Infection of Potato Tubers with the Common Scab Pathogen Streptomyces scabiei in a Soil‐less System

A novel soil‐less method was developed to define susceptibility of developing potato tubers accurately to infection with Streptomyces scabiei the causal agent of common scab disease. Hydroponic production enabled precise identification of individual tuber development. Direct inoculation of tubers with a spore suspension of S. scabiei resulted in disease development, demonstrating that infection could be initiated in a soil‐less media. Tubers were most susceptible to infection between 3 and 20 days after tuber initiation, confirming that this early period of tuber formation is critical to disease development.     

Staining Streptomyces Scabies in Lesions of Common Scab of Potato

Toluidine blue can be used to stain Streptotnyces scabies distinctively in slide cultures or in the lesions of common potato scab. This staining method is based on the metachromaticism of volutin, a constant constituent of the spores and mycelium of S. scabies. Either sections or smears are fixed in FPA (formalin, 5 parts; propionic acid, 7.5; 50% alcohol, 87.5), stained in a 1:100 dilution of saturated aqueous toluidine blue from 20 minutes to 24 hours, dehydrated in an acetone-xylene series and mounted. Cellular constituents of the potato tuber stain blue or are colorless whereas the mycelium of Streptomyces appears as a series of red volutin spheres in the blue stained cytoplasm. The criteria of volutin and cytoplasmic staining along with the 1 µ diameter of the mycelium make it possible to distinguish Streptomyces from the other microorganisms and cells in the lesion region.     

Growth-Regulated Expression of a Bacteriocin,Produced by the Sweet Potato Pathogen Streptomyces ipomoeae,That Exhibits Interstrain Inhibition

Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes.Streptomyces bacteria are gram-positive spore-forming soil bacteria which display a complex life cycle (4, 6). Spore germination leads to vegetative growth as tangled masses of substrate mycelia on solid surfaces. Nutrient limitation triggers production of secondary metabolites as well as growth of vertically extending aerial hyphae, which are morphologically distinct filaments that derive nutrients from the dying substrate cell layer. Eventually, individual aerial hyphae differentiate into chains of spores. Streptomyces mycelia growing in liquid media display a multiphasic growth curve similar to that of unicellular bacteria. As the culture enters stationary phase, Streptomyces bacteria produce secondary metabolites but do not typically differentiate morphologically (2).Streptomyces ipomoeae is the causative agent of soil rot, the widespread and destructive disease of sweet potatoes. The disease results in decay of fibrous feeder roots and development of necrotic lesions on the edible storage roots (9). Prevention of soil rot currently relies solely on development of resistant sweet potato cultivars; however, alternative approaches, including the use of biocontrol methods, are now beginning to be investigated.Previously, 36 strains of S. ipomoeae were divided into three groups based on interstrain inhibition during their cocultivation on agar plates (7). The group III strains were found to produce a diffusible 10-kDa bacteriocin-like protein (ipomicin), which is inhibitory to group I and II members and which is stable in culture supernatants for at least 1 h at 40°C (in a pH range of 6 to 10) (26). Sequence analyses of the ipomicin structural gene (ipoA) and its protein product revealed that ipomicin is initially expressed in precursor form, which upon processing of an N-terminal signal sequence becomes the 10-kDa mature form (26).Given its potential as a biocontrol agent, we were interested in elucidating the expression characteristics of ipomicin. It was noted previously that TTA, the rarest codon found within the GC-rich Streptomyces genus, is present once in ipoA within the region that designates the ipomicin signal sequence (26). TTA codons in Streptomyces bacteria are recognized by a single leucyl tRNA species encoded by the gene bldA, which is required for normal morphological and physiological differentiation in these organisms (5, 14). The latter effect is due in part to the fact that regulatory genes of antibiotic clusters and aerial hyphae development also contain TTA codons (5). In the model streptomycete, Streptomyces coelicolor, bldA has been shown to be an effective growth phase regulator because it is expressed initially in liquid and surface cultures as an inactive precursor and, upon processing, begins to temporally accumulate as a mature tRNA, at least under certain physiological conditions (15, 24). Interestingly, TTA-containing genes show variation in their dependence on bldA for expression, with some genes demonstrating strong dependence while others show only partial or no apparent dependence (14, 23).Here, stable ipomicin protein is shown to be temporally produced during S. ipomoeae growth in a manner inconsistent with ipoA mRNA levels. Similar contrary results for ipomicin protein and ipoA mRNA concentrations were observed during growth of the heterologous host S. coelicolor expressing a cloned version of the S. ipomoeae ipoA gene, and this effect was shown to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The data here suggest that regulation by bldA leads to temporal production of the ipomicin precursor, which when processed to the mature form is secreted to the external environment, where it accumulates.     

Determination of Factors Associated with Natural Soil Suppressivity to Potato Common Scab

Common scab of potatoes is a disease, which is difficult to manage due to complex interactions of the pathogenic bacteria (Streptomyces spp.) with soil, microbial community and potato plants. In Bohemian-Moravian Highlands in the Czech Republic two sites (Vyklantice and Zdirec) were selected for a study of common scab disease suppressivity. At both sites, a field with low disease severity occurs next to one with high severity and the situation was regularly observed over four decades although all four fields undergo a crop rotation. In the four fields, quantities of bacteria, actinobacteria and the gene txtB from the biosynthetic gene cluster of thaxtomin, the main pathogenicity factor of common scab, were analyzed by real-time PCR. Microbial community structure was compared by terminal fragment length polymorphism analysis. Soil and potato periderm were characterized by contents of carbon, nitrogen, phosporus, sulphur, calcium, magnesium, and iron. Quality of organic matter was assessed by high performance liquid chromatography of soil extracts. The study demonstrated that the suppressive character of the fields is locally specific. At Zdirec, the suppressivity was associated with low txtB gene copies in bulk soil, while at Vyklantice site it was associated with low txtB gene copies in the tuberosphere. The differences were discussed with respect to the effect of abiotic conditions at Zdirec and interaction between potato plant and soil microbial community at Vyklantice. Soil pH, Ca soil content or cation concentrations, although different were not in the range to predict the disease severity. Low severity of common scab was associated with low content of soil C, N, C/N, Ca and Fe suggesting that oligotrophic conditions may be favorable to common scab suppression.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Effect of Pathogen Isolate, Potato Cultivar, and Antagonist Strain on Potato Scab Severity and Biological Control

Antibiotic-producing Streptomyces spp. have shown potential in biocontrol of potato scab caused by Streptomyces scabies. However, results have been inconsistent among field trials. Pathogen isolate, antagonist strain and potato cultivar were investigated as potential sources of variation in the efficacy of potato scab biocontrol. Biocontrol success varied significantly among pathogen isolates and was not correlated with in vitro sensitivity to antibiotic inhibition. Antagonists also varied in their effectiveness as biocontrol agents, and the relative effectiveness of different antagonists varied among growing seasons. Finally, biocontrol varied among potato cultivars in the field. The diverse origins of significant variation in potato scab biocontrol suggest that consistent control in the field is likely to be difficult to achieve.     

An In Vitro Inhibition Test That Predicts Toxicity of Bacterial Pathogen Combinations in the Colorado Potato Beetle

Many insect bacterial pathogens are not toxic enough for field control. Combinations of bacteria may increase toxicity. Bacteria toxic to Colorado potato beetle , Photorhabdus luminescens, Chromobacterium violaceum and Serratia marcescens , were tested in pair-wise combinations in an in vitro double streak test to determine bacterial compatibility. Only C. violaceum and S. marcescens grew to confluency. Their combined toxicity in vivo was additive. Other bacterial combinations had clear zones between bacterial streaks indicating inhibition. In the insect, the combined toxicity was less than the most toxic bacteria of the pair. For these strains, this in vitro test predicted compatibility in the insect.     

试管内双歧杆菌、复方双歧杆菌制剂对肠道致病菌的拮抗作用

试管内,两歧双歧杆菌(6-1)株对福氏志贺氏痢疾杆菌和侵袭性大肠杆菌都具有一定的拮抗作用。复方双歧杆菌制剂对福氏志贺氏痢疾杆菌具有很强的拮抗作用,甚至能完全抑制其生长繁殖。提示,在抑制肠道致病菌方面,复方双歧杆菌制剂要比单一的双歧杆菌具备更强的效能和更多的优越性。     

Reduction of Yield Depressions in High Frequency Potato Cropping Soil after Seed Tuber Treatments with Antagonistic Fluorescent Pseudomonas spp.

Severe yield depressions observed in high frequency potato cropping caused by microbial factors other than eelworms or known pathogens (narrow rotation effect) were reproduced in pot experiments. Total plant weight in a soil continuously cropped with potatoes was only 69% of that in a similar soil continuously cropped with wheat. Tuber weight in potato-soil was only 13 % and number of tubers only 10% of that in wheat-soil. Seed tuber treatment with cell suspensions of fluorescent antagonistic pseudomonads in carboxymethylcellulose (CMC) raised tuber yield in potato-soil up to 70% (550 % increase), number of tubers up to 93% (934 % increase) and total plant weight up to 90% (31 % increase) as compared with CMC-treatment in wheat-soil. Antagonistic fluorescent Pseudomonas isolates WCS 365, WCS 358 and WCS 374 producing mainly siderophores, with little or no production of other inhibitory substances, were more effective in diminishing the narrow rotation effect than the isolates that produced mainly inhibitory substances of different nature.     

Interstrain Inhibition in the Sweet Potato Pathogen Streptomyces ipomoeae: Purification and Characterization of a Highly Specific Bacteriocin and Cloning of Its Structural Gene

Strains of the sweet potato soil rot pathogen Streptomyces ipomoeae had previously been divided into three groups based on their ability to inhibit one another during pairwise cocultivation. While group I strains are not antagonistic to members of the other groups, group II and group III strains produce separate substances that are inhibitory to strains outside their respective cognate groups. Here, we purified the group III inhibitory substance from the culture supernatant of a representative strain and found that it consists of a single 10-kDa cationic protein which is bacteriolytic for S. ipomoeae group I and II strains but which showed no inhibitory function against other streptomycetes or other bacterial genera tested. The structural gene for the inhibitor was cloned from a chromosomal library of the producing strain, and while the gene sequence revealed that the inhibitor is initially made in a larger precursor form, the deduced mature protein showed no significant homology to other known proteins. Our results demonstrate that S. ipomoeae group III inhibitory activity is manifested in the form of a highly specific, potentially novel bacteriocin, which we have designated ipomicin.     

Selection of Antagonistic Fluorescent Pseudomonas spp. and their Root Colonization and Persistence following Treatment of Seed Potatoes

One hundred and twelve fluorescent Pseudomonas isolates from potato periderm were screened for antagonistic activity in vitro against 5 pathogenic and saprophytic fungi, 5 Gram-positive bacteria including Streptomyces scabies and 4 Gram-negative bacteria including Erwinia car . var. carotovora and Erwinia car . var. atroseptica . Fifty-seven percent of the isolates showed wide spectrum inhibitory activity, mostly due to the production of fluorescent siderophores. Only 4 % of the isolates were very strong antagonists. By adding iron (Fe³+) to the test medium, the antagonistic activity drastically diminished but revealed some strong antagonists of which the antagonism was not affected. Apparently growth-inhibiting substances other than siderophores were involved. Antibiotic-resistant mutants of the antagonists were used to establish that the bacteria on the seed tubers migrated to the roots under different conditions. Drying of the treated potatoes gradually diminished the number of viable cells, but subsequent planting in moist soil led to substantial increases in the first three days. Initially high numbers of the antibiotic-resistant antagonists on the roots tended to drop quickly in time, probably due to loss of resistance to the antibiotics in the selective medium.     

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.     

Growth Promotion of Fluorescent Pseudomonads and Control of Potato Common Scab in Field Soil with Non-antibiotic Actinomycete-biofertilizer

Field studies on the control of potato common scab were done with non-antibiotic Actinomycete biofertilizer, which was manufactured from swine feces with coprophilous actinomycetes and acidified to pH 5.0 with sulfuric acid. The field soil at pH 5.0 was mixed with 0.56 kg of the acidified biofertilizer per subplot (1.0 x 1.2 m) to a depth of the 15 cm. The scab severity, in terms of the percent coverage of the total surface area of potato tubers, was 10.7%, in the control, while the lesions were slight in the biofertilizer subplot, scab severity being only 2.30/0, and the potato production was increased 3 times with the biofertilizer. The viable counts of Streptomyces scabies in the biofertilizer subplot decreased to 1 × 10³ per gram soil from 5 × 10⁵ by 12 weeks, while the inhabitant fluorescent pseudomonads increased to 2.4 × 10⁷ per gram soil from 3 × 10⁴, and the increased fluorescent pseudomonads were antagonistic to the pathogenic strains.     

Global Genetics and Invasion History of the Potato Powdery Scab Pathogen,Spongospora subterranea f.sp. subterranea

Spongospora subterranea f. sp. subterranea (Sss) causes two diseases on potato (Solanum tuberosum), lesions on tubers and galls on roots, which are economically important worldwide. Knowledge of global genetic diversity and population structure of pathogens is essential for disease management including resistance breeding. A combination of microsatellite and DNA sequence data was used to investigate the structure and invasion history of Sss. South American populations (four countries, 132 samples) were consistently more diverse than those from all other regions (15 countries, 566 samples), in agreement with the hypothesis that Sss originated in South America where potato was domesticated. A substantial genetic differenciation was found between root and tuber-derived samples from South America. Estimates of past and recent gene flow suggested that Sss was probably introduced from South America into Europe. Subsequently, Europe is likely to have been the recent source of migrants of the pathogen, acting as a “bridgehead” for further global dissemination. Quarantine measures must continue to be focussed on maintaining low global genetic diversity and avoiding exchange of genetic material between the native and introduced regions. Nevertheless, the current low global genetic diversity of Sss allows potato breeders to select for resistance, which is likely to be durable.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star‐shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.     

Variability within Clones of Potato cv. Russet Burbank to Infection and Severity of Common Scab Disease of Potato

Significant variability in the level of resistance to common scab disease (to both disease incidence – tuber surface area affected, and severity – lesion depth) within potato ( Solanum tuberosum ) cv. Russet Burbank clonal lines and other selected processing cultivars was shown. Across two trials, cvs. Russet Nugget and Shepody and Russet Burbank clone British Columbia were consistently grouped within the cluster of least disease incidence whereas cv. Russet Norkotah and Vancouver unit four were grouped within the cluster showing greatest disease incidence. Russet Nugget and British Columbia were also consistently grouped within the cluster showing least disease severity, whereas Shepody and Russet Burbank clone Starks were grouped within the cluster with greatest severity scores. For some clones, consistency of rating across trials was not always apparent. Of note for the Tasmanian industry, there were differences within the five commercial Vancouver clonal units used interchangeably, with unit 4 showing significantly greater disease severity and incidence than some of the other units. However the extent of increased disease never exceeded 1.75 times the best of the other Vancouver units and the relevance of these results to the relative field performance of unit 4 remains to be tested.