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.     

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.     

The incorporation of pathogen spores into rain-splash droplets: a modelling approach

Simple, theoretical, physical principles and existing experimental data were used to derive an analytical model to describe the incorporation of plant pathogen spores into splash droplets. Data were obtained from experiments on splash dispersal of spores of Pseudocercosporella herpotrichoides (cereal eyespot), Pyrenopeziza brassicae (oilseed rape light leaf spot) and Septoria nodorum (wheat glume blotch). In these experiments, incident drops of diameter 4–5 mm were allowed to fall onto spore suspensions 0.5 mm deep with 1.2 × 10⁵ to 6.5 × 10⁵ spores/mL. The analytical model was constructed as the product of three functions of droplet diameter which described, respectively, the frequency distribution of droplet diameters, the proportion of droplets carrying spores and the mean number of spores in spore-carrying droplets in each diameter category. The frequency distribution of droplet sizes was described by a log-normal distribution, the proportion of droplets carrying spores was described by an exponential function and the adimensional spore concentration in spore-carrying droplets was described by a power law. The cumulative proportions of spores in droplets in diameter categories of increasing diameter were calculated to compare observed and fitted data.     

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 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.     

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.     

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.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

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 .     

Pathology of cortical invasion by Plasmodiophora brassicae in clubroot resistant and susceptible Brassica oleracea hosts

The differences in physiological response of Brassica oleracea to infection and growth of Plasmodiophora brassicae in infected tissue were studied in clubroot resistant and susceptible hosts, grown in sand solution culture artificially inoculated with spores of P. brassicae (10⁸ spores mL⁻¹). Primary (root hair) and secondary (cortical) stages of P. brassicae occurred in both resistant and susceptible hosts. Symptoms of cortical invasion by P. brassicae in resistant and susceptible hosts included cell wall breaks, presence of vesicles or inclusion bodies within the cell walls, cell wall thickening in association with plasmodesmata and enlarged and/or disorganized host nuclei. The main difference between the resistant and susceptible host reaction was the absence of degradation of the secondary thickening and cell walls of the xylem in the resistant host. This study supports the existence of an amoeboid form of the pathogen in addition to the recognized two-phase life history of P. brassicae. Furthermore, it suggests that resistance in B. oleracea does not prevent the development of this amoeboid form. A reduced number of cell wall breakages suggests movement of the amoeboid form, may have been restricted but not prevented in the resistant host.     

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.     

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.     

Sporulation of Pyrenopeziza brassicae (light leaf spot) on oilseed rape (Brassica napus) leaves inoculated with ascospores or conidia at different temperatures and wetness durations

In controlled environment experiments, sporulation of Pyrenopeziza brassicae was observed on leaves of oilseed rape inoculated with ascospores or conidia at temperatures from 8 to 20°C at all leaf wetness durations from 6 to 72 h, except after 6 h leaf wetness duration at 8°C. The shortest times from inoculation to first observed sporulation ( l ₀), for both ascospore and conidial inoculum, were 11–12 days at 16°C after 48 h wetness duration. For both ascospore and conidial inoculum (48 h wetness duration), the number of conidia produced per cm² leaf area with sporulation was seven to eight times less at 20°C than at 8, 12 or 16°C. Values of Gompertz parameters c (maximum percentage leaf area with sporulation), r (maximum rate of increase in percentage leaf area with sporulation) and l ₃₇ (days from inoculation to 37% of maximum sporulation), estimated by fitting the equation to the observed data, were linearly related to values predicted by inserting temperature and wetness duration treatment values into existing equations. The observed data were fitted better by logistic equations than by Gompertz equations (which overestimated at low temperatures). For both ascospore and conidial inoculum, the latent period derived from the logistic equation (days from inoculation to 50% of maximum sporulation, l ₅₀) of P. brassicae was generally shortest at 16°C, and increased as temperature increased to 20°C or decreased to 8°C. Minimum numbers of spores needed to produce sporulation on leaves were ≈25 ascospores per leaf and ≈700 conidia per leaf, at 16°C after 48 h leaf wetness duration.     

Sensitivity of Phytophthora infestans to mandipropamid and the effect of enforced selection pressure in the field

Sensitivity to the new carboxylic acid amide (CAA) fungicide mandipropamid (MPD) in Phytophthora infestans was measured for isolates collected between 1989 and 2002 in Israel prior to the commercial use of MPD (baseline sensitivity, 44 isolates), and from MPD-treated (25 isolates) and untreated fields (215 isolates) in nine European countries and Israel between 2001 and 2005. All isolates were sensitive to MPD, with EC₅₀ values ranging between 0·02 and 2·98  µ g mL⁻¹. Plastic-tunnel (UK), shade-house (Israel) and field experiments (Israel) conducted during 2001–05 showed that enforced selection pressure, applied preventively or curatively, imposed by repeated sublethal (5  µ g mL⁻¹) or excessive (500–1000  µ g mL⁻¹) doses of MPD on mixed isolates of P. infestans produced no isolates resistant to the compound. The results of this study indicate that the probability of a buildup of resistant sub-populations of P. infestans to mandipropamid in the field is low.     

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.     

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.     

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.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

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.     

Pyrethroid sensitivity monitoring in Germany of oilseed rape pest insects other than pollen beetle

Pyrethroid resistant pollen beetles ( Meligethes aeneus ) are widely distributed in Germany and other European countries. Other insect pests on oilseed rape are also exposed to pyrethroids which often have more than one application per season. In spite of this scarce information about the sensitivity of other oilseed rape pest insects is available. To address this monitoring of pyrethroid sensitivity of different oilseed rape pest insects has been carried out since 2005. Laboratory experiments were conducted using an adult-vial-test design with samples collected in different areas of Germany.
Most samples of Ceutorhynchus napi , C. assimilis , C. pallidactylus , Dasineura brassicae , Phyllotreta spp. and Psylliodes chrysocephala tested from 2005–2007 showed 100% mortality at 0.003 and 0.015 µg/cm² lambda-cyhalothrin used as reference test pyrethroid. Some samples especially of C. pallidactylus , C. napi and C. assimilis showed a lower mortality with alive and healthy beetles at 0.015 µg/cm² lambda-cyhalothrin. For all samples tested from 2005–2007 100% mortality was found only at higher rates of 0.0375 or 0.075 µg/cm² lambda-cyhalothrin.
To date, no clear pyrethroid resistance has been detected in other oil seed rape pest insect populations in Germany.