Influence of light on the Monilinia laxa–stone fruit interaction

Light represents a signal for the regulation of virulence in many microbial pathogens. Two stone fruits, nectarines and cherries, were used to investigate the influence of light on brown rot caused by Monilinia laxa. Three single-spore isolates were inoculated on nectarines and incubated under different white lights, red light, blue light, green light, and black light with two photoperiods. In addition, to understand the effect of daylight irradiance on brown rot, M. laxa was inoculated on different cherry cultivars and incubated under two simulated solar irradiations. Significantly higher disease severity and sporulation were reported on inoculated nectarines incubated under 58 W white light for 12 hr light/12 hr darkness than on nectarines incubated in continuous darkness. Only red light caused a significant increase in the incidence and severity of the disease in nectarines inoculated with the three M. laxa isolates, compared to fruit incubated under white light. High light intensity (185.45 W/m²), caused greater brown rot severity in all cherry cultivars, both early and late varieties, than low irradiance (145.85 W/m²). Significant up-regulation of the pathogenicity-related MlPNL2 gene was observed as an early response after cherry inoculation under high-intensity light, especially in late cherry cultivars, while MlPG1 expression did not show any changes under different light irradiances. M. laxa was shown to be a light-responsive fungal pathogen, and light seemed to play an active role in brown rot development.     

UV protectants for Candida oleophila (strain O), a biocontrol agent of postharvest fruit diseases

This study investigated the influence of UV‐B radiation (280–320 nm) on survival of Candida oleophila strain O, an antagonist yeast that prevents postharvest diseases caused by Botrytis cinerea and Penicillium expansum on apple and pear fruits. Lethal doses (LD₅₀ and LD₉₀) were, respectively, 0·89 and 1·45 Kj m^?2 for in vitro exposure and 3·06 and 5·5 Kj m^?2 for in vivo exposure. A screening test of UV‐B protectants for strain O was also evaluated under in vitro and in vivo conditions. The in vitro results showed that sodium ascorbate (0·1% and 0·01%), riboflavin (0·1%) and uric acid (0·1% and 0·01%) were the most effective and most suitable protectants. However, only riboflavin (0·1%) and uric acid (0·1%) were effective under in vivo conditions. The efficacy obtained with strain O against P. expansum, when subjected to UV‐B radiation, was 75·0% and 49·2% for pathogen concentrations of 10⁵ and 10⁶ spores mL^?1, respectively. Adding riboflavin to strain O gave a similar efficacy (64·2%). Applying strain O together with uric acid (0·1%) was less active (47·7%). Nonetheless, its efficacy when applied with the antioxidants sodium ascorbate (71·1%) or ascorbic acid (82·5%) was the greatest. Riboflavin and uric acid were the most cost‐effective protectants, and could be included in the final formulation of strain O when applied preharvest.     

Preventive and post‐infection control of Botrytis cinerea in tomato plants by hexanoic acid

The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration‐ and pH‐dependent manner, and stopped spore germination at a very early stage, preventing germ‐tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 mm . Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 mm . Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4‐week‐old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea, which is responsible for many economic losses on fruits, vegetables and flowers.     

Temporal development and relationship amongst brown rot blossom blight,fruit blight and fruit rot in integrated and organic sour cherry orchards

The aim of this 4‐year study was to characterize temporal development of brown rot blossom blight and fruit blight (caused by Monilinia spp.) and their sporulating areas in sour cherry orchards; and to determine the relationships amongst incidence and sporulating area of blossom blight, fruit blight and fruit rot. The study was performed in integrated and organic orchard blocks on two cultivars (Újfehértói fürtös and Érdi b?term?). On both cultivars, disease progress on flowers and fruits was 2–10 times slower in the integrated than in the organic management system. The peak incidence values were 9 and 31 days after petal fall for blossom blight and fruit blight, respectively. After these dates, no new blight symptoms on flowers and/or fruits appeared and the disease was levelling off. Final blossom blight incidence ranged from 1 to 5% and from 12 to 34%, and fruit rot incidence from 2 to 6% and from 11 to 26% in the integrated and the organic orchards, respectively. The sum of fruit blight incidence ranged from 9 to 22% for the organic system, but was below 5% for the integrated system, while the final sporulating area was 5–16 mm² and <3 mm², respectively. Among the five highest Pearson's correlation coefficients, relationships between blossom blight and early fruit blight stage (r = 0·845, P = 0·0087 integrated; r = 0·901, P = 0·0015 organic), and between sporulating area and fruit rot (r = 0791, P = 0·0199 integrated; r = 0·874, P = 0·0039 organic) were the most significant relationships from an epidemic standpoint as they indicated a connection between different brown rot symptom types.     

Resistance to Rhizoctonia solani AG‐2‐2 (IIIB) in creeping bentgrass plants transformed with pepper esterase gene PepEST

A pepper esterase (PepEST) gene was introduced into creeping bentgrass (Agrostis stolonifera) by Agrobacterium‐mediated transformation. Purified recombinant PepEST proteins were sufficient to inhibit the growth of the fungal pathogens Rhizoctonia solani AG2‐2 (IIIB) (causing brown patch) and Sclerotinia homoeocarpa (dollar spot), but not the oomycete responsible for pythium blight, Pythium aphanidermatum. PepEST proteins were most effective against R. solani. After genetic transformation of creeping bentgrass with PepEST, the genomic integration of transgenes bar and PepEST was confirmed by Southern blot analysis, and their expression was also validated by northern blot and western blot analyses. Disease severity on R. solani‐inoculated leaves of transgenic plants was <10% compared to ca. 50% in non‐transgenic plants. Microscopic observation of infected leaves indicated that PepEST inhibited the growth of hyphae upon fungal infection.     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

Frequency of brown rot fungi on blossoms and fruit in stone fruit orchards in Greece

Brown rot is a devastating disease of stone fruits caused by Monilinia spp. This study was conducted to investigate the disease aetiology on blossoms and fruit in peach, apricot, sweet cherry and plum orchards, in Greece. In total, 1433 isolates obtained from orchards located in the main stone fruit production regions of Greece were identified to species based on the presence/size of a cyt b intron. Monilinia laxa and M. fructicola were detected at frequencies of 59 and 41%, respectively, while M. fructigena was absent. Monilinia fructicola was more common on fruit whereas M. laxa occurred in similar frequency on blossoms and fruit. Monilinia laxa was replaced by M. fructicola in fruit infections of peach in both regions investigated and in fruit infections of plum in the Imathia region. Assessments of aggressiveness of 30 isolates of both species on the petals and fruits of the hosts showed that M. fructicola isolates were more aggressive. This suggests that the predominance of M. laxa on the blossoms cannot be explained by higher aggressiveness. Measurements of the effect of temperature on mycelial growth showed that M. laxa isolates had a higher growth rate than M. fructicola at the lowest temperature tested of 5°C, whereas M. fructicola isolates showed higher growth rates at higher temperatures. The observed high frequency of M. fructicola in Greece represents a major threat for stone fruit production. Furthermore, the information obtained about delineation of species and plant organ preference could be useful for the implementation of disease management strategies.     

Colletotrichum acutatum interactions with unripe and ripe strawberry fruits and differential responses at histological and transcriptional levels

Microscopic investigations were conducted into the interaction of Colletotrichum acutatum on white and red strawberry (Fragaria ×ananassa) fruit surfaces. The results showed that, whilst the early interaction events were similar in both white and red fruits, after 24 h fungal colonization dramatically varied: in white fruits C. acutatum became quiescent as melanized appressoria, but on red fruits it displayed subcuticular necrotrophic invasion. A microarray analysis of white and red strawberries after 24 h of interaction with C. acutatum was performed, in order to reveal differences in gene expression possibly related to the different susceptibility of unripe and ripe fruits. Epi/catechin‐related genes and fatty acid metabolism genes, involved in the production of quiescence‐related molecules such as flavan‐3‐ols, proanthocyanidins and antifungal dienes, were found to be regulated during strawberry ripening, supporting a role for these molecules as preformed defence mechanisms. Besides several genes commonly regulated upon pathogen interaction, different genes were specifically transcribed only in white or red challenged fruits; a number of these, such as those coding for lectin and polyphenol oxidase, possibly account for specific pathogen‐induced responses. The putative biological role of these genes in the different susceptibility of fruits to C. acutatum is discussed.     

Resistance to Oculimacula yallundae and Oculimacula acuformis is conferred by Pch2 in wheat

The recent report of a differential response of wheat lines containing the Pch2 gene to infection with the eyespot pathogens Oculimacula yallundae and O. acuformis has prompted this re‐examination of the response to these fungi by the recombinant lines used to map Pch2. Homozygous recombinant substitution lines (RSL) derived from the hybridization of Chinese Spring (CS) and the CS chromosome substitution line Cappelle Desprez 7A (CS/CD7A), previously evaluated for response to glucuronidase (GUS)‐transformed O. yallundae, were evaluated for response to infection with GUS‐transformed O. acuformis. Based on visual scores and on GUS expression level, which reflects fungal colonization of seedling plants, evidence of a quantitative trait locus (QTL) conferring resistance to O. acuformis was found in two separate growth chamber experiments (logarithm of the odds, LOD, = 2·7 and 6·7 at 305 and 289 cM, respectively) that was equivalent in location to that for resistance to O. yallundae (LOD = 13·2 and 11·4 at 289 and 304 cM, respectively). These results confirm that Pch2 confers some degree of resistance against both O. yallundae and O. acuformis under these conditions.     

Biocontrol traits of two Paenibacillus polymyxa strains SQR‐21 and WR‐2 in response to fusaric acid,a phytotoxin produced by Fusarium species

The aim of the present study was to investigate the effects of the phytotoxin fusaric acid (FA) on the biocontrol traits of two biocontrol strains Paenibacillus polymyxa WR‐2 and SQR‐21. The results showed that the growth of both WR‐2 and SQR‐21 decreased with increasing FA concentration, and at 70 and 80 μg mL^?1 FA, respectively, the strains were unable to grow. The biocontrol traits of both strains were negatively affected by FA concentration higher than 2·5 μg mL^?1. However, at 2·5 μg mL^?1 FA, biofilm formation and root colonization were not affected and there was even a positive effect on the production of spores and hydrolytic enzymes (protease and β‐l,3‐glucanase). The production of fusaricidin‐type antifungal compounds was increased with an increase in FA concentration up to 50 and 60 μg mL^?1 for WR‐2 and SQR‐21, respectively. The production of antifungal volatile organic compounds by WR‐2 and SQR‐21 was increased only at 2·5 μg mL^?1 FA. The effect of FA on the overall metabolic activity of WR‐2 and SQR‐21 was also determined. This study will help to understand the response of P. polymyxa strains to FA and will help to improve their biocontrol efficiency.     

Long‐term viability of the northern anthracnose pathogen,Kabatiella caulivora,facilitates its transportation and spread

The conidia and resting hyphae of the northern anthracnose pathogen of Trifolium species, Kabatiella caulivora, were effectively carried by, and maintained long‐term viability on, a range of materials, including metals, fabrics, woods and plastics. Conidia and hyphae became thick‐walled and melanized with time. There were significant (P < 0.001) differences in conidia/resting hyphae survival between carrier materials and between temperature regimes. At 23 °C/8 °C day/night, conidia and resting hyphae remained viable on steel, corrugated iron, galvanized steel, all tested fabrics, wood and random mixed materials for up to 8 months. At 36 °C/14 °C day/night, conidia and resting hyphae remained viable for up to 8 months, but only on cotton, denim, fleece, silk, leather, paper, plastic and all wood materials. At 45 °C/15 °C day/night, conidia and resting hyphae remained viable up to 8 months only on fleece wool, Eucalyptus marginata (jarrah wood) and paper. There were significant differences between carrier materials in their abilities to retain conidia and resting hyphae after washing (P < 0.001). Metabolic activity was confirmed for conidia and resting hyphae recovered after 8 months and K. caulivora colonies successfully re‐established on potato dextrose agar. Findings confirmed the critical importance of materials as long‐term carriers of viable K. caulivora conidia and resting hyphae, highlighting the potential for spread of a highly virulent K. caulivora race within and outside Australia via farming equipment, clothing and other associated materials. Results also have wider biosecurity implications for the transportation of fungal‐infested carrier materials previously considered as low risk.     

Diversity and population dynamics of Penicillium spp. on apples in pre- and postharvest environments: consequences for decay development

Isolates of Penicillium spp. were collected regularly from 2001 to 2003 from the surfaces of apple fruit pre- and postharvest, and from the atmosphere of orchards and storage rooms in France. Penicillium spp. were not detected from the atmosphere of conventional orchards, while their density did not exceed 50 spores m⁻³ in the atmosphere of organically managed orchards. Penicillium spp. were seldom detected on apple surfaces in the orchard. The density of Penicillium on apples increased from 10 to 50 spores cm⁻² after 1 month in storage to 300–400 spores cm⁻² after 6 months. The level of airborne Penicillium increased by up to 2 × 10⁴ and 2·5 × 10³ spores m⁻³ within nondisinfected and previously disinfected warehouses, respectively. Penicillium expansum (30–62%) and P. solitum (6–45%) were the most prevalent species on apple or in storage rooms. Other species of Penicillium isolated included P. commune, P. verrucosum, P. chrysogenum, P. rugulosum and P. digitatum. Apple fruit were also surveyed for wounds and the number of open lenticels using the sulphur dioxide test. The incidence of wounding at harvest varied from 12 to 36%, depending on cultivar and locality. When apples were inoculated at harvest by either aqueous or aerial inoculum of P. expansum, the decay incidence was constantly higher than the incidence of wounding. The number of open lenticels per cm² of apple surface varied from 0·5 on cv. Boskoop to 4·4 on cv. Golden Delicious. An average of 13 and 2·1% of lenticels, respectively, were infected when they were inoculated by P. expansum and P. verrucosum. Cultivars of apple fruit that showed a greater number of open lenticels, combined with a large diameter varying from 100 to 200 µm, were more susceptible to P. expansum.     

Wheat leaf resistance to Pyrenophora tritici‐repentis induced by silicon activation of phenylpropanoid metabolism

Tan spot caused by Pyrenophora tritici‐repentis is a disease present in all wheat‐producing countries and silicon (Si) treatment of wheat plants has been shown to increase plant resistance to tan spot. In this study, the effect of phenylpropanoid metabolism on resistance to tan spot was evaluated and some phenolic compounds that accumulated in response to P. tritici‐repentis attack were identified. Furthermore, the effect of Si on phenylalanine ammonia‐lyase (PAL) activity and phenolic compound accumulation were determined in situ. Antifungal activity of differentially accumulated phenolic compounds was also evaluated in in vitro tests. Results showed that the increase in concentration of phenolic compounds was greatest at the onset of infection, and that some compounds showed fungitoxic effects including fungal tip swelling, granulation of germ tube and hyphae, and hyphal hyperbranching. Silicon‐induced reduction in both lesion size and tan spot disease progression were associated with activation of phenylpropanoid metabolism. PAL activity and accumulation of antifungal phenolic compounds were greater in pathogen‐inoculated plants supplied with Si. In these plants, fluorescence indicative of accumulation of phenolic compounds occurred early in epidermal cells and its intensity increased during the evaluation period, showing higher numbers of fluorescent cells around infected cells. Thus, the combined responses of cell fluorescence at sites of infection, increased PAL activity and accumulation of phenols indicate that Si strengthened wheat defence responses to infection by P. tritici‐repentis, reducing the severity of tan spot.     

The effect of environmental factors on infection of blueberry fruit by Colletotrichum acutatum

Anthracnose fruit rot of blueberries caused by Colletotrichum acutatum is a serious problem in humid blueberry‐growing regions of North America. In order to develop a disease prediction model, environmental factors that affect mycelial growth, conidial germination, appressorium formation and fruit infection by C. acutatum were investigated. Variables included temperature, wetness duration, wetness interruption and relative humidity. The optimal temperature for mycelial growth was 26°C, and little or no growth was observed at 5 and 35°C. The development of melanized appressoria was studied on Parafilm‐covered glass slides and infection was evaluated in immature and mature blueberry fruits. In all three assays, the optimal temperature for infection was identified as 25°C, and infections increased up to a wetness duration of 48 h. Three‐dimensional Gaussian equations were used to assess the effect of temperature and wetness duration on the development of melanized appressoria (R² = 0·89) on Parafilm‐covered glass slides and on infection incidence in immature (R² = 0·86) and mature (R² = 0·90) blueberry fruits. Interrupted wetness periods of different durations were investigated and models were fitted to the response of melanized appressoria (R² = 0·95) and infection incidence in immature (R² = 0·90) and mature (R² = 0·78) blueberry fruits. Additionally, the development of melanized appressoria and fruit infection incidence were modelled in relation to relative humidity (R² = 0·99 and 0·97, respectively). Three comprehensive equations were then developed that incorporate the aforementioned variables. The results lay the groundwork for a disease prediction model for anthracnose fruit rot in blueberries.     

Synthesis, characterization, absolute structural determination and antifungal activity of a new chlorinated aromatic avenaciolide analogue

BACKGROUND: Avenaciolide, a natural product isolated from Aspergillus avenaceus H. Smith, possesses several interesting biological properties, such as antifungal and antibacterial activities and inhibition of glutamate transport in mitochondria. In a study aiming to discover new compounds with antifungal activity, a bis‐γ‐lactone analogous to avenaciolide was prepared and characterized by elemental analysis, mass spectrometry, and infrared and NMR spectroscopy. RESULTS: The absolute structures of this compound and of the synthetic precursor (also a bis‐γ‐lactone) were determined by X‐ray diffraction analysis. The bis‐γ‐lactones synthesized crystallize in the orthorhombic space group P2₁2₁2₁, and the crystal packings are supported by C? H···O hydrogen bonds. The compound showed antifungal activity against Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., while the synthetic precursor was inactive under the in vitro test conditions employed. CONCLUSION: The results indicate that it is not only the bis‐γ‐lactone skeleton that is important to antifungal activity. The latter also depends on the presence of the exocyclic double bond possibly due to a Michael addition type reaction with the fungal enzymes. Copyright © 2008 Society of Chemical Industry     

Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide (PMA) and qPCR

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and M. fructicola. Conventional methods used to identify M. fructicola are mainly based on phenotypic characteristics and pathogen quantification is not always accurate. In contrast, methodologies based on molecular tools improve pathogen characterization and identification but are not able to differentiate between live and dead conidia. In this study the PMA‐qPCR methodology was optimized, validated and applied to quantify viable cells of M. fructicola in artificially and naturally infected samples. qPCR methodology showed good primer efficiency and sensitivity with quantification limits lower than obtained using a plate count method. The conditions of propidium monoazide (PMA) pretreatment were 60 μm PMA for 20 min incubation and 30 min of light‐emitting diode (LED) exposure that, combined with qPCR, measured live cells accurately without overestimation of dead cells. Using this methodology in naturally infected samples, M. fructicola live cells were quantified specifically, in contrast to other traditional methodologies that cannot distinguish among Monilinia spp. The developed methodology based on combined PMA‐qPCR will be a new tool to quantify viable M. fructicola in further epidemiological and ecological studies of this fungus.     

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND: Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS: None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group‐I‐like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS: Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143‐associated intron. Copyright © 2010 Society of Chemical Industry