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.     

Degradation of fluridone in submersed soils under controlled laboratory conditions

The experimental, aquatic herbicide fluridone (1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone) was degraded in two submersed soils and in the water above those soils to one acidic metabolite (identified as 1,4-dihydro-1-methyl-4-oxo-5-[3-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid by mass spectrometry). A sandy and a silt loam soil were treated with [¹⁴C]fluridone, immersed in water, and analyzed after 1, 3, 5, 7, 9, and 12 months. Seven to fifteen percent of the ¹⁴C applied to the soils was recovered in the water on each of the various collection dates. The acidic metabolite accounted for 86 to 93% of the radioactivity in the water fraction 7 months after treatment. The metabolite was absorbed strongly by both soils and comprised about 60% of the total ¹⁴C in each soil after 12 months. The remainder of the ¹⁴C in the soils after 12 months was either the parent compound (～30%) or an undefined insoluble residue (～10%).     

A TaqMan real-time PCR assay for <Emphasis Type="Italic">Rhizoctonia cerealis</Emphasis> and its use in wheat and soil

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays.     

Suppression of Rhizoctonia solani in potato fields. 1. Occurrence

A search was made forRhizoctonia solani-suppressive soils by establishing many small experimental plots, half of which were planted withRhizoctonia-infected seed potatoes and the other half with disinfected seed stock. The sclerotium index of the harvested tubers was compared witht that of the seed potatoes. In suppressive soils, the sclerotium index of the harvest is much lower than that of the seed potatoes. None of the plots on holocene marine soils (loamy sand, sandy loam, clay loam and clay) proved to be suppressive in 1978 and 1979. Only on pleistocene, slightly acid sandy soil suppressiveness was observed. In 1978, four out of twelve plots showed suppressiveness when the plots were planted with seed potatoes produced on a sandy soil. In 1979, only two out of thirtyone plots were slightly suppressive when planted with seed potatoes produced on a young clay loam from a new polder. A higher percentage of sclerotia on tubers from sandy soils proved to be infected with antagonistic fungi (73%) than of those on tubers from marine clay or loam soils (25%). Factors that influence suppressiveness are suggested.     

A new quantitative real-time PCR assay for Rhizoctonia solani AG3-PT and the detection of AGs of Rhizoctonia solani associated with potato in soil and tuber samples in Great Britain

Rhizoctonia solani is an important pathogen of potatoes causing stem canker and black scurf. The fungus is a species complex comprised of 13 known anastomosis groups (AGs). AG3-PT is the anastomosis group frequently associated with disease in potatoes. A real-time PCR assay was designed to the rDNA ITS region of AG3-PT isolates to enable the pathogen to be detected directly in tuber and soil samples. The resulting assay was highly specific for AG3-PT, and did not amplify DNA from isolates from other AGs or subgroups of AG3. Using a bulk DNA extraction method capable of extracting from up to 250 g of soil, the assay could detect one individual sclerotium of AG3-PT (weighing 200 μg) in 250 g of soil. The AG3-PT assay was used, with assays for AG2-1, AG5 and AG8 to determine the prevalence of those AGs in UK potato soils and tubers. AG2-1 and AG3-PT were the predominant groups in tubers and soils, although AG3-PT was more frequently isolated from tubers, highlighting its importance as a potato pathogen. AG3-PT was also detected in more than half of the tuber samples tested suggesting the importance of seed borne inoculum.     

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.     

Suppression of Rhizoctonia solani in potato fields. II. Effect of origin and degree of infection with Rhizoctonia solani of seed potatoes on subsequent infestation and on formation of sclerotia

The seed potatoes used in these experiments had been grown in a slightly acid pleistocene sandy soil or in a marine, holocene sandy loam. They were free of sclerotia ofR. solani or lightly or moderately speckled with them. Seed potatoes from the sandy soil produced plants that suffered less fromRhizoctonia than plants from seed potatoes that had been grown on the marine sandy loam. Similarly harvested tubers had, in a non-conducive soil and in conducive soils with a (very) low inoculum density ofR. solani, fewer sclerotia when they came from seed potatoes grown in an acid sand. In each soil, the degree of infestation of the crop not only depended on the severity of infection of the seed potatoes, but also on their origin. With regard to sclerotia production on tubers, three types of soil were distinguished: suppressive, conducive with a high, and conducive with a very low inoculum density ofR. solani. The differences in infestation and in the amounts of sclerotia on tubers between the crop grown from seed potatoes from the sandy soil and that from seed potatoes from the marine sandy loam soil, is attributed to a richer load of antagonists on the former and possibly to a larger proportion of saprophyticRhizoctonia strains among their sclerotia. The antagonists seem to be inhabitants of the subterranean parts of the plant and to function independently of the soil. This implies possibilities for their use in biological control.     

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。     

Characterization of Pectobacterium carotovorum subsp. carotovorum and brasiliense from diseased potatoes in Kenya

Using a DNA-based typing method, 48 bacterial strains isolated from infected potato (Solanum tuberosum) tubers originating from Kenya were characterized. The pel gene specific primers showed that all the 48 bacterial strains were pectolytic. Subspecies-specific primers EXPCCF/EXPCCR and Br1f/L1r identified 66 % of the strains as Pectobacterium carotovorum subsp. carotovorum while 34 % were identified as Pectobacterium carotovorum subsp. brasiliense based on their characteristic band sizes of 550 and 322 bp, respectively. Amplification of the 16S-23S rDNA (ITS) region did not yield observable differences in banding patterns between the Kenyan strains. However, PCR-RFLP analysis together with partial nucleotide sequences of the housekeeping mdh and gapA genes confirmed the results obtained by the specific primers. Phylogenetic analysis of the concatenated partial gene sequences grouped Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliense Kenyan strains together with those identified in other parts of the world with 90 % and 99 % bootstrap support values, respectively. Pathogenicity assays using representative Kenyan strains demonstrated varied levels of tuber maceration ability. The Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. brasiliense Kenyan strains were shown to be less aggressive in causing soft rot when compared to type strains. This study describes for the first time the genetic diversity of pectolytic bacteria causing soft rot disease of potatoes in Kenya.     

Biochemical,physiological, and molecular characterization of <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> (formerly named <Emphasis Type="Italic"> Erwinia chrysanthemi</Emphasis>) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.     

A paucity of bacterial root diseases: Streptomyces succeeds where others fail

There are relatively few bacterial diseases of roots, in comparison to those of aerial plant tissues. Numerous species and pathovars of Pseudomonas,Erwinia and Xanthomonas are important pathogens of leaf and stem tissue on dozens of plant families but these bacterial genera only infrequently attack roots or other underground plant structures. In contrast, there is a growing list of Streptomyces species that are very effective root pathogens. These filamentous, Gram-positive bacteria can cause scab, rot and gall diseases of plant roots and other underground plant structures. The best known pathogenic Streptomyces species is S. scabiei. Horizontal transfer of pathogenicity genes among diverse scab-causing streptomycetes appears to explain the emergence of several new plant pathogens over the last half century. It is proposed that the ability to penetrate plant tissue is essential for successful root infection as there are few natural openings in roots. In contrast, leaves have many natural openings that allow bacteria access to the interior tissues. Thaxtomin, a phytotoxin produced by many plant pathogenic streptomycetes, appears to aid penetration of developing plant tissues by inhibiting primary cell wall development.     

Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum

The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.     

First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Japan

In October 2010, a bacterial disease produced flecks and spots on leaves of Chinese cabbage, cabbage and Japanese radish in Nagano Prefecture, Japan. The symptoms started on the abaxial surface of leaves as angular, water-soaked flecks of 1–2 mm in diameter with a yellow halo of 3–4 mm width. These flecks then became visible on both leaf surfaces, enlarged and coalesced into large blight lesions. The symptoms were similar to bacterial leaf spot caused by Pseudomonas syringae pv. maculicola. The bacterium isolated from leaf lesions formed a white colony and produced polysaccharides on YP agar. The isolates were identified as P. syringae group by LOPAT tests and the 16S rDNA sequence. Moreover, the results of pathogenicity on cruciferous plants, bacteriological characteristics, rep-PCR and the sequences of rpoD and gyrB showed that the isolates should be identified as P. cannabina pv. alisalensis (recently transferred from P. syringae pv. alisalensis). This is the first report of P. cannabina pv. alisalensis isolated from diseased crucifers in Japan.     

Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Host and biotic factors affecting sporulation ofstemphylium botryosum f. sp. lycopersici on tomatoes and ofalternaria porri f. sp. sol ani on potatoes

Factors known to inhibit sporulation of bio trophic fungal pathogens were found to enhance sporulation of two necrotrophic fungi. The sporulating potential ofStemphyliurn botryosum f. sp.lycopersici on tomatoes and ofAlternaria porri f. sp.solani on potatoes increased with necrotization, reaching a maximum on dead leaves. Wetting the dead leaves for the whole period of incubation with increasing concentrations of glucose resulted in progressively decreasing sporulation of both pathogens. However, application of glucose during the first half of the incubation period inhibited sporulation ofS. botryosum f. sp.lycopersici on tomatoes only a little, and increased that ofA. porri f. sp.solani on potatoes. The capacity ofS. botryosum f. sp.lycopersici to sporulate on leaves lasted for 12 weeks at 29°C, and ofA. porri f. sp.solani for 12 weeks at 29°C and for over 21 weeks at 20°C. The results emphasized basic differences in sporulation between biotrophic and necrotrophic parasites. Specific techniques useful for studying sporulationin vivo are discussed.     

Temporal association of potato tuber development with susceptibility to common scab and Streptomyces scabiei‐induced responses in the potato periderm

Using hydroponics and novel non‐destructive pot culture systems which enable inoculation at specific tuber development stages, the dynamics of common scab infection patterns in potato were studied in order to provide more precise identification of tuber physiological factors associated with susceptibility. At the whole‐tuber level, infection percentages were greatest when Streptomyces scabiei inoculation occurred early; at 2 weeks after tuberization (WAT) 68% of tubers became infected, contrasting with late inoculation (8 WAT), when only 4% infection occurred. The first‐formed internodes were most susceptible to infection, whilst later‐forming and slower‐expanding internodes were less susceptible. Detailed tuber physiological examination of internode 2 showed that pathogen‐induced changes, including increased phellem (periderm) thickness, cell layers and phellem suberization (key physiological features believed critical to S. scabiei infection) were promoted through S. scabiei inoculation. Sequential harvesting showed enhanced phellem suberization (28% greater than the control) within 7 days of pathogen exposure, while phellem thickness and layer responses were also initiated early in the infection process (10–14 days after pathogen exposure) and these responses were independent of symptom expression. Differences in cultivar response were observed, with greater phellem suberization observed 10 days after tuberization (DAT) in the common‐scab‐tolerant cv. Russet Burbank than in the susceptible cv. Desiree. Likewise, Russet Burbank had thicker and more numerous cell layers in the phellem (up to eight cell layers) during early tuber growth (20–30 DAT) than Desiree (up to six cell layers).     

Accumulation of hyperparasites of Rhizoctonia solani by addition of live mycelium of R. solani to soil

Addition of live mycelium ofRhizoctonia solani to three slightly, acid sands collected in winter from a field after a previous potato crop resulted in accumulation of hyperparasites and less infestation of sprouts of infected seed potatoes. A similar effect was observed in alkaline marine sandy loam soils, but only after a second addition of live mycelium. The predominant hyperparasite in these experiments wasVerticillium biguttatum.     

Occurrence of black rot of cultivated mushrooms (Flammulina velutipes) caused by Pseudomonas tolaasii in Korea

From black spots on winter mushroom (Flammulina velutipes), fluorescent bacteria were repeatedly isolated during surveys at places of production in the years 2009–2010 in Korea. From these lesions three bacterial strains (designated CHM13, CHM16, CHM17) were isolated which, following inoculation of mushroom stipes and caps, yielded characteristic black spots and sunken lesions, which developed into a severe black rot. Results of Gram stain and biochemical tests preliminarily identified these isolates as Pseudomonas tolaasii. This was confirmed by pathogenicity to winter mushroom, physiological and biochemical properties, analysis of the 16S rRNA and rpoB gene sequences, fatty acids profile, specific and sensitive PCR assays and, lipopeptide detection. This is the first report of the isolation of Pseudomonas tolaasii from cultivated winter mushroom in Korea.     

Seedling blight of Glycyrrhiza uralensis caused by Pythium myriotylum,P. aphanidermatum and P. spinosum and identifying primary inoculum sources using multiplex PCR detection

Pythium species, isolated from seedlings of Glycyrrhiza uralensis with blight, were identified as P. myriotylum, P. aphanidermatum, and P. spinosum on the basis of morphological characteristics and sequences of the internal transcribed spacer regions of rDNA. In pathogenicity tests, the isolates of the three Pythium species caused blight, producing the original disease symptoms. The primary inoculum source was determined using a multiplex PCR to detect the pathogen. All the Pythium species were detected in the soils of fields with the diseased plants and in soils of adjacent field soils.     

The effect of different ways of watering pots after the application of aldicarb on control of the potato cyst-nematode

The multiplication ofHeterodera rostochiensis on potatoes in pots was reduced more strongly by aldicarb mixed with the soil than by aldicarb applied to the top of the soil. Excessive amounts of water strongly reduced the effectiveness of the first method of application; moderate amounts of water did not improve the second method sufficiently.