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.     

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.     

Variability in the reaction of squash (Cucurbita pepo) to inoculation with Sphaerotheca fuliginea and methodology of breeding for resistance

Reaction to inoculation with powdery mildew caused by Sphaerotheca fuliginea was observed on leaf discs and young plants of eleven representatives of seven edible cultivar groups of Cucurbita pepo. Disease intensity (i.e. number of infections per leaf) was highly correlated ( r ²=0·863, P <0·0001) with spore yield per leaf. Spore yield per leaf and frequency of sporulation on leaf disks were moderately ( r ²=0·505), but significantly correlated ( P <0·01), suggesting that frequency of sporulation can be used for initial screening against susceptibility in a breeding programme. Spore yield per leaf and spore yield per artificially inoculated leaf disc were highly correlated ( r ²=0·87, P <0·0001); this suggests that counting of spores on leaf discs, a laborious but accurate procedure, could be used on the remaining plants as a second step in selection for resistance of the variation in the response of edible C. pepo to the pathogen, 85·8% was attributed to differences between the edible groups and only 14·2% to individual cultivars within a group. Cultivars of the cocozelle and vegetable marrow groups were the most susceptible, whereas relatively resistant cultivars were found in the scallop and straightneck groups.     

Effects of bean rust (Uromyces appendiculatus) epidemics on host dynamics of common bean (Phaseolus vulgaris)

Epidemics of bean rust ( Uromyces appendiculatus ) and their effects on host dynamics of common bean ( Phaseolus vulgaris ) were studied in three controlled greenhouse experiments, with and without fungicide sprays, on two susceptible bean cultivars, Dufrix and Duplika. Bean plants were artificially inoculated with a suspension of 10⁵ U. appendiculatus urediniospores mL⁻¹ water and temporal disease progress, as well as host growth dynamics (leaf size and defoliation), were monitored on a leaflet basis in comparison with non-inoculated plants, which were sprayed with deionized water. Progress curves of bean rust, expressed as the proportion of leaf area occupied by pustules (uredinia), or as the proportion occupied by total lesion area (= halo areas + pustule area), were well described by logistic functions with maximum disease levels clearly lower than 1. Bean rust epidemics substantially affected host growth by reducing the total leaf area formed by 17·4–35·6% and 35·3–46·2% compared with healthy plants for cvs Dufrix and Duplika, respectively. Fungicide sprays mitigated the negative effect of bean rust, leading to a gain in leaf area of 17–21% compared with unsprayed plants in both cultivars in two experiments, while in another experiment, disease control had no effect in Dufrix, but a clear effect in Duplika. In addition to the growth depression, bean rust also led to pronounced losses of leaf area as a result of reduced leaf size (leaf shrivelling) and accelerated defoliation.     

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.     

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.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Host–parasite relationships of Meloidogyne incognita on spinach

Host–parasite relationships in root-knot disease of spinach caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Nematode-induced mature galls were large and usually contained one or more females and egg masses with eggs. Feeding sites were characterized by the development of giant cells containing granular cytoplasm and many hypertrophied nuclei. The cytoplasm in these giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P _i) in a series from 0–128 eggs and second-stage juveniles per cm³ soil and growth of spinach cv. Symphony F₁ seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^P–T ] was fitted to fresh top- and total plant-weight data for inoculated and control plants. Tolerance limits ( T ) of spinach cv. Symphony F₁ to M. incognita race 1 for fresh top and total plant weights were 0·25 and 0·5 eggs and second-stage juveniles per cm³ soil, respectively. The minimum relative values for fresh top and total plant weights were zero in both cases at P _i ≥ 32 eggs and second-stage juveniles per cm³ soil. Root galling was least at low initial population densities and greatest at 16 eggs and second-stage juveniles per cm³ soil. Maximum nematode reproduction rate was 33·1-fold at the lowest P _i.     

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.     

Genetic variation in Danish populations of Erysiphe graminis f.sp. hordei: estimation of gene diversity and effective population size using RFLP data

Genetic variation of the barley powdery mildew fungus ( Erysiphe graminis f.sp. hordei ) was estimated in three Danish local populations. Genetic variation was estimated from the variation amongst clones of Egh , and was therefore an estimate of the maximum genetic variation in the local populations. The average gene diversity, Ĥ , was estimated as 0.84. The effective population size was estimated as: log₁₀ ( N^ _e ) = 0.64 − log₁₀(μ), or 4.4 × 10⁹, assuming a nucleotide mutation rate ( μ) of 10⁻⁹ per base per generation. There was no significant genetic differentiation between locations.     

CHLORAMBEN (AMIBEN) DEGRADATION JN SOIL

Summary. When columns of three soil types were perfused with a microbial medium containing 5·7 and 26·9 ppm ¹⁴COOH-labelled chloramben, there was a rapid decrease in the ¹⁴C activity remaining in solution, attributable to soil adsorption. Soil pH and organic matter content were the primary factors controlling chloramben adsorption. Soil pH may enhance adsorption through its effect on the number of soil anion exchange sites, and sites for polyvalent cation bridging and H bonding. Following adsorption, liver decreases in solution ¹⁴C-chloramben activity were attributed to degradation; ¹⁴CO2 evolved during perfusion amounted to 25–30% of the total decrease in ¹⁴C-chloramben from solution. It was concluded that more than 90% of the ¹⁴C-activity remaining in solution could be attributed to intact chloramben. Thus, decarboxylation appeared to be the primary mechanism of chloramben degradation in soils. Lag phases observed during degradation and subsequent investigations with re-amended solutions implicated the soil microflora; even at chloramben concentrations of 26.9 ppm no toxic effect on the chloramben-degrading micro-organisms was apparent.
Dégradation du chlorambène [amibène) dans le sol     

Effects of the mycoparasite Verticillium biguttatum on barley stunt disease caused by Rhizoctonia solani anastomosis group 8 in a model system

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.     

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.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

A RAPID BIOASSAY FOR SIMULTANEOUS IDENTIFICATION AND QUANTITATJON OF PICLORAM IN AQUEOUS SOLUTION

Summary. When seeds of lettuce ( Lactuca sativa L.) were placed on small squares of filter paper moistened with solutions of picloram (4-amino-3,5,6-trichloropicolinic acid), the degree of inhibition on of root and hypocotyl elongation 72 hr later was related to picloram concentration. Inhibition was a useful parameter in determining quantities of picloram ranging from 0·03 to 7·2 μg. (Growth stimulation occurred from 0·00072 to 0·0072 μg. After paper chromatography of plant exudates containing ¹⁴C-labelled picloram, location and quantities of picloram on the chromatograms were determined by the lettuce bioassay and compared with determinations by the methods of ultraviolet light sensitivity, ¹⁴C 4π strip scanning and 14 C dilution calculations. R_f values determined lay all methods were identical, and the quantitative determination by the bioassay agreed closely with the calculations based on the amount of ¹⁴C detected by liquid scintillation counting.
Un essai biologique rapide d'idntification et de dosage de piclorame en solution aqueuse     

Management of phytophthora root rot in radiata pine seedlings

Chemical and biological agents were evaluated for their ability to suppress root rot, caused by Phytophthora cactorum , in field-grown radiata pine seedlings in New Zealand. Trials were conducted over two seasons in an area of a forest nursery with a natural infestation of P. cactorum , and a history of root rot. In each season, symptoms of root rot developed during April, one month after root pruning, when seedlings were approximately six months old. In trial one, root rot incidence by mid July 2007 was 9·1% in untreated plots and 8·4% in plots that had been treated with metalaxyl-M/mancozeb (14 kg ha⁻¹) at seedling emergence. Disease incidence was lowest (2·1%) in plots that received seven monthly applications of phosphorous acid (6·5 L ha⁻¹). Other treatments, including seed coating with thiram or Trichoderma spp., and foliar applications of methyl jasmonate, did not control disease. In trial two, effects of treatment timing relative to root pruning were investigated. By late June 2008, three months after root pruning, root rot incidence was 22·2% in the untreated plots. Phosphorous acid was the most effective treatment and almost completely suppressed disease (0·1% incidence) when applied fortnightly from February until May (seven applications). Metalaxyl-M/mancozeb (15 kg ha⁻¹) was not effective (21·4% incidence) when applied five months before root pruning. However, disease incidence was reduced when the chemical was applied one week after root pruning (14·9% incidence) and greater control was achieved (8·2% incidence) when the application rate was increased to 50 kg ha⁻¹.     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

The role of phytoalexins in resistance to fusarium wilt in pigeon pea (Cajanus cajan)

Mechanisms of resistance to fusarium wilt (Fusarium udum) were investigated in pigeon pea cultivars from Malawi. Wilt-susceptible (Malawi local) and wilt resistant (ICP 9145) plants were stem-inoculated with a spore suspension containing 2·10⁶ conidia/ml of the pathogen. Occlusion of a small proportion of infected vessels was observed, but the resistant reaction appeared to depend mainly on rapid phytoalexin synthesis. Four fungitoxic isoflavonoid phytoalexins—hydroxygenistein, genistein, cajanin and cajanol-were isolated from plants 15 days after inoculation. Cajanol was identified as the main antifungal compound. The concentration of cajanol was 329·4 μg/g in the resistant cultivar as against 88·6 μg/g in the susceptible cultivar 15 days after inoculation. Crude extract from the resistant plants sampled at 24 h after inoculation contained 34·8 μg ml of cajanol. The LD₅₀ value of cajanol for spore germination was determined as 35μg/ml. The cajanol content of fungus-infected ICP 9145 10 days after inoculation totally inhibited conidial germination of F. udum.     

A South African isolate of ryegrasss mosaic virus

An isolate of ryegrass mosaic virus (RMV-SA) was found in Italian ryegrass plants in two areas in South Africa. Preliminary characterization indicated that the virus exhibited the same symptoms and host range as RMV strains previously found in Canada and the UK. The virus was transmitted by the eriophyid mite Abacarus hystrix. Particle structure and size were comparable to the USA and UK strains. The coat protein and RNA were found to be slightly larger than previously reported, being 32kDa and 2·8 × 10⁶, respectively. The virus exhibited serological cross-reactivity with antisera to RMV-Wales and RMV-Canada but was not serologically related to other Rymovirus members.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.