A method for detection of seedborne bacterial diseases in tomato seeds

A method for detection and quantitative estimation of tomato seedborne pathogenic bacteria has been developed. It enables detection in a 7 g tomato seed sample of as few as ten colony-forming units per gram tomato seeds of the following seedborne pathogens of tomato:Pseudomonas syringae pv. tomato,Pseudomonas corrugata, Xanthomonas campestris pv.vesicatoria, andClavibacter michiganense subsp.michiganense. With representative seed samples, the method employs dry grinding, weighing, bacterial extraction and quantitative calculation on selective or semi-selective medium. The efficiency of this method was tested by diluting pathogen-free seed lots with naturally or artificially infested tomato seeds. This procedure enables one to determine the minimal threshold of pathogen which can be detected by this method on media, in comparison with the percentage of diseased seedlings developed from the same seed lots in the growth chamber or in the greenhouse.     

Complementary bacterial enrichment techniques for the detection ofPseudomonas syringae pv.tomato andXanthomonas campestris pv.vesicatoria in infested tomato and pepper seeds

A scheme for routine seed testing forXanthomonas campestris pv.vesicatoria andPseudomonas syringae pv.tomato in pepper and tomato seeds was developed. The scheme is based on different bacterial enrichment techniques. As few as 1000 and 10–100 colony forming units per gram of seeds were detected using a liquid enrichment technique or leaf enrichment technique, respectively. Relatively large amounts of saprophytes on the seed surfaces did not interfere with the detection of the pathogens.     

Reduction of Bacterial Speck (Pseudomonas syringae pv. tomato) of Tomato by Combined Treatments of Plant Growth-promoting Bacterium, Azospirillum brasilense, Streptomycin Sulfate, and Chemo-thermal Seed Treatment

Inoculation of tomato seeds with the plant growth-promoting bacterium Azospirillum brasilense, or spraying tomato foliage with A. brasilense, streptomycin sulfate, or commercial copper bactericides, separately, before or after inoculation with Pseudomonas syringae pv. tomato, the casual agent of bacterial speck of tomato, had no lasting effect on disease severity or on plant height and dry weight. Seed inoculation with A. brasilense combined with a single streptomycin foliar treatment and two foliar bactericide applications at 5-day intervals (a third or less of the recommended commercial dose) reduced disease severity in tomato seedlings by over 90% after 4 weeks, and significantly slowed disease development under mist conditions. A. brasilense did not induce significant systemic resistance against the pathogen although the level of salicylic acid increased in inoculated plants. Treatment of tomato seeds that were artificially inoculated with P. syringae pv. tomato, with a combination of mild chemo-thermal treatment, A. brasilense seed inoculation, and later, a single foliar application of a copper bactericide, nearly eliminated bacterial leaf speck even when the plants were grown under mist for 6 weeks. This study shows that a combination of otherwise ineffective disease management tactics, when applied in concert, can reduce bacterial speck intensity in tomatoes under mist conditions.     

Evaluation of seedborne bacterial pathogens on common bean cultivars grown in central Anatolia region,Turkey

Bacterial diseases of bean cause economically important losses worldwide. The most important method for managing bacterial diseases on bean is the use of pathogen-free seed. In this study, 198 different dry bean seed samples of six different cultivars including Dermason, Cali, Sira, Battal, Bombay and Seker, were collected from 12 provinces of Central Anatolia Region of Turkey. All were tested to investigate the seedlots as primary inoculum sources of the major bacterial diseases. The data revealed that 22,72 %, 13,63 %, 11,11 %, 1,51 % and 0.5 % of seed samples tested were contaminated with five seedborne bacterial pathogens, Pseudomonas savastanoi pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss), Xanthomonas axonopodis pv. phaseoli (Xap), X. axonopodis pv. phaseoli var. fuscans (Xapf) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), respectively. All bacterial strains isolated were identified based on morphological, physiological, biochemical, molecular and pathogenicity tests. The results showed that Psp and Pss were found together on cv. Cali; Psp and Xap on cv. Dermason and cv. Sira; and Pss and Xap on cv. Seker, cv. Dermason, and cv. Cali. Therefore, the results in the present study suggested that evaluation and selection of pathogen-free seeds are very important for preventing the spread of pathogens and effective management of seed borne bacterial diseases prevalent in bean growing regions; in addition to implementation of integrated crop production strategies such as crop rotation, sanitation, seed treatment, tolerant/resistant cultivar selection and proper bactericide application.     

Flower infection of Brassica oleracea with Xanthomonas campestris pv. campestris results in high levels of seed infection

During seed production, Brassica seed may become infected with Xanthomonas campestris pv. campestris after systemic colonization of plants upon leaf infection, or alternatively, after flower infection. Polytunnel experiments were conducted in 2007 and 2008 to study the relative importance of these colonization routes resulting in seed infection. Cauliflower plants (Brassica oleracea) were spray-inoculated at the 8-leaf stage, after formation of cauliflowers or during flowering, at which stage leaves or blossoms were inoculated. Inoculation at all stages resulted in a relatively high percentage of systemic infection; the average estimated infection incidences for stem base and peduncle infections were 16 % and 19 %, respectively. When seed samples were examined by dilution plating for deep-seated infection following hot water treatment, Xcc was detected in 61 % of the 23 seed samples harvested from plants with inoculated flowers. However, symptom development in seedlings raised from the seeds could not be confirmed in a grow-out test under favourable conditions for Xcc infection at a high RH (>95 %) and a relative high temperature (28 °C). Xcc was not detected in 59 seed samples harvested from leaf-inoculated plants with the exception of one sample from plants inoculated at peduncle formation. In a third polytunnel experiment carried out in 2009, the population dynamics of Xcc on inoculated flowers was investigated. Following spray-inoculation of flowers, 52 % of the flowers were infected with Xcc. During development of siliques, infection incidence decreased slowly and at 56 dpi, 20 % of the superficially disinfected siliques were infected with Xcc. It was estimated that 0.18 % of the seeds was infected and that 1–10 % of the infected siliques contained infected seeds. The implications of these results for control of Xcc in a seed production crop are discussed.     

Epidemiology of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> on tomato

Leaf spot of tomato, incited by Pseudomonas syringae pv. syringae, has been reported recently in Italy on grafted and non-grafted tomato plants (scion Cuore di Bue, rootstock Solanum lycopersicum x Solanum hirsutum cv. Beaufort). In some greenhouses, more than 80% of plants were affected, with a marked reduction in yield. This work was undertaken in order to understand the effect of the number of hours of incubation at high relative humidity (r.h.) and temperature as well as the effect of the presence of wounds at infection time on the development of leaf spot. A difference in sensitivity to leaf spot was observed in the various cultivars tested, in terms of severity of P. syringae pv. syringae, with “Cuore di Bue” being the most susceptible of these cultivars. The development of leaf spot is mostly favored by the presence of wounds, at temperatures between 15 and 20°C. The severity of the disease is lower at 10 and 25°C and very low at 30°C. Under the most favorable temperature conditions, the presence of wounds is sufficient to allow the development of the pathogen immediately upon incubation at high r.h. The effect of wounds and the relatively low requirement of hours of incubation at high r.h. suggest the need for careful management and handling of plants when temperatures range between 15 and 25°C, and particularly within 15 and 20°C. All operations carried out, particularly at transplant and immediately after, should avoid the creation of wounds.     

The effect of plant extracts as seed treatments to control bacterial leaf spot of tomato in Tanzania

Bacterial leaf spot (BLS) caused by seed-borne xanthomonads is a serious disease of tomato (Solanum lycopersicum L.), causing significant losses in both yield and quality. To identify more effective control measures, we evaluated crude extracts from 84 plant species in in vitro and in planta assays for antibacterial activity against BLS of tomato. In the in vitro assays, 20.2?% of the tested plant extracts totally inhibited growth of bacteria when seed washings from treated seeds were plated on nutrient agar medium. In the in planta assays, 17.8?% of the tested plant extracts reduced BLS incidence by 100?% in tomato seedlings. The most effective seed treatments were obtained with extracts from Aloe vera, Betula pendula, Coffea arabica, Glycyrrhiza uralensis, Juniperus communis, Ocimum basilicum, Quercus robur, Rheum palmatum, Rosmarinus officinalis, Ruta graveolens, Sinapis alba, Yucca schidigera and Salvia officinalis. Seed treatment of tomato with these extracts completely inhibited Xanthomonas perforans in both in vitro and in planta assays. Extracts from A. vera, C. arabica and Y. schidigera were tested three times using tomato seeds of cultivars Tanya, Cal-J and Moneymaker in Tanzania. Treatment of tomato seeds with these extracts had a positive effect on the number of normal seeds and had no effect on seedling vigor, height and weight. These results indicate that plant extracts from A. vera, C. arabica and Y. schidigera are potential candidates for seed treatment against seed-borne xanthomonads of tomato in Tanzania.     

Detection and identification of seed-borne pathogenic bacteria of imported tomato seeds in Egypt

Imported tomato seed lots of different cultivars were assayed for the presence of seed-borne bacterial pathogens. The liquid assay method was used for detection of the bacteria, and seed extracts were plated on different semi-selective media. Pseudomonas corrugata and Xanthomonas campestris pv. vesicatoria were detected in 14.7% and 12% of the seed samples tested respectively. These pathogens were identified by means of biochemical, physiological and pathogenicity tests as well as the Biolog GN Microplate System for X. campestris pv. vesicatoria. Both P. corrugata and X. campestris pv. vesicatoria were more easily identified on Tween B and CKTM media than on other media. This is the first report of the occurrence of these important pathogens on tomato seeds in Egypt.     

Solar UV-B radiation limits seedborne anthracnose infection and induces physiological and biochemical responses in Lupinus mutabilis

The aim of this study was to test whether solar UV-B radiation and temperature have an effect on infection of lupin seeds by Colletotrichum acutatum. Samples of infected seed were placed in a solar oven and exposed on sunny days for 15, 30, 45, 60, 75, 90 and 120 min. The degree of reduction in disease incidence and seed germination was dependent on the exposure time. Exposure times of 75 min (UV-B 4.41 kJ m⁻², ≈76 °C) and higher reduced incidence from 5% to undetectable levels, but also reduced seed germination by around 10% compared with untreated seed. Therefore, in a second experiment, infected seeds were exposed for 45 or 60 min in the solar oven (UV-B 2.83 or 3.75 kJ m⁻², respectively, ≈76 °C), for 60 min at ambient temperature (UV-B 3.75 kJ m⁻², ≈21 °C) or to dry heat for 60 min at 75 °C. Exposure for 60 min in the solar oven reduced seed infection by 99%, while UV-B-radiated seed at ambient temperature or dry-heat reduced infection by 60% or 32%, respectively. To evaluate the effect of UV-B plus high temperature on seedlings, lupin seed exposed for 45 or 60 min (UV-B 2.83 or 3.75 kJ m⁻², respectively, at ≈76 °C) were grown and physiological and biochemical responses of the seedlings were assessed. Seedlings from exposed seed had higher total concentrations of chlorophyll, protein and peroxidase activity than those grown from unexposed infected seed.     

Seed germination, seedling establishment and growth patterns of wrinklegrass (Ischaemum rugosum Salisb.)

Several laboratory and glasshouse experiments were conducted to assess seed germination, seedling establishment and growth patterns of wrinklegrass (Ischaemum rugosum Salisb.) influenced by temperature and light regimes, and chemical media. Wrinklegrass was a positively photoblastic species, and seed germination was temperature‐dependent and light‐mediated. Seeds soaked in distilled water for 24 h, or oven‐dried at the respective temperature regimes of 15, 20, 25, 30, 35, or 40°C prior to treatment in distilled water and incubated in darkness, failed to germinate. Likewise, no germination prevailed when the seeds were exposed to similar temperature regimes and treated with 0.2 m KNO₃, 5% H₂O₂ or 0.01 m HNO₃, and incubated under continuous darkness. Seeds treated with 5% H₂O₂ at 30°C, or oven‐dried and treated with 0.01% M HNO₃ at 35°C registered 10 and 20% germination. Approximately 75 and 90% of the light‐exposed seeds for all treatments germinated in the first three and six days at 25°C. No germination occurred at 15°C in the first three days after treatment. Seeds subjected to 40°C for six days after treatment recorded 36% germination. The optimum temperatures for seed germination were 25–30°C. Seed drying and soaking treatments widened the windows of the optimal temperatures for wrinklegrass germination. The acidic media of KNO₃, H₂O₂ or HNO₃ favored seed germination. Less than 5% of seed germination occurred with burial or water inundation at depths exceeding 2 cm. Seed burial or inundation at ≥2 cm depths inhibited seed germination. Seeds sown onto moist paddy soils registered ca. 50% germination. Free‐floating seeds on the water surface registered ca. 98% germination within the first six days after seeding. The mean number of seedlings that survived was inversely proportional to water depths, with close to 100% mortality at the 14 cm depths of inundation. Both plant height and seedling survival were linearly proportional to the amount of root mass of seedlings which penetrated the soil. The weed was a prolific seed producer (ca. 6000 seeds/genet or 18 000 seeds/genet per year). The vegetative and reproductive efforts of each wrinklegrass plant registered values of 0.68 and 0.32, respectively.     

Infection studies ofPseudomonas Tomato,causal agent of Bacterial speck of Tomato

Pseudomonas tomato, the causal agent of bacterial speck of tomato (Lycopersicon esculentum), was isolated on King B medium supplemented with penicillin and cycloheximide. An inoculum concentration of 10⁴-10⁵ colony-forming units per ml was used to obtain typical symptoms. Wounding of leaves by rubbing them with carborundum powder, or spraying with diluted wax solvent, significantly increased disease incidence. The best inoculation was obtained by subjecting the plants to 40°C for 30 min to 3 h or by incubating them for 24 h under mist prior to inoculation. The physiological age of the plants did not affect disease development. The bacteria survived in and were reisolated from soils, commercial seeds and plants after periods varying from 2 months to one year.     

Dormancy and viability of Phalaris minor seed in a rice–wheat cropping system

Seed placement, soil temperature and soil moisture content influenced the process of after-ripening in Phalaris minor seeds. Seeds of P. minor collected from the soil just after wheat harvesting exhibited higher germination than seeds from P. minor threshed directly. There was a pronounced impact of periodic inhabitation of seed into the soil on germination after its dispersal. Germination was strongly inhibited when the seed was kept in soil at more than field capacity (FC) or in water. Maximum germination of seed incubated in soil at FC occurred at 30°C while a temperature of 40°C favoured after-ripening of seed when mixed with dry soil or kept dry without any medium. Release from conditional dormancy was quicker in the seed retrieved from the soil kept at 20°C than at 10°C. Seed release from conditional dormancy and germination increased with a rise in temperature from 30 to 40°C when the seed was retrieved from incubation in soil at FC for 70 days. The seed kept immersed in water was least responsive to a rise in temperature. Seed recovered from dry soil, or kept without any medium, responded quickly at both temperatures. Light enhanced the germination of Phalaris minor seed. The seedbank subjected to rice (Oryza sativa) field management conditions lost vigour in comparison with the seed stored in laboratory. There was significant variability in seed viability when exposed to differential water management conditions in rice.     

Germination of proximal and distal seeds of Poa trivialis L. from contrasting habitats

Seeds of Poa trivialis L. were collected from one grassland and two arable habitats. Seeds from the grassland population were less dormant than the arable populations. Distal (upper) seeds were consistently more dormant than proximal (basal) seeds. Dry storage at 4°C and 15°C for 4 weeks after shedding resulted in a slightly greater loss of dormancy than storage at 23°C. Germination was enhanced by subjecting seeds stored at 15°C to repeated hydration and dehydration cycles. Germination of P. trivialis seeds was density-dependent. Germination of distal seeds was particularly impaired at high densities. The ecological implications of these results are discussed in relation to seed survival strategies.     

Detection of Clavibacter michiganensis ssp. michiganensis in tomato seeds by immunofluorescence microscopy and dilution plating

A method for detectingClavibacter michiganensis ssp.michiganensis in tomato seeds was evaluated. The method is based on rapid screening of tomato seed lots using indirect immunofluorescence staining (IF), followed by dilution plating of IF positive seed lots. Different polyclonal antisera, prepared againstC. michiganensis ssp.michiganensis were tested for their specificity using IF. All strains ofC. michiganensis ssp.michiganensis tested reacted with the polyclonal antisera. Two of nine saprophytic isolates from tomato seeds were positive with the antisera as well as with the control normal serum, but cells of these isolates were distinct in shape from cells ofC. michiganensis ssp.michiganensis.For extraction of the pathogen from the seed, seeds were either blended with a stomacher or soaked at 4–6 °C. The stomacher method yielded more fluorescent cells in IF than 24 h soaking of seed samples. However, soaking of seeds for 48 h generally yielded less saprophytes and overall higher numbers ofC. michiganensis ssp.michiganensis colonies in dilution plating when compared to blending by a stomacher. SCM medium was generally more selective than KBT and modified CNS medium. However, the efficacy of the medium was dependent on the seed lot and/or extraction method used. Confirmation of suspected colonies with YDC (yeast-dextrose-carbonate medium), IF and a pathogenicity test on tomato seedlings proved to be highly reliable (P>0.95). For routine testing of seed lots it is recommended to screen tomato seed lost after soaking seeds for 24 h at 4–6 °C with IF, followed by plating of IF-positive seed lots on modified CNS and SCM after soaking seeds for an additional 24 h.     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Transmission of <Emphasis Type="Italic">Colletotrichum coccodes via</Emphasis> tomato seeds

Anthracnose of tomato caused by Colletotrichum coccodes is a devastating disease of ripe fruits. This pathogen may also infect tomato roots, stems and leaves. In the present study, C. coccodes is shown to be capable of contaminating seeds collected from artificially inoculated tomato fruits. Seedlings germinating from these infected seeds exhibited disease symptoms and therefore may transmit the pathogen to the next crop. The proportion of infected seeds ranged between 20% and 63% in all C. coccodes isolates tested and correlated with the aggressiveness of the isolates to tomato fruits. Fungicidal treatment of the collected seeds reduced, but did not eliminate, seed infection. A transgenic C. coccodes isolate expressing green fluorescent protein was used to visualize the pathogen. Mycelium was observed both on surfaces of the seed coat and within 1% of the embryos.     

Effect of temperature during burial on dormant and non-dormant seeds of Lamium amplexicaule L. and ecological implications

Spring-produced seeds of Lamium amplexicaule L. were dormant at maturity in May and after-ripened when buried and stored over a range of temperatures, becoming conditionally dormant at low (5, 15/6 and 20/10°C) and non-dormant at high (25/15, 30/15 and 35/20°C) temperatures. Conditionally dormant seeds germinated to high percentages at 5 and 15/6°C, and non-dormant seeds germinated to high percentages at 5, 15/6, 20/10, 25/15 and 30/15°C. Seeds that became conditionally dormant at 5°C afterripened completely (i.e. became non-dormant) after transfer to 30/15°C. Buried seeds that became non-dormant in a non-temperature-controlled glasshouse during summer were still non-dormant after 12 weeks of storage at 30/15°C, while those stored at 5°C for 12 weeks had entered conditional dormancy. Thus, low temperatures cause reversal of the afterripening that takes place at high temperatures, but not that which takes place both at low and at high temperatures. Low winter temperatures cause dormant autumn-produced seeds and non-dormant seeds in the soil seed pool to become conditionally dormant. The ecological consequences of these responses to temperature are discussed in relation to the timing of seed germination in nature.     

A high temperature requirement for after ripening of imbibed dormant Poa annua L. seeds

Freshly harvested seeds of Poa annua L. collected in south Louisiana were stored in moist soil at seven temperatures between 5°C and 35°C. At monthly intervals, seed lots were removed and germinated at each of the seven temperatures. Seed were dormant for at least 1 month at all test temperatures. Seeds stored for 2 months at 30 and 35°C showed conditional dormancy; there was 100% germination at 10 or 15°C, and poorer germination at 5 or 20°C. Seeds started to lose viability after 2 months at 35°C and were dead after 7 months. In seeds stored at 10–30°C, there were increased percentages and a wider range of germination temperatures as storage time or storage temperatures increased. Seeds stored at 10°C remained dormant for 9 months, but by 12 months of storage the seeds germinated only at 5 or 10°C. Nearly all seeds stored at the same temperatures in air dry soil remained dormant for 6 months, regardless of storage temperature. These results differ from other reports of low temperatures breaking seed dormancy in Poa annua L. and suggest an adaptation to subtropical climates.     

Comparison of immunofluorescence staining and indirect isolation for the detection of Corynebacterium flaccumfaciens in bean seeds1

Corynebacterium flaccumfaciens, causing bacterial wilt in bean, is a quarantine organism for Europe. This paper compares two methods for detecting C. flaccumfaciens in seed lots on the market. Experimentally contaminated bean seeds were homogenized and a standard extraction and concentration procedure was used. The final pellets were used for immunofluorescence staining and indirect isolation (through bean seedlings). The immunofluorescence staining sensitivity threshold was 1.23 × 10⁷, corresponding to 1.82 × 10⁴ fluorescent cells per ml of final concentrate. The risk of false negative reactions to immunofluorescence staining caused by serologically unrelated C. flaccumfaciens strains was also examined. The indirect isolation sensitivity threshold was 1.23 × 10⁶ producing 43% symptomatic plants and 53% positive reisolation. There was no serious interference between C. flaccumfaciens and Pseudomonas syringae pv. phaseolicola in mixed inoculations and both pathogens were successfully reisolated. The comparative results are discussed in relation to the applicability of the methods to commercial lots of bean seeds.     

Dormancy,germination and emergence of Urochloa panicoides regulated by temperature

Urochloa panicoides is an annual weed of summer crops. In Argentina, in subhumid areas with monsoon rainfall, it germinates and establishes in a single flush. To (i) identify the environmental factors that modify its seed dormancy level and germination and (ii) quantify the parameters describing the thermal behaviour of the germination and emergence dynamics of this weed under non‐limiting water conditions, we established a set of germination experiments performed (i) under controlled conditions using seeds after ripened for 3 or 6 months in different thermal and hydric conditions and (ii) under field conditions, where the soil temperature was modified by applying different shading levels. Seed dormancy level remained high with 3 months after ripening in all treatments. After 6 months, seeds stored at 4°C in dry conditions did not germinate at any temperature, while seeds stored at 25°C in dry conditions and in situ germinated c. 20% and 60% respectively. Germination percentage was higher in seeds harvested before their natural dispersal. The base, optimum and maximum temperatures for seed germination were 6, 35 and 45°C respectively. Shading reduced the number of emerged seedlings, possibly by reducing the soil thermal amplitude. The results explained the dormancy‐breaking mechanism of U. panicoides that allows a high germination rate in the field when rainfall occurs.