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1.
Populations of total bacteria and Erwinia herbicola were monitored on apple blossom in selected orchards at two different geographical regions in New Zealand (Canterbury and Hawke's Bay). In all four orchards surveyed E. herbicola populations remained negligible (less than 50cfu/blossom) throughout flowering, increasing rapidly at petal drop to reach levels of 1 × 103 cfu/blossom (Hawke's Bay) to 1 × 105 cfu/blossom (Canterbury). Total bacterial populations increased 100-fold at petal drop in both locations. Pseudomonas populations were predominant in Canterbury throughout all flowering stages and in Hawkes Bay after early flowering. When screened for inhibitory activity to E. amylovora , 26% of all isolates from Canterbury showed some inhibition in vitro , but none of the isolates from Hawke's Bay showed inhibition of E. amylovora in vitro. Two Canterbury isolates with strong in vitro inhibitory activity also inhibited E. amylovora in immature pear fruit. One isolate was identified as E. herbicola and one isolate as Pseudomonas fluorescens.  相似文献   

2.
Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 103 cells mL−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.  相似文献   

3.
The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel-end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony-forming units per ml. Following concentration by high-speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi-selective media, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre-enrichment of samples in semi-selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi-selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.  相似文献   

4.
Small plots of potatoes were inoculated with a mixture of Erwinia carotovora (E. c.) subsp. carotovora and E. carotovora subsp, atroseptica strains resistant to rifampicin. Subsequently the population off, c. subsp, carotovora and E. c. subsp, atroseptica (rifampicin-resistant and wild types) present as epiphytes on the surface of potato leaves was assessed using three methods, qualitative, semi-quantitative and quantitative, during 1986 and 1987. The population was generally low (< 102 colony forming units (> 104cfu/g leaves) but reached higher levels (> 104 cfu/g) on occasions later in the growing season, Rifampicin-resistant erwinias were reisolated only infrequently throughout this study. Different methods of haulm destruction (herbicide, pulverization, sulphuric acid treatment and natural senescence) greatly influenced the number of erwinias present in the resulting plant debris. Pulverization resulted in the highest population (106-107 wild-type cfu/g) in both seasons. In 1987. the wettest of the two seasons of this study, herbicide treatment resulted in similarly high populations. The results suggest that the high numbers of erwinias found in the haulm debris were probably derived from the generally low populations of epiphytic bacteria previously present on healthy leaves, E. c. subsp, carotovora was the most frequent subspecies in the rotting plant debris; E. c. subsp, atroseptica was more commonly found on healthy leaves. The implications of the results are discussed in relation to the production of seed potatoes with a low risk of blackleg.  相似文献   

5.
A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 102 and 1 × 103 colony forming units (CFU) mL−1 and detection sensitivity was increased to as few as 2–4 CFU mL−1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.  相似文献   

6.
Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 103 or 4 × 104 spores mL−1, rather than 4 × 102 or 4 × 105 spores mL−1, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.  相似文献   

7.
An enzyme-linked immunosorbent assay (ELISA) was standardized for detecting Xanthomonas campestris pv. undulosa (Xcu) in plant tissues. Antiserum prepared against somatic antigens of Xcu reacted with cells of pathovars undulosa, cerealis, translucens and phleipratensis , but not with other bacterial species belonging to the genera Xanthomonas, Pseudomonas, Agrobacterium, Clavibacter , and Erwinia. The lower limit of detection of pure cultures was 5 × 103 cfu/ml. A semi-selective enrichment broth (SSEB) improved the recovery of Xcu in cultures mixed with contaminating bacteria commonly found on wheat seeds. In ELISA tests the enriched samples gave two- to three-fold increases in A405nm readings when viable cells of Xcu were present. By enrichment, X. campestris pathovars undulosa, cerealis, translucens and phleipratensis were detected in samples that originally had less than 5 × 102 cfu/ml. Semi-selective enrichment combined with ELISA (SSEB-ELISA) allowed for determination of the percentages of infestation of wheat seed lots. Potential seedling infection (PSI) of naturally infested wheat seed lots was obtained by growing seed samples in the greenhouse under conditions optimal for disease development. Three methods were evaluated for their capacity to estimate the PSI: ELISA, combined SSEB and ELISA, and direct plating onto semi-selective XTS agar. Percentages of seed infestation determined by combined SSEB and ELISA resulted in a highly significant correlation with the PSI (r = 0·87, P × 005), whereas determinations made by ELISA or direct plating onto XTS did not significantly correlate with the PSI determined in the greenhouse. This test may constitute a convenient tool for fast initial screening of wheat seed lots in wheat certification programmes.  相似文献   

8.
Polyacrylic acid (PAA) protected Xanthi-nc tobaccos against necrosis provoked by Pseudomonas syringae (hypersensitive reaction). When highly concentrated bacterial suspensions (1 × 108 b/ml) were injected into PAA-treated leaves, tissue collapse was delayed; with more dilute bacterial suspensions (3 × 107 or 1 × 107 b/ml), a significant reduction or a total suppression of necrosis was observed. This phenomenon occurred when the PAA treatment was applied 4 days prior to injection of the bacteria, but not when it was applied 3 h prior to injection. No such protection occurred in Samsum NN tobaccos. PAA had no toxic effect on P. syringae when added to the injected suspensions, or in vitro in culture media. The bacterial population remained constant in protected tissue, but declined sharply in necrosing tissues. These results suggest that host metabolism is involved in PAA-induced suppression of necrosis.
Induced suppression of necrosis due to bacteria shows some analogies with induced resistance to viruses. The possibility of common steps in their biochemical mechanism is discussed.  相似文献   

9.
Real-time PCR was used for quantitative detection of Erwinia amylovora , the causative agent of fireblight. Specific primers were created from a DNA fragment of the common plasmid pEA29, successfully used for standard PCR identification of the pathogen. The primers amplified DNA from various E. amylovora strains, but not from other plant-associated bacteria. DNA of E. amylovora was also amplified from field samples and from inoculated apple leaves or flowers. Neither the presence of other bacteria nor low amounts of tissue extracts from bark or leaves changed the signal threshold. Assays with SYBR Green I instead of the Taqman probe showed a similar sensitivity, detecting 50 cells per assay. Real-time PCR could be especially useful for mass screening of commercial products and for resistance studies of transgenic host plants, in breeding experiments and after treatments to control fireblight.  相似文献   

10.
Root systems of tobacco dipped in suspensions containing 2 × 109 colony forming units (CFU)/ml of avirulent bacteriocin-producing strains (ABPS) of Pseudomonas solanacearum and assayed immediately after planting in steam-sterilized soil had 8 × 106 CFU/root system of ABPS. The bacterial population declined to an average of 5·3 × 105 CFU/root system after 30 days. Roots of seedlings dipped in bacterial suspensions of ABPS were more effectively protected against wilt caused by P. solanacearum than those dipped in suspensions of an avirulent nonbacteriocin-producing strain (ANBPS). Lipopolysaccharide (LPS) isolated from one ABPS (121) inhibited the attachment of bacteria on roots by 70% but had no effect on the reduction of wilt, whereas bacterial cells significantly reduced the disease severity as compared to LPS or water treatment. In steam-sterilized soil containing a 1:1 mixture (5 × 105 CFU/g of oven-dried soil) of ABPS 121 or 237 and the virulent strain K-60, ABPS 121 reduced multiplication of the virulent strain in soil and in the rhizosphere of seedlings. When roots of seedlings were dipped in a suspension of 2 × 109 CFU/ml of ABPS before planting, root colonization by the virulent strain added to steam-sterilized soil at 2 × 106 CFU/g of oven-dried soil was significantly reduced. When roots were dipped in a suspension of ABPS and assayed 20 days after planting, 98% of the bacterial population was found in the original zone of inoculation and only 2% was detected in new growths of the root system. Plants which were grown in soil infested with ABPS 121 or K-60 had both strains present at variable populations along all sections of roots.  相似文献   

11.
The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 106 spores mL−1 in 2004/05 and 0, 1 × 102 or 1 × 105 spores mL−1 in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 105 spores mL−1.  相似文献   

12.
利用GFP标记和平板分离法研究了拮抗内生细菌Bacillus mojavensisZA1在马铃薯根内及根际的定殖动态及其对土著微生物的影响。结果表明,标记菌株ZA1-gfp在土壤中定殖数量为3.7×104~7.3×104cfu/g;马铃薯根际为3.0×103~1.5×105cfu/g,根内为49~630cfu/g;马铃薯的根部横切面、根毛和根表皮等部位均观察到发绿色荧光的菌株ZA1-gfp菌体或聚集菌落。利用灌根法施入生防菌后,土壤中细菌、放射菌数量显著增加(P<0.05),土壤真菌数量在中期下降;土壤中细菌、真菌和放线菌的数量分别在相对稳定范围内(数据之间差异不显著)消长。该结果说明菌株ZA1-gfp能够在马铃薯根内及根际定殖且不影响土壤中土著微生物的群落稳定性。  相似文献   

13.
Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real-time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S-23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real-time PCR was 103 colony forming units per mL (cfu mL−1) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.  相似文献   

14.
A spherical virus–like agent ($$32 nm in diameter) was isolated from a plant of Brachypodium sylvaticum (Huds.) Beauv. growing in a botanic garden in England and showing yellow streaks in the leaves. The agent was readily purified and sedimented as a single component in sucrose density rate gradients. The particles had a sedimentation coefficient at infinite dilution of $$122S and a buoyant density of 1.35 g/cm3 in CsCl and 1·33 in CS2SO4. The particles were stable at acid pH but above pH 7·0 in the presence of EDTA dissociated. A protein having a major polypeptide with a molecular weight of $$3·76 × 104 and a species of single stranded RN A with a MW of 1·67 × 106 were detected in the particles. The agent was not transmitted by manual inoculation, by the insects Myzus persicae Sulzer, Rhopalosiphum padi L. or Nephotettix virescens Distant, through soil by leakage from roots or by seed. The particles had physicochemical properties in common with tombus– and sobemoviruses but were not serologically related to 10 members of these groups or to 57 other small spherical RNA plant viruses.  相似文献   

15.
The pattern of Cylindrocladium pteridis adhesion, germination and penetration in eucalypt leaves was assessed using scanning electron microscopy. The effects of inoculum concentration, leaf wetness period, plant age and branch position of cylindrocladium leaf blight and defoliation severity were assessed in greenhouse studies using two Eucalyptus grandis × E. urophylla hybrid clones. Penetration occurred through stomata, and there was no difference in the number of penetrations between young and old leaves. Percentage leaf area with lesions and defoliation increased with the increase in inoculum concentration (1 × 102 to 105 conidia mL−1), duration of leaf wetness period (6 to 48 h) and plant age (60 to 180 days). Branch position in plants also significantly affected the percentage leaf area with lesions and defoliation, the latter variable being significantly higher at the stem base. The highest values of lesion area were also observed on leaves at the stem base in both clones. The Pearson correlation between defoliation and leaf area with lesions was significant in all experiments ( r  > 0·9) indicating a high association between these two variables.  相似文献   

16.
J. D. JANSE 《EPPO Bulletin》1988,18(3):343-351
A detection method is described for latent infections of Pseudomonas solanacearum race 3 in potato. This method is based on extraction of 200 heel ends of potato, followed by screening with an indirect antibody stain (IFAS) and (in IFAS-positive cases) a pathogenicity test on tomato (PT). Using the method in some sensitivity and specificity tests with more than 600 samples it appears that: (a) its detection level is 104 cells ml-1 in IFAS and 102— 104 cells ml-1 in PT; (b) per cent recovery is 0.1–10% (10% at lower contamination levels); (c) false-positive samples due to cross-reacting bacteria in IFAS were limited to only 2–3%, using two polyclonal antisera against whole cells of P. solanacearum. The merits of the method in comparison with others (ELISA, selective media) are discussed.  相似文献   

17.
The height of barley stunted by Rhizoctonia solani anastomosis group 8 was significantly increased by up to 72·8% after incubation for 8 days at 20°C in seedling tray tests following application of the mycoparasite Verticillium biguttatum. The pathogen and mycoparasite were applied at the rate of 1% Perlite maizemeal inoculum (w/w potting mixture) resulting in propagule densities of approximately 24·0 and 6·6 × 105 colony-forming units (cfu) per g potting mixture, respectively. Sieving (2 mm) R. solani inoculum prior to dilution in potting mixture increased the recovery of propagules from 1·2 × 2·1 × 103 cfu per g inoculum compared with recovery when inoculum was sieved after dilution. Applications of a V. biguttatum isolate from the UK (vbl) and a Dutch isolate (M73) reduced stunting to a similar extent but did not stimulate the growth of healthy plants. The height of stunted plants was significantly increased after application of V. biguttatum inoculum after 6 days if inoculated trays were preincubated for 1 day prior to planting but a similar increase was only detected after 7 days if seeds were planted immediately. The number of stunted plants which emerged after 4 days was significantly increased by treatment with V. biguttatum but preincubation had no additional effect. These results suggested that control of R. solani was effected both before and after the initiation of disease.  相似文献   

18.
Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 107 rather than 1 × 107 spores mL−1. Infection frequencies of pruning wounds declined as wounds aged.  相似文献   

19.
The production of ascochitine by seven isolates of Ascochyta fabae accounted for the toxicity of culture filtrates of the fungus to cells isolated from leaves of Vicia faba. The LD50 value for cells from cultivars that were susceptible, tolerant or resistant to the fungus was similar i.e. 3·0 × 10−5 m , 3·8 × 10−5 m and 2·9 × 10−5 M, respectively. Ascochitine affected neither the germination of seeds nor the growth of mature plants at 5·17 × 10−4 m but caused necrosis and wilting of plant cuttings at 2·5 × 10−4 m and 5·10−4 m . There was no association between virulence of 16 isolates of A. fabae for three cultivars of V.faba and the production of ascochitine in vitro. One isolate produced no ascochitine in vitro and yet was the most virulent for two of three cultivars. The toxin could not be extracted from infected plants.  相似文献   

20.
Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 103 culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.  相似文献   

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