无致病力产细菌素拮抗菌nQE-104在番茄植株上定殖能力的研究

 本文报道青枯菌的无致病力产细菌素拮抗菌nOE-104番茄根表和体内的定殖能力及其对致病菌LE-101的抑制作用.浸根和茎部针刺接种试验结果表明:nOE-104可以在番茄根表定殖,并且能进入部分番茄植株体内沿维管束扩展.拮抗菌nOE-104菌液浸根处理后5天,nOE-104在番茄根表的数量稳定在10⁴~10⁵(CFU/主根),两周以后其数量逐渐下降.浸根处理后5天,nOE-104在番茄体内的数量稳定在10⁵~10⁶(CHU/克组织)。番茄苗用拮抗菌nOE-104菌液浸根后移栽,然后再在土壤中添加致病菌LE-101处理的植株,其根表和体内的nOE-104对LE-101的增殖有抑制作用.     

无致病力产细菌素拮抗菌MA-7防治番茄青枯病研究初报

用无致病力产细菌素拮抗菌MA-7和nOE-104,分别对细菌素敏感菌LE-101用病土在室外进行盆栽试验,结果表明:MA-7和nOE-104都能推迟并减轻发病,而MA-7的防效显著优于nOE-104。MA-7对不敏感菌LE-8也有一定的防治效果。     

Biocontrol of seedborne Botrytis cinerea in chickpea with Gliocladium roseum

An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea . Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 10⁷ and 10⁸ conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×10⁷ conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×10⁸ conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum , and treatment with G. roseum at 10⁸ conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.     

Cultivar dependence of polyacrylic acid effects on Pseudomonas syringae in Nicotiana tabacum

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.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).     

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

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.     

PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

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.     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

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.     

A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity

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.     

Some properties of a virus-like agent found in Brachypodium sylvaticum in the United Kingdom

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.     

Effect of method of inoculation, inoculum density and seedling age at inoculation on the expression of resistance of cocoa (Theobroma cacao L.) to Verticillium dahliae Kleb.

Soil drench and stem puncture inoculation were compared as methods for selecting cocoa cultivars with resistance to Verticillium dahliae. Disease progress was more rapid and induced symptoms were more severe following stem puncture and, under glasshouse conditions, differences between cultivars were detected 15 days after inoculation. Moreover, using stem puncture, inoculum densities of 10⁴ conidia/ml were sufficient to differentiate resistant and susceptible cultivars, whereas with the soil drench method, inoculum densities of 10⁷ conidia/ml were necessary. Although a substantially higher proportion of plants were affected by stem puncture inoculation, the resistance of cultivar Pound-7 remained effective at high inoculum densities of 10⁸ conidia/ml. With either method, older seedlings were more susceptible to V. dahliae than younger ones. However, with stem puncture, 15-day-old seedlings were sufficiently susceptible for a valid disease assessment. In contrast, with soil inoculation, 60-day-old plants were required. In a nursery trial with 15-day-old seedlings, seven cocoa genotypes previously selected as resistant, moderately resistant or susceptible to Verticillium dahliae , on the basis of root inoculation, were ranked in the same order when stem punctured. Stem puncture inoculation of young seedlings is cost-effective in terms of time and space, and is therefore recommended for screening of cocoa for wilt resistance, especially in large-scale breeding programmes.     

Biological control of southern blight disease of tomato caused by Sclerotium rolfsii with simplified mycelial formulations of Trichoderma koningii

Two simple formulations of an antagonistic strain of Trichoderma koningii were employed against southern blight disease caused by Sclerotium rolfsii in seedling, potted outdoor and field-grown tomatoes (cvs. Ife No. 1 and Ibadan Local). Corn cob germling inoculum and mycelium powder of T. koningii significantly controlled ( P ≤0·05) symptoms of damping off, blight and wilting in both tomato cultivars. The populations of the antagonist increased from an initial 1 × 10⁴ to about 1 × 10⁶ colony-forming units per g of soil in the protected plants. Moreover, sclerotial counts decreased significantly ( P ≤0·05) in these soils and those sclerotia found had been parasitized by T. koningii. Trichoderma -protected plants were more vigorous than those in the other treatment categories. The significance of these results is discussed in relation to the use of Trichoderma in appropriately simplified formulations.     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: control of sclerotinia disease in glasshouse lettuce

The effects of Coniothyrium minitans inoculum quality and an 8-week interval between inoculum application and crop planting on sclerotinia ( Sclerotinia sclerotiorum ) disease in three successive lettuce crops were investigated in a glasshouse trial. Spore suspensions of three isolates of C. minitans (Conio, IVT1 and Contans) applied at 10⁸ CFU m⁻² and a standard Conio maizemeal–perlite application (06 L m⁻², 10¹¹ CFU m⁻²) were assessed for their ability to control S. sclerotiorum . Only the maizemeal–perlite inoculum (isolate Conio) consistently reduced sclerotinia disease. In the third lettuce crop only, isolates IVT1 and Contans formulated by Prophyta and isolate IVT as an oil–water formulation, all applied as spore suspensions, reduced disease at harvest compared with the untreated control. Recovery, viability and C. minitans infection of sclerotia buried during the 8-week period prior to each of the three lettuce crops, and of sclerotia formed on the crop, were tested. Only the maizemeal–perlite inoculum (isolate Conio) reduced the recovery of sclerotia buried in soil for weeks between inoculum application and crop planting, reducing their viability and increasing infection by C. minitans . Eight weeks was sufficient to enable C. minitans to infect sclerotia of S. sclerotiorum , and may account for disease control. After harvest of the second and third crops, maizemeal–perlite treatment (isolate Conio) reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore-suspension treatments. The effect of inoculum concentration and the influence of soil temperature (varying with time of year) on infection of sclerotia by C. minitans are discussed.     

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壤微生物的影响

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。     

Factors affecting the control of Pythium ultimum damping-off of sugar beet by Pythium oligandrum

Application of Pythium oligandrum to a soil-based compost as a mycelial suspension (5 × 10² CFU g⁻¹ of dry compost) and oospore alginate pellets (10⁵ oospores/g of dry compost) controlled pre- and postemergence damping-off of sugar beet caused by Pythium ultimum to a level similar to metalaxyl seed treatment. Oospore seed treatments and aqueous suspensions of oospores applied to compost failed to control disease. Problems in the use of P. oligandrum oospore inocula for the control of damping-off were highlighted. It was shown that treatment of oospores with cellulase (20 g L⁻¹) increased germination approximately three-fold in comparison to untreated spores. Untreated and cellulase pretreated oospores were subsequently evaluated as seed treatments for their ability to control damping-off of sugar beet. The highest rate of pretreated oospores (10⁴ oospores/seed) gave levels of emergence and establishment in infested compost that were not significantly different from the uninfested controls, whereas seed treatment with untreated oospores gave no significant reduction in disease. In a trial carried out in a controlled environment to assess the effect of pH (4.5–8.0), P. oligandrum (10⁴ cellulase pretreated oospores/seed) was shown to control pre- and postemergence damping-off of sugar beet at pH 7.0 and 7.5 only.     

Effect of Gliocladium and Trichoderma on damping-off and blight of snapbean caused by Sclerotium rolfsii in the greenhouse

Aqueous suspensions of conidia of 285 wild-type strains and mutants of Gliocladium virens, Trichoderma hamatum, T. harzianum and T. viride were tested against Sclerotium rolfsii in the greenhouse. Ten strains of G. virens and four strains of T. harzianum suppressed damping-off of snapbean by 30-50% and blight by 36-74%. All strains of T. hamatum and T. viride tested as conidia were ineffective. In general, strains of G. virens were more effective in suppressing disease in the greenhouse than strains of T. harzianum. Several strains of G. virens and T. harzianum used alone were equal to or more effective than double and triple mixtures of such strains in disease suppression. Of four formulations of G. virens tested, germlings, alginate-bran-fermenter biomass pellets, and Pyrax-fermenter biomass mixtures reduced disease considerably and all three formulations were more effective than conidia in aqueous suspension. Strain G1-3 of G. virens added to soil as Pyrax-fermenter biomass mixtures in amounts to provide colony-forming units ranging from 1 -5 × 10³ to 1 -2 × 10⁴ per g soil provided statistically significant protection of the host at all concentrations. The extent of biological control with strains G1-3 and G1-21 of G. virens also depended on the strain of the pathogen used. Both strains effectively suppressed disease caused by strain Sr-1 (small sclerotia) of S. rolfsii. They were partially effective against strain Sr-116 (medium size sclerotia) and ineffective against strain Sr-3 (large sclerotia). Although strains G1-3 and G1-21 colonized the sclerotia of all three strains of S. rolfsii in soil effectively, they only reduced germinability of sclerotia of strain Sr-1.     

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer , but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro , as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 10⁸ CFU mL⁻¹, growth of the pathogen was completely limited in vitro , and no infection was found in peach and nectarine fruits treated with or without calcium.     

Allelopathy of the moss Hypnum plumaeforme by the production of momilactone A and B

Extract of soil under colonies of Hypnum plumaeforme inhibited the growth of roots and shoots of cress, lettuce, lucerne, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli . Increasing the extract concentration increased the inhibition, which suggest that the soil may contain growth inhibitory substances and possess allelopathic potential. The extract of the soil under H. plumaeforme was purified and two main inhibitory substances were isolated and determined by MS and ¹H- and ¹³C-NMR spectral data as momilactone A and B. Momilactone A and B inhibited hypocotyls and roots of cress seedlings at concentrations >10 and 1 μ m respectively. The endogenous concentration of momilactone A and B in H. plumaeforme was 58.7 and 23.4 μg g⁻¹ dry weight respectively and the concentration of momilactone A and B in MS growth medium of H. plumaeforme was 4.3 and 6.4 μg g⁻¹ dry weight of H. plumaeforme , respectively. These results suggest that momilactone A and B were probably secreted into the medium during the incubation and momilactone A and B found in the soil under H. plumaeforme may have been released by the moss. Therefore, growth inhibitory activity of the soil under H. plumaeforme may be caused by momilactone A and B, which may act as allelopathic agents of H. plumaeforme .     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

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.