Dispersal of Pseudocercosporella herpotrichoides and Pyrenopeziza brassicae spores in splash droplets

Splash dispersal of conidia which differ in shape and size, those of Pseudocercosporella herpo ichoides (needle-shaped, 52×2 μm) and Pyrenopeziza hrassicae (cylindrical, 12×3 μm), was studisd by allowing 5 mm drops to fall 13 m on to suspensions (depth 0.5 mm, concentration 120, 000 spores/ml) of each. The resulting splash droplets were collected on photographic film. The pattern of spore dispersal with distance and the distribution of spore-carrying droplets and spores within droplet-size categories were similar for both fungi. Regressions of square root spore number) on droplet diameter gave lines with slopes of 0.0138 for P. herpotrichoides and 0.0167 for P. brassicae.     

Measurement of spore-carrying splash droplets using photographic film and an image-analysing computer

An improved method for the sampling of splash droplets carrying fungal spores uses fixed photographic film. Droplets left clear, permanent traces within which spores were easily visible. The droplet spread factor was constant because the gelatine layer was uniform. A comparison of estimates of the numbers of splash droplets, spore-carrying droplets and spores dispersed by a 5 mm drop falling onto a spore suspension (depth 0.5 mm) with those obtained by other workers demonstrated the reliability of this method. The accuracy was improved by semi-automatic analysis of the spore-carrying splash droplet traces with an image-analysing computer.     

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.     

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.     

Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

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.     

Splash dispersal of Pseudocercosporella herpotrichoides spores in wheat monocrop and wheat–clover bicrop canopies from simulated rain

Simulated rain was allowed to fall onto spore suspensions of Pseudocercosporella herpotrichoides . The resulting splash droplets were collected on horizontal traps at the bottom of a canopy and at 12 cm above treatments comprising no-crop, wheat monocrop and a wheat–clover bicrop. The number of conidia collected on horizontal traps declined exponentially with distance from the inoculum source. The mean number of spores collected in the absence of any crop was twice that in a monocrop; in the monocrop it was twice that in the bicrop. Both splash droplet and spore deposition gradients were steeper in the monocrop treatment than in no-crop, and shallower in bicrop than in monocrop. Evidence is presented that suggests the clover canopy acts as a secondary source for the redistribution of previously dispersed droplets and spores.     

Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere

To elucidate the mechanism of clubroot suppression under neutral soil pH, a highly reproducible germination assay system under soil culture conditions was designed based on the hypothesis that germinated spores of Plasmodiophora brassicae could be identified by the absence of a nucleus (i.e. having released a zoospore to infect a root hair of the host plant). Brassica rapa var. perviridis seedlings were inoculated with a spore suspension of P. brassicae at a rate of 2·0 × 10⁶ spores g⁻¹ soil and grown in a growth chamber for 7 days. The spores were recovered from rhizosphere and non-rhizosphere soils and stained with both Fluorescent Brightener 28 (cell-wall-specific) and SYTO 82 orange fluorescent nucleic-acid stain (nucleus-specific stain). Total numbers of spores were counted under UV-excitation, and spores with a nucleus that fluoresced orange under G-excitation were counted. The significant increase in the percentage of spores without a nucleus (germinated spores) in the rhizosphere after 7 days' cultivation and the correlation with root-hair infections validated the assay system. Applications of calcium-rich compost or calcium carbonate to neutralize the soil significantly reduced the percentage of germinated spores in the rhizosphere, as well as the number of root-hair infections. The present study provides direct evidence that the inhibition of spore germination is the primary cause of disease suppression under neutral soil pH.     

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%).     

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.     

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum , the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L⁻¹ AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 10³ conidia mL⁻¹ to almost 80% at 1 × 10⁶ conidia mL⁻¹. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum , reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum .     

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.     

Real-time PCR assay for quantification of Tilletia caries contamination of UK wheat seed

A quantitative real-time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat-seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log₁₀ (number of spores per seed + 1) on mean log₁₀ DNA ( µ g) produced a coefficient of determination ( R ²) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real-time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed.     

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.     

Evaluation of cacao (Theobroma cacao) clones against seven Colombian isolates of Moniliophthora roreri from four pathogen genetic groups

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1·2 × 10⁵ spores mL⁻¹): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri . Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.     

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.     

Estimating relative crop yield loss resulting from herbicide damage using crop ground cover or rated stunting, with maize and sethoxydim as a case study

The research goal was to determine whether crop damage from herbicides measured early in the growing season soon after treatment could be used to estimate relative crop yield loss. Percentage stunting was rated visually and percentage crop ground cover (i.e. percentage of the ground surface covered by vegetation) was determined from video photographs taken 2–4 weeks after sethoxydim-susceptible maize ( Zea mays L.) was sprayed with sethoxydim at various rates plus crop oil concentrate. Averaged over 3 years, relative percentage maize yield was a negative sigmoidal function of relative sethoxydim rates from 0.065× to 0.5×, where the 1× rate was 420 g a.i. ha⁻¹ ( r ² = 0.80). Relative maize yield was positively linearly related to percentage crop ground cover and negatively linearly related to rated percentage stunting averaged over 3 years. Linear regression models of relative maize yield vs. percentage maize ground cover explained only slightly more data variability ( r ² = 0.86) than did rated stunting ( r ² = 0.82) over 3 years. The advantages and disadvantages of rated stunting and crop ground cover as scientific measurements are discussed.     

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.     

Factors influencing infection of eucalypts by Cylindrocladium pteridis

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.     

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.     

Differential susceptibility of barnyard grass (Echinochloa crus-galli var. crus-galli) ecotypes to Exserohilum longirostratum

Ten barnyard grass ( Echinochloa crus-galli var. crus-galli ) ecotypes collected from several locations in Malaysian and Indonesian granaries were tested for variation in their susceptibility to the leaf blight pathogen ( Exserohilum longirostratum ). Four phenological growth stages of the ecotypes were sprayed with E. longirostratum at the rate of 1 × 10⁷ conidia mL⁻¹ under glasshouse conditions. The results of the study showed that plants inoculated with 1 × 10⁷ conidia mL⁻¹ and exposed for 24 h dew periods were severely infected and the percentage dry weight reduction of all ecotypes significantly increased. The 1–2 and 2–3-leaf stage plants were completely killed within 6 days after inoculation. However, susceptibility reactions among the ecotypes were observed at the 4–5-leaf stage, while the 6–7-leaf stage was resistant. The ecotypes, K-02, PK-04, KN-02, CJ-01, and L-01, were the most susceptible ecotypes as indicated by a higher area under disease progress curve value. The apparent disease progress rates for these ecotypes were significantly faster compared to the other ecotypes. The dry weight reduction among the four growth stages was variable. These findings show that the 1–2 and 2–3-leaf stages are the most susceptible, while the 6–7-leaf stage was resistant. This study has confirmed that a variable response to the fungal pathogen occurred within an intraspecific barnyard grass collection.