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.     

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.     

A chemi-thermal treatment for control of seedborne bacterial pathogens of tomato

A 6-year study led to the development of a tomato seed treatment that controlledPseudomonas syringae pv.tomato (PT),Pseudomonas corrugata (PC),Xanthomonas campestris pv.vesicatoria (XV), andClavibacter michiganense subsp.michiganense (CM). Tomato seeds were immersed, at a ratio of 1:4 (w/v) seeds: chemical, in a solution containing cupric acetate, acetic acid, pentachloronitrobenzene, 5-ethoxy-3(trichloromethyl)-l,2,4-thiadiazole, and Triton x-100, for 1 h at 45±0.1 °C. The treatment was carried out in a thermostat-controlled bath circulator. The pathogens PT, XV and CM were almost eradicated after immersion of seeds for 30 min at 25 °C, whereas PC was controlled only after 1 h of treatment at 45 °C. The effectiveness of the treatment was related to the formation of a Cu²⁺ organic complex in the solution. The treatment did not affect seed germination or seedling vigor. Most of the tomato seeds produced or imported by Israel are now successfully treated by this method.     

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.     

Implications of bacterial contaminated seed lots and endophytic colonization by Pseudomonas fuscovaginae on rice establishment

The economic impact of seedborne bacterial diseases on rice production provides a major motivation for research on seed health. This paper reports on the endophytic growth of a rifampicin‐marked strain of the seedborne rice pathogen Pseudomonas fuscovaginae. The bacterium was found in most tested seeds indicating that, even without visible discolouration, seed transmission is possible. Crushed discoloured seeds contained more bacterial cells than did non‐crushed discoloured seeds. These bacteria were released during seed soaking, contaminating clean seed and lowering seed germination. Cells of a rifampicin‐resistant strain of P. fuscovaginae, which had been inoculated onto rice seeds, were subsequently recovered from different growth stages and from different rice tissues, thereby indicating endophytic colonization. These results have implications for seedling establishment, as symptomless seeds do not assure disease‐free seeds, and the presence of seedborne bacteria results in poor germination and poor seedling establishment. Elimination of seedborne bacteria by soaking in sodium hypochlorite can increase seed germination. This could be used in developing control strategies, and, if practised regularly, reduce entry of seedborne disease‐causing organisms into crops, resulting in lower disease pressure.     

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.     

Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real-time PCR

Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beacon-PCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real-time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10² CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large-scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.     

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.     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media‐based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10¹ to 10⁵ CFU per seed; one contaminated seed was mixed into 1000‐seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).     

Characteristics of immunofluorescence microscopy and of dilution-plating to detect Pseudomonas syringae pv. phaseolicola in bean seed lots and for risk assessment of field incidence of halo blight

Routine laboratory testing of 710 bean seed lots from various origins forPseudomonas syringae pv. phaseolicola (Psp) with immunofluorescence microscopy (IF) showed that 27.5% of the seed lots (five subsamples of 1000 seeds tested per sample) contained two or more IF-positive cells in a total of 500 microscope fields (magnification 500×). Simultaneously performed dilution-platings of IF-positive subsamples on King's medium B confirmed presence of Psp for one-third of these IF-positive seed lots. The grey area of disagreement between both laboratory tests was studied by comparison of test data and by field trials.The number of IF-positive cells per subsample was positively correlated with isolation and identification of Psp (R=0.85). The detection level of IF was ca. 10² Psp cells per ml of undiluted subsample extract. The detection level of Psp by isolation on King's medium B was variable, being inversely related with the saprophyte to Psp ratio. The high sensitivity of IF was in part due to high percentages of dormant or dead IF-positive cells in the sample extract. Field trials over two years with 10 000 seeds per seed lot, showed disease incidence for 9 of the 22 seed lots. Of ten IF-positive lots with five positive subsamples per sample, nine were positive in the field test plot (the negative lot gave primary infection spots of Psp when used for commercial growing). By isolation, seven of these ten IF-positive lots were positive. Of the five IF-positive lots with two or less positive subsamples, isolation and field trial were both negative. Based on data on seed transmission from literature, field incidence was unlikely for these five samples in a 10 000 seeds field trial. All seven IF-negative lots were negative in the field trial. The value of IF and isolation for indexing bean seed lots for Psp is discussed.This study was carried out at the Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Binnenhaven 1, P.O. Box 16, 6700 AA Wageningen the Netherlands, to which address correspondence should be addressed.     

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil,lettuce seeds and plants using loop‐mediated isothermal amplification

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.     

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.     

Identification of genomic regions associated with resistance to blackleg (Leptosphaeria maculans) in canola using genome wide association study

Black rot of crucifers is one of the most important diseases of wild rocket (Diplotaxis tenuifolia L. (D.C.)) caused by the seedborne pathogen Xanthomonas campestris pv. campestris. From 2005, it frequently affected this cultivation in the south of Italy, leading to heavy crop losses. In the present work, we aimed to describe the physiological and molecular characteristics of twenty X. campestris pv. campestris strains isolated from plants and seeds. Ten Xanthomonas spp. strains contaminating seeds were identified on the basis of molecular characterization and in vivo pathogenicity on a discriminating host range. Some of seed-borne isolates were ascribed to the species Xanthomonas campestris pv. raphani and X. campestris pv. incanae, indicating the occurrence of non-host pathogenic Xanthomonas on wild rocket seeds. As well as the presence of pathogenic bacteria, even non-pathogenic Xanthomonas spp. strains were detected on the seeds, underlying the importance of identifying them to evaluate the suitability of lots intended for sowing. A phylogeny using 69 Gyrase B (gyrB) sequences retrieved from the literature, was also carried out, highlighting species relatedness. Overall, this study provides a comprehensive framework for Xanthomonas species affecting wild rocket in Southern Italy.

     

Catalog of Micro-Tom tomato responses to common fungal, bacterial, and viral pathogens

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.     

菜豆种子普通细菌性疫病菌检测

 由Xanthomonas axonopodis pv. phaseoli和Xanthomonas fuscans subsp. fuscans引起的菜豆普通细菌性疫病是严重影响菜豆生产的限制因子之一,可造成严重的产量和品种损失。染菌种子是病原菌传播的主要途径。本研究对5个菜豆主要产区的60份菜豆种子样品进行普通细菌性疫病菌检测。在MT选择性培养基上有36份种子样品浸提液检测到目标病原菌, 种子样品带菌量为2.49×10²~5.20×10⁷ CFU/粒。选择36个分离物接种感病品种“英国红”植株,所有分离物均引起接种植株发病。特异PCR检测结果表明,有20个分离物为X. fuscans subsp. fuscans,16个分离物为X. axonopodis pv. phaseoli。试验结果表明,我国一些菜豆主产区商业种植和研究用种子多数污染普通细菌性疫病菌,建议建立无菌种子生产区和加强种子管理,以有效控制病害发生。     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

Determination of seedborne fungi in Onion and their transmission to Onion sets

Samples of onion (Allium cepa L.) seeds were obtained from seven regions in Turkey. The seed coat, embryo and endosperm were cultured, the seedborne fungi were determined and their transmission to onion sets was investigated in both sterile and field soils. Among the fungi determined,Aspergillus alutaceus Berk, and Curt.,Beauveria bassiana (Bals.) Vuill.,Cladosporium cladosporioides (Fres.) de Vries,Geotrichum sp.,Humicola fuscoatra Traaen,Trichoderma harzianum Rifai andT. pseudokoningii Rifai in onion seeds, andFusarium culmorum (W.G.Sm.) Sacc,F. graminearum Schwabe andF. sambucinum Fuckel in onion sets, were recorded for the first time.Aspergillus niger v. Tieghem was found at the highest rate in seed samples (especially in the seed coat), and in bulbs and roots of onion sets that developed from these seeds, whether in sterile or field soil.Fusarium oxysporum Schlecht was isolated at a higher rate from onion sets grown in sterile or field soil, than from seeds.F. acuminatum Ellis and Everhart,F. sambucinum, F. equiseti (Corda) Sacc. andF. graminearum were isolated only from onion sets grown in sterile soil. In dual culture tests, theseFusarium isolates were inhibited byA. niger and thus, exceptfor F. oxysporum, could not develop in agar plate. TheFusarium spp. appeared in onion-sets grown in sterile soil and were inhibited by other fungi in field soil. It was concluded that all fungi were seedborne and thatA. niger andFusarium spp., but not the other fungi, were transmitted from the seeds to onion sets.A. niger andF. oxysporum were also transmitted through the soil.