas around the world.     

Increased abundance of patatins,lipoxygenase and miraculins in a thaxtomin A-habituated potato Russet Burbank somaclone with enhanced resistance to common scab

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A-habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label-free quantitative proteomic analysis revealed changes in the accumulation of defence-related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz-type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf-bud cuttings. S. scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.     

Phytotoxin Production and Aerial Mycelium Formation by Streptomyces scabies and S. acidiscabies in Vitro

Culture conditions (pH, Ca²⁺, phosphate, and temperature) on phytotoxin production (thaxtomin A and concanamycins A and B) and aerial mycelium formation were examined for two strains each of Streptomyces scabies and S. acidiscabies. Thaxtomin production decreased at pH 7.5 and increased at 15°C in S. scabies and increased at 30 mM phosphate in S. acidiscabies. Concanamycin production was stimulated by the addition of CaCl₂ and was correlated with formation of aerial mycelium. Aerial mycelium formation in S. scabies was greater the higher the pH, Ca²⁺ concentration and temperature, whereas that in S. acidiscabies was unaffected. Received 2 November 2000/ Accepted in revised form 27 April 2001     

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.     

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。     

Factors affecting the incidence and severity of Spongospora subterranea infection and galling in potato roots

The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g⁻¹ soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g⁻¹ to 59% (15 sporosori g⁻¹) and 73% (50 sporosori g⁻¹) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.