Impact of time between field application of Bacillus subtilis strains SB01 and SB24 and inoculation with Sclerotinia sclerotiorum on the suppression of Sclerotinia stem rot in soybean

This study evaluated the impact of time between the application of cell suspensions or cell-free filtrates of Bacillus subtilis strains SB01 or SB24 on soybean plants under field conditions and inoculation with Sclerotinia sclerotiorum on their effectiveness for suppression of S. sclerotiorum. The results showed that the cell suspensions of two strains provided greater effectiveness than the cell-free filtrates, but the suppression effectiveness decreased as the time between application in the field and S. sclerotiorum inoculation increased. The B. subtilis cell suspensions applied on soybean leaves for up to 10 days under field conditions were able to provide a significant (P < 0.01) reduction in disease severity by approximately 20–90% at 5 days after the S. sclerotiorum inoculation. When rated 15 days after S. sclerotiorum inoculation, plants treated with bacterial cells for ≤6 days reduced Sclerotinia stem rot severity by 15–70%. Most effectiveness was provided by the cell suspensions present on soybean leaves for <3 days under field conditions, which significantly (P < 0.01) reduced disease severity by 40–70% over 15 days. In comparison, the cell-free filtrates remaining on leaves for <6 days significantly (P < 0.01) reduced disease severity during the first 5 days after the inoculation, while the best cell-free filtrate treatments were those with ≤1-day intervals, which significantly (P < 0.01) reduced disease severity by 10–40% during 15 days after the inoculation. The effectiveness of B. subtilis was reduced when it rained after application.     

Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a serious fungal disease of soybean. Senescing petals provide a starting nutrient source for the invasion of healthy tissue by the advancing oxalic acid secreting fungal hyphae. Since oxalic acid is a major pathogenicity factor of SSR, transgenic soybean capable of degrading oxalic acid may be resistant to the pathogen. Transgenic soybean plants were produced byAgrobacterium -mediated transformation with the wheat germin gene (gf-2.8) encoding an oligomeric protein, oxalate oxidase (OxO), which oxidizes oxalic acid to carbon dioxide and hydrogen peroxide (H₂O₂). Transgenic soybean homozygous for 35S- gf-2.8 produced an approx. 130 kDa protein indistinguishable from wheat germin, and with OxO activity. OxO activity was prominent in cell walls proximal to the site of pathogen attack. The transgenics had greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum indicating that the germin gene product conferred resistance to SSR. This is the first report of plant resistance to the fungal pathogen S. sclerotiorum in transgenic plants expressing OxO.     

Temperature adaptation in isolates of Sclerotinia sclerotiorum affects their ability to infect Brassica carinata

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a serious disease in oilseed Brassica crops worldwide. In this study, temperature adaptation in isolates of S. sclerotiorum collected from differing climatic zones is reported for the first time on any crop. Sclerotinia sclerotiorum isolates from oilseed rape (Brassica napus) crops in warmer northern agricultural regions of Western Australia (WW3, UWA 7S3) differed in their reaction to temperature from those from cooler southern regions (MBRS‐1, UWA 10S2) in virulence on Brassica carinata, growth on agar, and oxalic acid production. Increasing temperature from 22/18°C (day/night) to 28/24°C increased lesion diameter on cotyledons of B. carinataBC054113 more than tenfold for warmer region isolates, but did not affect lesion size for cooler region isolates. Mean lesion length averaged across two B. carinata genotypes (resistant and susceptible) fell from 4·6 to 2·4 mm for MBRS‐1 when temperature increased from 25/21°C to 28/24°C but rose for WW3 (2·35 and 3·21 mm, respectively). WW3, usually designated as low in virulence, caused as much disease on stems at 28/24°C as MBRS‐1, historically designated as highly virulent. Isolates collected from cooler areas grew better at low temperatures on agar. While all grew on potato dextrose agar between 5 and 30°C, with maximum growth at 20–25°C, growth was severely restricted above 32°C, and only UWA 7S3 grew at 35°C. Oxalate production increased as temperature increased from 10 to 25°C for isolates MBRS‐1, WW3 and UWA 7S3, but declined from a maximum level of 101 mg g^?1 mycelium at 20°C to 24 mg g^?1 mycelium at 25°C for UWA 10S2.     

Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin

The polygalacturonases (PG) and oxalic acid produced by Sclerotinia sclerotiorum in infected soybean hypocotyls were investigated as elicitors of the phytoalexin glyceollin I.Purification to homogeneity through isoelectrofocusing and ion-exchange fast protein liquid chromatography revealed three endo-PG isoenzymes (PG-I, PG-II and PG-IV) and one exo-PG (PG-III) in 6-day-old etiolated soybean hypocotyls infected with the B-24 isolate of S. sclerotiorum.PG-I and PG-III, in the range of concentrations tested (0·15–1·2 reducing units ml⁻¹), did not act as elicitors of glyceollin I synthesis. Some elicitor activity was shown by PG-II at 0·6–1·2 reducing units ml⁻¹. PG-IV, at lower doses (0·038–0·30 reducing units ml⁻¹), was even more effective in inducing phytoalexin synthesis. However higher concentrations of PG-IV induced tissue softening and decreased phytoalexin accumulation.PG-II and PG-IV released heat-stable elicitors from purified soybean cell walls supporting the evidence that uronides are intermediate inducers in elicitation by endo-PGs. Oxalic acid was an active elicitor of glyceollin I over the range of concentrations tested (0·31–20 m ) with the maximum at a concentration of 5 m . The inability of oxalic acid to release uronides from purified cell walls makes it unlikely that uronide intermediate elicitors are involved in elicitation by oxalic acid.     

In vitro Effect of the Phytoalexin Glyceollin I on Polygalacturonase and Oxalic Acid Production ofSclerotinia sclerotiorum

The polygalacturonase (PG) activity in culture filtrates fromSclerotinia sclerotiorum is reduced when glyceollin I, the major soybean phytoalexin, is present in the culture medium. When the enzyme activities in the culture filtrates are expressed per unit of fungal growth, PG activity decreases with increasing concentration of glyceollin I in the culture medium. The phytoalexin does not influence the isoenzyme pattern. This suggests that glyceollin I may inhibit quantitative but not qualitative enzyme production. Only the highest glyceollin I concentration tested inhibits oxalic acid production. The inhibitory effect on mycelial growth is confirmed. The data suggest a further hypothesis about the role of phytoalexin during pathogenesis.     

Effectiveness of Coniothyrium minitans and Trichoderma atroviride in suppression of sclerotinia blossom blight of alfalfa

The effects of the mycoparasites Coniothyrium minitans and Trichoderma atroviride on the suppression of alfalfa blossom blight caused by Sclerotinia sclerotiorum were evaluated under indoor and field conditions. When T. atroviride (9·0 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) or C. minitans (9·0 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) were applied to detached young alfalfa florets, T. atroviride effectively inhibited saprophytic growth of S. sclerotiorum, whereas C. minitans showed no inhibition under the same conditions. When T. atroviride (6·9 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) or C. minitans (6·9 × 10⁴ conidia/floret) + S. sclerotiorum (6·0 × 10³ ascospores/floret) was applied to young alfalfa petals in vivo just after pollination, the percentage of pod formation was higher for T. atroviride+S. sclerotiorum than that for C. minitans+S. sclerotiorum, and the percentage of pod rot was lower for T. atroviride+S. sclerotiorum than that for C. minitans+S. sclerotiorum. However, when they were applied to senescent petals attached to developing pods of alfalfa at 9·2 × 10⁴ conidia/floret together with S. sclerotiorum at 4·5 × 10³ ascospores/floret at 14 days after pollination, C. minitans was more effective than T. atroviride in suppressing sclerotinia pod rot and seed rot of alfalfa. Field experiments showed that three applications of C. minitans (5·4 × 10⁶ conidia mL⁻¹) or T. atroviride (5·4 × 10⁶ conidia mL⁻¹) at a 7-day interval to blossoms of alfalfa effectively suppressed sclerotinia pod rot in two out of three annual trials. Coniothyrium minitans effectively suppressed sclerotinia seed rot in all three years, whereas T. atroviride was not effective against seed rot in any of the trial years. The efficacy of C. minitans was not significantly different (P > 0·05) from benomyl (250 µg ai mL⁻¹). This study suggests that C. minitans has potential as a biocontrol agent to control blossom blight of alfalfa caused by S. sclerotiorum.     

Suppression of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> by antifungal substances produced by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in MCD (MCD_cm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v⁻¹) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCD_cm. Sclerotia of Ss soaked in the filtrates of MCD_cm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCD_cm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCD_cm. Treatment of leaves of oilseed rape with cultural filtrates of MCD_cm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

Ortho-aminoacetophenone,a non-lethal repellent: The effect of volatile cues vs. direct contact on avoidance behavior by rodents and birds

Taxonomic differences in responsiveness to chemosensory irritants are prevalent among avian and mammalian species and represent a major obstacle to the development of general vertebrate repellents. We evaluated the effect of ortho-aminoacetophenone (OAP), a potent avian repellent, on ingestive behavior of two rodent species, Prairie vole (Microtus ochrogaster), Deer Mouse (Peromyscus maniculatus Wagn.), and an avian species, European starling (Sturnus vulgarus L.) utilizing similar experimental conditions to facilitate interspecies comparisons. All three species avoided OAP-treated food. Apple consumption by voles was decreased from a baseline of theoretical zero% by OAP (0·01–10·0 ml liter⁻¹), P <0·000 01, while mice avoided all but the lowest concentration of OAP (0·01−10 ml liter⁻¹), P <0·000 01. A repellent should elicit avoidance behavior prior to the animal having physical contact with the commodity, ideally producing aversion via volatile cues rather than through direct contact. Therefore, we utilized two delivery methods for presentation of the test solutions to evaluate the repellency of OAP in the presence and absence of direct contact. Apple consumption by birds following exposure to OAP by either direct contact or volatile cues differed from a baseline of theoretical zero% consumption, P < 0·001. When birds had access to OAP through both direct and volatile exposures, reduction in apple consumption by European starlings was greater than observed following contact with the compounds volatile cues alone, P < 0·03. These findings argue against a major role for olfaction or nasotrigeminal chemoreception in avoidance of OAP-treated food. Instead, taste or oral trigeminal chemoreception appear to mediate responding.     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

Effects of temperature,inoculum concentration,leaf age,and continuous and interrupted wetness on infection of olive plants by Spilocaea oleagina

Experiments were conducted on olive plants in controlled environments to determine the effect of conidial concentration, leaf age, temperature, continuous and interrupted leaf wetness periods, and relative humidity (RH) during the drier periods that interrupted wet periods, on olive leaf spot (OLS) severity. As inoculum concentration increased from 1·0 × 10² to 2·5 × 10⁵ conidia mL^?1, the severity of OLS increased at all five temperatures (5, 10, 15, 20 and 25°C). A simple polynomial model satisfactorily described the relationship between the inoculum concentration at the upper asymptote (maximum number of lesions) and temperature. The results showed that for the three leaf age groups tested (2–4, 6–8 and 10–12 weeks old) OLS severity decreased significantly (P < 0·001) with increasing leaf age at the time of inoculation. Overall, temperature also affected (P < 0·001) OLS severity, with the lesion numbers increasing gradually from 5°C to a maximum at 15°C, and then declining to a minimum at 25°C. When nine leaf wetness periods (0, 6, 12, 18, 24, 36, 48, 72 and 96 h) were tested at the same temperatures, the numbers of lesions increased with increasing leaf wetness period at all temperatures tested. The minimum leaf wetness periods for infection at 5, 10, 15, 20 and 25°C were 18, 12, 12, 12 and 24 h, respectively. The wet periods during early infection processes were interrupted with drying periods (0, 3, 6, 12, 18 and 24 h) at two levels of RH (70 and 100%). The length of drying period had a significant (P < 0·001) effect on disease severity, the effect depending on the RH during the interruption. High RH (100%) resulted in greater disease severity than low RH (70%). A polynomial equation with linear and quadratic terms of temperature, wetness and leaf age was developed to describe the effects of temperature, wetness and leaf age on OLS infection, which could be incorporated as a forecasting component of an integrated system for the control of OLS.     

Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape

Sclerotinia stem rot (SSR) is a severe disease of oilseed rape, which severely impacts the crop productivity worldwide. Sclerotinia sclerotiorum causes SSR, resulting in the secretion of oxalic acid (OA), which can be further degraded to carbon dioxide (CO₂) and hydrogen peroxide (H₂O₂) by oxalate oxidase (OXO). In the present investigation, the barley oxalate oxidase (BOXO, Y14203) gene was introduced into oilseed rape by Agrobacterium‐mediated transformation to investigate the mechanism by which OXO promotes resistance to S. sclerotiorum. Compared to the control 72 h post‐inoculation, there were c. 15–61% fewer lesions on leaves of the transgenic oilseed rape, which thus exhibited a detectable level of partial resistance in leaf tissue to S. sclerotiorum. Transgenic oilseed rape also showed decreased oxalate and increased hydrogen peroxide levels compared to the control, and the expression of defence response genes involved in the hydrogen peroxide signalling pathway was also induced. Therefore, the improved resistance of oilseed rape could be attributed to the enhanced OA metabolism, production of hydrogen peroxide and the hydrogen peroxide‐mediated defence levels during infection.     

Biological control of sclerotinia stem rot of canola using antagonistic bacteria

Stem rot caused by Sclerotinia sclerotiorum is a major fungal disease of canola worldwide. In Australia the management of stem rot relies primarily on strategic application of synthetic fungicides. In an attempt to find alternative strategies for the management of the disease, 514 naturally occurring bacterial isolates were screened for antagonism to S. sclerotiorum. Antifungal activity against mycelial growth of the fungus was exhibited by three isolates of bacteria. The bacteria were identified as Bacillus cereus (SC‐1 and P‐1) and Bacillus subtilis (W‐67) via 16S rRNA sequencing. In vitro antagonism assays using these isolates resulted in significant inhibition of mycelial elongation and complete inhibition of sclerotial germination by both non‐volatile and volatile metabolites. The antagonistic strains caused a significant reduction in the viability of sclerotia when tested in a greenhouse pot trial with soil collected from the field. Spray treatments of bacterial strains reduced disease incidence and yielded higher control efficacy both on inoculated cotyledons and stems. Application of SC‐1 and W‐67 in the field at 10% flowering stage (growth stage 4·00) of canola demonstrated that control efficacy of SC‐1 was significantly higher in all three trials (over 2 years) when sprayed twice at 7‐day intervals. The greatest control of disease was observed with the fungicide Prosaro^® 420SC or with two applications of SC‐1. The results demonstrated that, in the light of environmental concerns and increasing cost of fungicides, B. cereus SC‐1 may have potential as a biological control agent of sclerotinia stem rot of canola in Australia.     

Relationship between disease severity and escape of Pseudoperonospora cubensis sporangia from a cucumber canopy during downy mildew epidemics

Fundamental to the development of models to predict the spread of cucurbit downy mildew is the ability to determine the escape of Pseudoperonospora cubensis sporangia from infected fields. Aerial concentrations of sporangia, C (sporangia m^?3), were monitored using Rotorod samplers deployed at 0·5 to 3·0 m above a naturally infected cucumber canopy in two sites in central and eastern North Carolina in 2011, where disease severity ranged from 1 to 40%. Standing crop of sporangia was assessed each morning at 07·00 h EDT and ranged from 320 to 16 170 sporangia m^?2. Disease severity and height above the canopy significantly (P < 0·0001) affected C with mean concentration (C_m) being high at moderate disease. Values of C_m decreased rapidly with canopy height and at a height of 2·0 m, C_m was only 7% of values measured at 0·5 m when disease was moderate. Daily total flux (F_D) was dependent on disease severity and ranged from 5·9 to 2242·3 sporangia m^?2. The fraction of available sporangia that escaped the canopy increased from 0·028 to 0·171 as average wind speed above the canopy for periods of high C increased from 1·7 to 3·6 m s^?1. Variations of C_m and F_D with increasing disease were well described (P < 0·0001) by a log‐normal model with 15% as the threshold above which C_m and F_D decreased as disease severity increased. These results indicate that disease severity should be used to adjust sporangia escape in spore transport simulation models that are used to predict the risk of spread of cucurbit downy mildew.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

Relationship between the extent of colonization by Verticillium dahliae and symptom expression in strawberry (Fragaria × ananassa) genotypes resistant to verticillium wilt

Eleven strawberry (Fragaria × ananassa) genotypes from the University of California breeding programme known to be resistant to verticillium wilt were inoculated with Verticillium dahliae. Individual plants were given a resistance score based on the severity of visual symptoms, and the extent of colonization was quantified as the percentage of petioles not colonized by the pathogen. Both resistance scores and the percentage of pathogen‐free petioles decreased significantly from May to June (P < 0·05) during each of two growing seasons, indicating a progression of both colonization and symptom expression. Even the most resistant genotypes had plants with some infected petioles, and manifested some symptoms of verticillium wilt. Significant (P < 0·05) genotypic variance was detected for the percentage of pathogen‐free petioles, but not for resistance score. The percentage of pathogen‐free petioles had a strongly positive genotypic correlation (r_g = 0·77, P < 0·01) with resistance score, indicating that about 60% of the genotypic variation for visual symptoms in this set of resistant genotypes was explained by the extent of colonization of individual plants by V. dahliae. Conversely, the genotypic correlation between the percentage of pathogen‐free petioles and the resistance score for plants sampled in May (r_g = 0·74, P < 0·01) was smaller than that for plants harvested in July (r_g = 0·93, P < 0·01). Together, these results suggest that the overall performance of strawberry genotypes in the presence of V. dahliae can be enhanced by both resistance and tolerance, but that tolerance may be less stable over the course of a season. Distinguishing between these two mechanisms may require evaluations that supplement visual assessments of resistance.     

o-Diphenol oxidase inhibition—an additional role for oxalic acid in the phytopathogenic arsenal of Sclerotinia sclerotiorum and Sclerotium rolfsii

Oxalic acid secreted by the phytopathogens Sclerotinia sclerotiorum and Sclerotium rolfsii and its effect on o-diphenol oxidase activity in infected apples and dwarf bean pods respectively has been investigated. Concentrations of oxalic acid in culture filtrates and in infected tissues were determined. Oxalic acid was found to suppress enzymic browning in apples by mixed-competitive inhibition of the o-diphenol oxidase. By contrast, in bean pods the secreted oxalic acid reduced the pH to a level where o-diphenol oxidase was inactive. It is postulated that this action enhances the pathogen's success by suppressing the host's defence mechanisms.     

Inhibition of Botrytis cinerea growth and suppression of botrytis bunch rot in grapes using chitosan

Chitosan inhibited growth of Botrytis cinerea in liquid culture and suppressed grey mould on detached grapevine leaves and bunch rot in commercial winegrapes. Germination of B. cinerea was completely inhibited in malt extract broth containing chitosan at concentrations greater than 0·125 g L^?1. However, treated conidia were able to infect detached Chardonnay leaves and pathogenicity was not affected, even after incubation for 24 h in chitosan at 10 g L^?1. When added after conidial germination, chitosan inhibited B. cinerea growth and induced morphological changes suggestive of possible curative activity. The effective concentration of chitosan that reduced mycelial growth by 50% (EC₅₀) was 0·06 g L^?1. As a foliar treatment, chitosan protected detached Chardonnay leaves against B. cinerea and reduced lesion diameter by up to 85% compared with untreated controls. Peroxidase and phenylalanine ammonia‐lyase activities were also induced in treated leaves. In vineyard studies, Chardonnay winegrapes exhibited 7·4% botrytis bunch rot severity at harvest in 2007 after treatment with an integrated programme that included chitosan sprays from bunch closure until 2 weeks preharvest, compared with 15·5% in untreated controls and 5·9% with fungicide treatment. In the following season, botrytis bunch rot severity was 44% in untreated Chardonnay at harvest and the integrated programme (21%) was less effective than fungicides (13·8%). However, in Sauvignon blanc winegrapes, the integrated and the fungicide programme each reduced botrytis bunch rot severity to 4% and were significantly different from the untreated control (11·5%). This study provides evidence that suppression of botrytis in winegrapes by chitosan involves direct and indirect modes of action.     

Corynelia uberata as a threat to regeneration of Podocarpus falcatus in Ethiopian forests: spatial pattern and temporal progress of the disease and germination studies

The order Coryneliales includes several fungi such as Corynelia spp. that are pathogenic to trees in the Podocarpaceae. The aim of this study was to assess the spatial pattern and temporal progress of disease caused by Corynelia uberata on Podocarpus falcatus in Ethiopian forests and to evaluate the germination potential of seed retrieved from fruit infected by C. uberata. Corynelia uberata was found on leaves, young stems and/or on fruit of P. falcatus in Ethiopian forests. Spatial analysis in the Adaba‐Dodola forest showed that disease intensity of C. uberata was significantly higher in non‐‘WAJIB’ blocks (disturbed forest) than ‘WAJIB’ blocks (sustainably managed forest) (P < 0·0001). In the temporal disease progress study, a significantly higher incidence and severity of disease on fruit was recorded during the wet season relative to dry season (P < 0·0001). The green milk stage of fruit exhibited significantly higher mean incidence (P < 0·0001) and severity (P < 0·0001) of disease compared to other growth stages of fruit. The disease incidence and severity in general, as well as on different fruit growth stages, were highly correlated (P < 0·0001, R² ≥ 0·95). Germination rate of seed decreased significantly with an increase in the level of fruit infection by C. uberata (P < 0·0001). Thus, C. uberata can apparently influence germination of seed and may pose a threat to the regeneration of P. falcatus from seeds in Ethiopian forests.     

Infection routes for Sclerotinia sclerotiorum in apetalous and fully petalled winter oilseed rape

Near‐isogenic lines (NILs) of apetalous (AP) and fully petalled (FP) winter oilseed rape were used to investigate infection by Sclerotinia sclerotiorum, which occurs mainly via infected petals adhering to leaves in FP oilseed rape. AP1 flowers had an average of 1·4 and 0·8 petals per flower in field and polytunnel experiments, respectively. In field experiments there were no significant differences between counts of FP1 petals, FP1 stamens and AP1 stamens adhered to leaves during flowering. At any one sample time, significantly more stamens tested positive for S. sclerotiorum on AP1 than FP1 NILs, e.g. in 2004, at early flowering 37·5% and 24·2% of stamens tested positive on AP1 and FP1 NILs, respectively. In polytunnel experiments, there were significantly more sclerotinia lesions per plant in the FP1 than in the AP1 NIL. The AP1 NIL did not avoid infection completely, probably because it produced some petals, and lesions were initiated from adhered stamens as well as petals. However, while 8·5% and 16·3% of petals initiated lesions in FP1 and AP1 NILs, respectively, only 2·5% and 1·0% of stamens initiated lesions in FP1 and AP1 NILs, which suggests stamens may be less infective than petals. In field experiments the AP1 NIL had significantly less incidence of sclerotinia stem rot than the FP1 NIL in 2004 (4·9% and 7·0%, respectively). However, there was no significant difference in stem rot incidence between AP and FP lines in 2005 (3·6% and 4·3%, respectively) or 2006 (5·5% and 3·9%, respectively).