Effects of avirulent bacteriocin-producing strains of Pseudomonas solanacearum on the control of bacterial wilt of tobacco

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.     

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.     

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.     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae

Several factors affecting the severity of bacterial canker of pear were studied. In the orchard, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 10⁶ colony-forming units/shoot. However, under favourable conditions in a growth chamber, cankers formed on detached shoots inoculated with 5 cfu/shoot. A second-order polynomial relationship was established between log₁₀ transformed canker length and log₁₀ transformed inoculum dose. In orchard and growth chamber experiments, shoots were susceptible from the time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at – 10°C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. The practical implications of these results are discussed as a basis for control of bacterial canker of pear.     

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.     

Detection of Xanthomonas campestris pv. undulosa infested wheat seeds by combined liquid medium enrichment and ELISA

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 A_405nm 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.     

Interaction of a bioherbicide and glyphosate for controlling hemp sesbania in glyphosate-resistant soybean

The bioherbicidal fungus, Colletotrichum truncatum (Schwein.) Andrus & Moore, was tested at different inoculum concentrations alone and in combination with, prior to or following treatment with different rates of glyphosate ( N -[phosphonomethyl]glycine) (Roundup Ultra) for the control of hemp sesbania ( Sesbania exaltata [Raf.] Rydb. ex A.W. Hill) in Roundup Ready soybean field plots. Colletotrichum truncatum and glyphosate were applied in all pair-wise combinations of 0, 1.25, 2.5, 5.0, and 10.0 × 10⁶ spores mL⁻¹ (i.e. 3.125, 6.25, 12.5, and 25 × 10¹¹ spores ha⁻¹), and 0.15, 0.30, 0.60, and 1.2 kg ha⁻¹, respectively. Weed control and disease incidence were enhanced at the two lowest fungal and herbicidal rates when the fungal spores were applied after glyphosate treatment. The application of the fungus in combination with or prior to glyphosate application at 0.30 kg ha⁻¹ resulted in reduced disease incidence and weed control regardless of the inoculum's concentration. At the highest glyphosate rates, the weeds were controlled by the herbicide alone. These results suggest that it might be possible to utilize additive or synergistic herbicide and pathogen interactions to enhance hemp sesbania control.     

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.     

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.     

Incidence of bacteria inhibitory to Erwinia amylovora from blossoms in New Zealand apple orchards

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.     

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%).     

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.     

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.     

Effectiveness of systemic resistance in bean against foliar and soilborne pathogens as induced by biological and chemical means

Unifoliate leaves of 9-day-old green bean, Phaseolus vulgaris cv. Redlands Pioneer, were inoculated with 10⁴ conidia/ml Colletotrichum lindemuthianum , causing local lesions, or sprayed with 20 μg 2, 6-dichloro-isonicotinic acid/ml formulated by Ciba-Geigy Ltd as CGA 41396. At various times afterwards (7–16 days), first, second or third trifoliate leaves of these plants were challenge-inoculated with 10⁵ conidia/ml C. lindemuthianum or with the rust pathogen, Uromyces appendiculatus. The numbers of anthracnose lesions or rust uredinia resulting from challenge-inoculation were reduced to similar extents by both pre-treatments compared with control plants. Halo blight, caused by Pseudomonas syringae pv. phaseolicola , was reduced in first trifoliates following treatment of unifoliate leaves 6 days earlier with CGA 41396. Induced resistance to root-infecting pathogens was not observed when stems of either 14- or 16-day-old plants were inoculated with mycelial plugs of Fusarium solani f. sp. phaseoli , or when 11- and 15-day-old plants were inoculated with Rhizoctonia sp., Treatment with CGA 41396 did not protect seedlings when they were transplanted into a mix containing the Fusarium sp. 1 day later.     

Differences in cerato-ulmin production between the EAN, NAN and non-aggressive subgroups of Ophiostoma ulmi

A test of comparative in vitro cerato-ulmin wilt toxin production in the aggressive and non-aggressive subgroups of the Dutch elm disease pathogen Ophiostoma ulmi was carried out by turbidity and ELISA methods. Ten non-aggressive, ten EAN aggressive and ten NAN aggressive isolates were tested from a range of geographical sources. In liquid shake cultures the non-aggressive isolates produced the greatest and the NAN aggressives the least mean biomass. Despite considerable variation in cerato-ulmin production by individual isolates in three separate experiments, both the turbidity and ELISA methods showed a clear separation of the non-aggressive and aggressive subgroups. Non-aggressive isolates produced little or no cerato-ulmin (ELISA range of means 0–56.0 ng/ml) and EAN and NAN aggressive isolates moderate to high levels (EAN 1.6–89.0 × 10⁴ ng/ml and NAN 0.2–300 × 10⁴ ng/ml). In the aggressive isolates no correlation was detected between cerato-ulmin production and either biomass or pathogenicity to clonal Commelin elm. The role of cerato-ulmin in the pathogenicity of O. ulmi is discussed.     

Virulence for faba bean and production of ascochitine by Ascochyta fabae

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.     

Controlled environment studies on the infection of Emex australis by Phomopsis emicis

The effect of temperature, dew period, inoculum concentration and host maturity on the growth and development of Emex australis after inoculation with Phomopsis emicis was studied in controlled environments. Disease was assessed by recording changes in the length of stems, number of fully expanded leaves, dry weights, number of fruits and disease severity index. Disease was as severe at 18°C as it was at 28°C. There was a trend of increased disease with longer dew period. Inoculum concentrations of 1·10⁶ and 1·10⁷ conidia per ml resulted in a significant net reduction in stem growth compared with lower concentrations. Progressively higher disease ratings occurred as the inoculum concentration increased to 1·10⁷ conidia per ml. There were no significant effects of inoculation with P. emicis on either seedlings, rosettes or flowering rosettes, but 10-week-old plants were highly susceptible to infection and stem collapse. The implications of these results for the potential development of P. emicis as a mycoherbicide are discussed.     

Current status of biological control of paddy weeds in Vietnam

Rice is a staple food in Vietnam and accounts for > 7.7 × 10⁶ cultivated ha, which provide 35.5 × 10⁶ t of rice, of which 4.2 × 10⁶ t were exported in 2004. The enlargement of the cropping area and the enhancement of rice yield have rapidly increased the amount of agrochemicals, including herbicides, in crop production in Vietnam. From 1990–2003, the percentage of herbicides in total pesticides has increased ≈ 10-fold to 30.2%. In addition, the improper use of herbicides caused environmental hazards, unsafe agricultural products, and human health problems. Biological management integrated with traditional weed control techniques might help to reduce the dependence on synthetic herbicides and build eco-friendly, sustainable agricultural production in Vietnam. This paper reviews the efforts in establishing a strategy for biological management of weeds that was conducted in recent years by Vietnamese weed scientists. This has included cropping system management, water and soil management, integrated pest management, and utilization of plant allelopathy as major components of the strategy. Many plants with strong allelopathic potential can be a source for biological weed suppression and soil fertility improvement. The utilization of allelopathic properties in rice might also help to provide new rice cultivars with weed-suppressing characteristics.     

Production of conidia by Peronosclerospora sorghi on sorghum crops in Zimbabwe

Factors affecting the production of conidia of Peronosclerospora sorghi , causing sorghum downy mildew (SDM), were investigated during 1993 and 1994 in Zimbabwe. In the field conidia were detected on nights when the minimum temperature was in the range 10–19°C. On 73% of nights when conidia were detected rain had fallen within the previous 72 h and on 64% of nights wind speed was < 2.0 m s⁻¹. The time period over which conidia were detected was 2–9 h. Using incubated leaf material, conidia were produced in the temperature range 10–26°C. Local lesions and systemically infected leaf material produced 2.4–5.7 × 10³ conidia per cm². Under controlled conditions conidia were released from conidiophores for 2.5 h after maturation and were shown to be well adapted to wind dispersal, having a settling velocity of 1.5 × 10⁻⁴ m s⁻¹. Conditions that are suitable for conidia production occur in Zimbabwe and other semi-arid regions of southern Africa during the cropping season.