Effect of Streptomyces melanosporofaciens strain EF-76 and of chitosan on common scab of potato

The efficiency of Streptomyces melanosporofaciens strain EF-76, a geldanamycin producer, and of chitosan, a polymer derived from chitin that elicits plant defense mechanisms, to protect potato tubers against common scab was evaluated under both controlled and field conditions (years 2000 and 2001). S. melanosporofaciens EF-76 reduced disease incidence in the greenhouse assay and in the 2001 field assay. EF-76 also reduced symptom severity on potato tubers grown under field conditions. Chitosan provided a protective effect against S. scabies,the causal agent of potato common scab during the 2000 field assay by reducing both disease incidence and symptom severity. Combination of S. melanosporofaciensEF-76 and chitosan ensures a level of protection that was at least equivalent to the protection conferred by one of the two products used alone. In some instances, an additive effect of protection was observed when both products were used in combination. Combination of S. melanosporofaciensEF-76 and of chitosan thus represents a promising method of biocontrol against common scab.     

Streptomycete spores entrapped in chitosan beads as a novel biocontrol tool against common scab of potato

Spores of Streptomyces melanosporofaciens EF-76, an actinomycete that inhibits the growth of several plant pathogens, were incorporated in beads of chitosan and polyphosphate using the entrapment technique called complex coacervation. The degradation of spore-loaded beads was monitored by measuring the residual amount of chitosan in soil and by enumerating the S. melanosporofaciens population over time. After the introduction of spore-loaded chitosan beads into soil, the amount of chitosan in sterile soil remained at 1.550 mg/g throughout the first week and diminished to 0.101 mg/g after 7 weeks. Bead degradation proceeded faster in non-sterile soil but a progressive release of both chitosan oligomers and the antagonistic microbial agent was nevertheless observed. Application of these spore-loaded chitosan beads to seed potato tubers protected progeny tubers against common scab.     

Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion

Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin. Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390 were characterized. FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them. FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76. Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54. Strain FP-60 showed no protective effect against common scab. The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.     

Inoculum Density and Population Dynamics of Suppressive and PathogenicStreptomycesStrains and Their Relationship to Biological Control of Potato Scab

SeveralStreptomycesstrains are capable of suppressing potato scab caused byStreptomyces scabies.Although these strains have been successful in the biocontrol of potato scab in the field, little is known about how populations of pathogenicStreptomycesin the potato rhizosphere are influenced by inoculation of the suppressive strains. The effects of inoculum densities of pathogenic and suppressiveStreptomycesstrains on their respective populations on roots and in rhizosphere soil were examined during the growing season. The relationships between inoculum density or rhizosphere population densities and disease severity were also investigated. Populations of suppressiveStreptomycesstrain 93 increased significantly on roots with increasing inoculum dose. At its highest inoculum dose, the suppressive strain reached a population density greater than 10⁶CFU/g root 14 weeks after planting. The ability of the suppressive strain to increase its populations with increasing inoculum density was hindered at high inoculum doses of the pathogen, suggesting that density-dependent competitive interactions may be occurring between the two antagonists. Strain 93 was most effective at preventing scab early in the growing season (8 weeks after planting), when tubers were most susceptible to the scab disease. Population densities of the suppressive strain in soil were more highly negatively correlated with scab severity than were populations on roots, suggesting that rhizosphere soil rather than potato roots may be the primary source of inoculum of the suppressive strain for tubers.     

Management of common scab of potato in the field using biopesticides,fungicides, soil additives,or soil fumigants

Common scab, caused by Streptomyces scabiei is an economically important potato disease worldwide. The potato industry in New Brunswick, Canada experience $1.2 million loss every year due to this disease. Superficial, raised, or deep-pitted brownish lesions on infected tubers reduce the quality and marketability of both fresh-market and processing potatoes, and hence, common scab is considered a priority disease for which adequate control measures are lacking. The objective of this research was to compare various potential treatments in suppressing the disease. Two field experiments were conducted at McCain's Research Farm, Florenceville-Bristol, New Brunswick, Canada, in 2008 and 2009 to assess the efficacy of Bacillus subtilis, Enterobacter cloacae, Chloropicrin, Pic-Plus, manganese sulphate and mustard meal in comparison with the chemical controls fludioxonil and mancozeb against common scab of potato. The disease incidence was significantly reduced by 36% due to the addition of mustard meal to the soil; 35.4% due to fludioxonil seed treatment; 30.0% due to soil fumigation with Pic-Plus; or by 27.2% due to soil fumigation with Chloropicrin. Potato tubers with scab severity ≥5% which are considered unmarketable in Canada were significantly reduced by 56.1% due to seed treatments with B. subtilis; by 57.8% due to fludioxonil; or by 63.1% due to the soil addition of mustard meal. The same treatments significantly increased marketable yield by 32.5%, 24.6%, and 24.6%, respectively. Soil fumigation with Chloropicrin or Pic-Plus increased marketable yield by 9.5% or 7.1%, respectively. These findings indicate that, in addition to the fludioxonil seed treatment, the seed treatment with the biopesticide containing B. subtilis and the soil addition of mustard meal treatments are potential alternatives for managing common scab of potatoes.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

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.     

Genome shuffling enhances biocontrol abilities of Streptomyces strains against two potato pathogens

Aims: To employ the genome shuffling technique for improving the phenotype of a biocontrol control agent of the genus Streptomyces. Methods and Results: Two rounds of genome shuffling (GS) were carried out with Streptomyces melanosporofaciens EF‐76, a geldanamycin producer. Six fusants that showed optimized in vitro antagonistic activity against Streptomyces scabies or Phytophthora infestans, two important pathogens of potato crops, were selected. All selected fusants retained the capacity to produce geldanamycin, but none overproduced this antibiotic. The higher antagonism ability appeared to result from a diversification of secreted metabolites. Seven or eight metabolites were detected in the HPLC profiles of parental strains, whereas 12–15 were detected in fusant strains. Biocontrol assays revealed that four of six fusants protected tubers more efficiently than parental strains. Conclusions: GS emerged as an elegant and rapid tool to optimize the antagonistic ability of Streptomyces strains. Optimization of the in vitro antagonistic activity against plant pathogens appears to be an effective approach to select for improved biocontrol agents. The enhanced phenotype did not depend on an overproduction of a specific antibiotic but rather on the secretion of a wider variety of secondary metabolites. Significance and Impact of the Study: Improved capacities of a biocontrol agent compensate for the lack of efficient chemical control of potato scab.     

Bacillus altitudinis strain AMCC 101304: a novel potential biocontrol agent for potato common scab

ABSTRACT

Potato common scab, caused by Streptomyces spp., is one of the leading causes of heavy commercial losses in the potato industry and is thus one of the most serious plant diseases worldwide. This study identified and assessed potential biocontrol agents against potato common scab. In total, 110 isolates were obtained through antagonistic tests; among which, Bacillus sp. strain AMCC 101304 was found to be most effective at inhibiting the potato common scab pathogen, Streptomyces scabies. Bacillus sp. strain AMCC 101304 was finally identified as Bacillus altitudinis by morphological observation, physiological and biochemical experimentation, as well as 16S rRNA sequencing and phylogenetic analysis. Pot experiments were conducted twice (in spring and autumn) to verify the biocontrol effect of B. altitudinis AMCC 101304 against potato common scab. In spring, the control efficiency reached 76.34%. In autumn, the disease incidence was reduced from 100% to 34.19% (one treatment with strain AMCC 101304) and 38.42% (two treatments with strain AMCC 101304), and the control efficiency reached 82.50% (one application) and 78.43% (two applications). The present study demonstrated the potential of an isolate, identified as B. altitudinis AMCC 101304, as an effective biocontrol agent for future use in the field.     

Effects of foliar sprays of daminozide on the incidence of potato common scab

Single foliar sprays of the growth retardant daminozide (1.5–12 g/l) approximately halved the incidence of common scab, caused by soil-borne Streptomyces scabies, on potted potato plants in the glasshouse. Two analogues of daminozide (N-dimethylaminomaleamic and N-(dimethylamino)-methylsuccinamic acids) also decreased scab, but others were inactive. Of 22 other unrelated growth regulators and translocated chemicals tested as foliar sprays, only gibberellic acid (0.1 g/l) decreased scab incidence, but many of the tubers were distorted. Chlormequat chloride and chlorphonium chloride, as root treatments, were inactive. In other experiments with daminozide, scab incidence was decreased after application to soil. In tests with two plants per pot, spraying one of each pair decreased its rate of stem extension, but did not affect the other, indicating that little or no daminozide passed into the soil from the roots of the sprayed plant. The decrease in scab brought about by foliar sprays was not altered by varying their timing during the period before symptom development (1 to 5 wk after potting). In agar plate tests, daminozide was only weakly toxic to S. scabies. It is concluded that daminozide probably decreased scab by altering the physiology of the plants, so that scab symptoms did not develop.     

Characterization of Streptomyces scabies mutants deficient in melanin biosynthesis

Streptomyces scabies, a causal agent of common scab, produces both melanin and a secondary metabolite called thaxtomin A. To establish a possible relation between melanin and thaxtomin A production in S. scabies, we carried out N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and isolated 11 melanin-negative mutants of S. scabies EF-35. These mutants were characterized for thaxtomin A production, pathogenicity, sporulation, and stress resistance. Nine of these mutants showed a significant reduction in thaxtomin A production when compared with the wild strain. However, only a few mutants exhibited a reduced level of virulence or a loss in their ability to induce common scab symptoms on potato tubers. Other pleiotrophic effects, such as higher sensitivity to heavy metals and incapacity to sporulate under certain stress conditions, were also associated with a deficiency in melanin production.     

Responses of potato cultivars to the common scab pathogens, Streptomyces scabies and S. turgidiscabies

A glasshouse experiment was conducted to study the responses to Streptomyces scabies and S. turgidiscabies in potato cultivars Bellona, Matilda and Sabina (Solanum tuberosum). Potatoes were grown in a peat‐sand mixture inoculated with one of the two strains of either S. scabies or S. turgidiscabies. Logit models were used to analyse the data on disease incidence and severity, whereas the data on emergence and yield were tested by analysis of variance. S. turgidiscabies, a recently described potato pathogen in Finland, possessed a high ability to cause superficial, raised and pitted lesions on all three cultivars tested. Symptoms induced by S. turgidiscabies were similar to those of S. scabies, regardless of the cultivar, which suggests that the two causal organisms of common scab cannot be distinguished based on symptoms. Infection by S. turgidiscabies and S. scabies delayed emergence, had the tendency to decrease the yield, and increased the proportion of small tubers in the yield, regardless of the potato cultivar. Differences in the levels of resistance to common scab were evident between potato cultivars, since cvs. Matilda and Bellona showed higher disease incidence and more severe scab symptoms than cv. Sabina.     

Effects of rotation length, fungicide treatment of seed tubers and nematicide on diseases and the quality of potato tubers

In 1982 – 88, potatoes were grown in 2-, 4- and 6-course rotations with spring barley on a field infested with Globodera rostochiensis. Severity of stem canker and black scurf increased with increasing frequency of previous potato crops, and seed tuber treatment with tolclofos-methyl became less effective in controlling diseases. This suggested that previous crops had increased the amounts of soil-borne inoculum of Rhizoctonia solani. Oxamyl soil treatment increased stem canker in one year and decreased black scurf in four years. Seed tuber treatment with imazalil or prochloraz decreased stem base infection by Polyscytalum pustulans and skin spot and silver scurf on tubers. Black dot was prevalent on tubers in all years and was not affected by seed tuber treatment or previous cropping. Oxamyl increased black dot and common scab in five years and decreased % tuber dry matter in six years. Cysts of G. rostochiensis were found attached to Désirée but not to Maris Piper tubers in August. At harvest tubers of both cultivars were affected by superficial pitting and its severity was related to soil populations of G. rostochiensis at planting. This damage was controlled by oxamyl. It is suggested that the pitting developed from holes made in the tuber skin at larval invasion. In 1989, Désirée seed tubers and healthy mini tubers were planted in all plots and severity of stem canker and black scurf increased with increasing proximity of previous potato crops and with the number of previous crops. Black dot on stems and tubers was not affected by previous cropping but was much less severe in a plot that had not grown potatoes during the seven years of the experiment. The severity of common scab generally decreased as the number of preceding potato crops increased.     

Green manures and crop sequences influence alfalfa root rot and pathogen inhibitory activity among soil-borne streptomycetes

A two-year trial was conducted to determine the effects of green manures and crop sequences on plant disease, streptomycete and bacterial densities, and inhibitory activity of indigenous streptomycetes against four target pathogens. Green manure treatments, buckwheat (Fagopyrum esculentum L.), canola (Brassica napus L.), sorghum-sudangrass (Sorghum bicolor) (L.) Moench × Sorghum sudanense (Piper) Stapf.), and fallow control were tested in conjunction with three crop sequences in a Phytophthora-infested soil placed in containers. Alfalfa (Medicago sativa L.), potato (Solanum tubersoum L.), or corn (Zea mays L.) was grown in the first year, and alfalfa was grown in all containers in the second year. Compared to fallow controls, alfalfa grown in sorghum-sudangrass- or buckwheat-treated soil had significantly greater stand counts and total biomass, respectively. In addition, alfalfa grown in fallow-treated soils had the greatest Phytophthora root rot as a function of stand count. Crop rotation also had a significant effect on alfalfa root rot and yield. Potato scab disease intensity was greatest on tubers grown in fallow-treated soils, while tubers grown in canola-treated soils had the highest yields (total tuber weight). Green-manure-treated soils tended to have greater streptomycete and bacterial densities than fallow-treated soils. In addition, buckwheat- or sorghum-sudangrass-treated soils had greater proportions of streptomycetes that were antagonistic against the target pathogens than fallow-treated soils. The proportion of antagonists in soil was negatively correlated with alfalfa root rot, and positively correlated with alfalfa stand counts. Inhibitory activity of the streptomycetes was also negatively correlated with potato scab and positively correlated with potato yield. These data suggest that green manures may provide a strategy for increasing pathogen inhibitory activity within the streptomycete community in soil, and, in conjunction with crop rotation, may contribute to the control of a diverse collection of soil-borne plant pathogens on multiple crop species.     

Chemical control of potato common scab disease under field conditions

The aim of this study was to evaluate certain fungicides against Streptomyces scabies (Thaxter), the main causal agent of common scab disease in potato and other crops, in vitro and in vivo. Fourteen isolates of S. scabies were isolated from naturally infected potato tubers showing common scab symptoms. All isolates were pathogenic to potato tubers and produced typical symptoms of common scab. Isolate (No. 11) caused highest disease index (DSI) followed by isolates 10, 8 and 5 (61.13%). Seven fungicides (Rizolex, Capitan, Moncut, Ridomil, Maxim, Topsin, and Oxyplus) were screened in vitro for their toxicity against the pathogen isolate (No. 11). Results showed that four of them (Rizolex, Capitan, Moncut, and Ridomil) exhibited inhibition zone ranging from 5.33 to 26.33?mm. Capitan, Ridomil, and Rizolex were able to reduce DSI under field condition but they varied in their effects. Capitan was the best fungicide which aids in the reduction of disease (33.8%) followed by Ridomil (31.5%) while Rizolex (21.2%) was the lowest one.     

The effects of soil moisture at the time potato tubers are forming on the incidence of common scab (Streptomyces scabies)

Two complementary field experiments at Rothamsted in 1965, with the potato variety Majestic, related the incidence of common scab (Streptomyces scabies) to rainfall, soil moisture and time of tuber formation. In plots where the soil was maintained at field capacity (less than 10 cm. Hg moisture tension) by watering, tubers had little or no scab; infection increased in amount on plots allowed to dry to 30 cm., 50 cm. or more Hg moisture tension during late June. The time tubers formed was varied by planting sprouted (chitted) and non-sprouted seed tubers, and tuber development and scab incidence were observed on sample plants lifted at frequent intervals from unwatered plots. Scab lesions were first seen on 12 July when the distribution of lesions on the surface of tubers was affected by size of tuber both within and between the different seed-tuber treatments. These differences were correlated with estimates of tuber size on 28 June, the beginning of the first dry period. Tubers 1.0 cm. or more in diameter on 28 June had few or no lesions at the stolon attachment (heel) end of tubers on 12 July, whereas tubers smaller than this on 28 June had many lesions in this region on 12 July. The larger the tuber was on 28 June, the greater was the area free from scab lesions on 12 July.     

<Emphasis Type="Italic">Streptomyces scabiei</Emphasis> and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.     

Thyme and suico essential oils: promising natural tools for potato common scab control

• Potato common scab is a worldwide disease mainly caused by Streptomyces scabiei. It seriously affects potato crops by decreasing tuber quality. Essential oils (EO) are natural products with recognised antimicrobial properties. In this research, the antibacterial activities of thyme, oregano, suico and mint EO against S. scabiei were analysed.
• Infected tubers and soil samples were used for bacterial isolation; the obtained isolates were genetically identified. The chemical composition of the EO was determined by GC‐MS. The broth microdilution method was used to analyse antibacterial properties of EO.
• Thirty‐one bacterial isolates were obtained. The isolate chosen for antibacterial assays was morpho‐physiologically and genetically identified as S. scabiei. Thyme EO was mainly composed of thymol and o‐cymene; suico EO of dihydrotagetone, trans‐tagetone and verbenone; oregano EO of trans‐sabinene hydrate, thymol and ?‐terpinene; and mint EO of menthone and menthol. All the EO tested were effective against S. scabiei, but thyme and suico EO were the most successful, with a minimum inhibitory concentration of 0.068 g·l^?1 and 0.147 g·l^?1, respectively, and a minimum bactericidal concentration of 0.137 g·l^?1 and 0.147 g·l^?1, respectively. Scanning electron microscopy showed similar damage caused by both thyme and suico EO to the bacterial envelope. Total phenolic content of EO was not related to their antibacterial activity.
• Thyme and suico EO are effective antibacterial agents against S. scabiei, impeding bacterial viability and disturbing the bacterial cell envelope. These EO are promising tools for control of potato common scab.

     

Decreased severity of potato common scab after foliar sprays of 3,5 -dichlorophenoxyacetic acid, a possible antipathogenic agent

Glasshouse tests on the potato cultivars Majestic and Maris Bard measured the effects of single early foliar sprays of ring-substituted phenoxyacetic acids (0.9 × 10–³ M) on the incidence of potato common scab, caused by soil-borne Streptomyces scabies. The most effective compound was 3,5-dichlorophenoxyacetic acid (3,5-D), which decreased scab by about 90%; its action was preventative, early sprays being more effective than late However, it slightly decreased yield, and increased the number of tubers per plant and the proportion of deformed tubers. The trichloro- and tetrachloro- acids containing the 3,5-dichloro- group also decreased scab, though to lesser extents; the most active of these was the 2,3,4,5-tetrachloro- acid, which had the same side effects as 3,5-D. The herbicide 2,4-D almost halved scab incidence, but seriously decreased yield. Other acids tested had little or no effect on scab. In tests against S. scabies in culture, 3,5-D was no more toxic than other disubstituted acids which were inactive, or weakly active, against the disease in vivo. 3,5-D may be an ‘antipathogenic agent’, preventing scab development by altering tuber metabolism.     

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.