Grape Black Rot Research Articles

Revisiting the intron hypothesis of QoI resistance in Phyllosticta ampelicida, the causal agent of grape black rot, and other Phyllosticta species

AbstractChemical control of grape black rot, caused by Phyllosticta ampelicida, relies mainly on the use of demethylation inhibitors (DMIs) and quinone outside inhibitors (QoIs). The effectiveness of QoI fungicides is influenced by alternative respiration activity, and the exon/intron structure and point mutations in the target protein's gene, the cytochrome b (cytb) gene. Our study aims to investigate the QoI fungicide sensitivity of 48 P. ampelicida isolates in vitro by measuring EC50 and the molecular characteristics of the cytb gene and its mRNA in P. ampelicida and other Phyllosticta species. Mycelial growth tests revealed that the P. ampelicida isolates were sensitive to both azoxystrobin and trifloxystrobin; baseline EC50 values were 0.029 and 0.022 μg/mL, respectively. Addition of salicylhydroxamic acid (SHAM) resulted in lower EC50 values (0.024 and 0.017 μg/mL, respectively). None of the typical point mutations conferring resistance to QoIs in some fungi were detected. A group I intron was present right after the 143rd codon in the cytb gene in four of the six Phyllosticta species examined. The sequence and exon/intron structure of the cytb gene of P. ampelicida isolated from Vitis vinifera is studied in detail and published here. Our results indicate a low risk of QoI resistance development via the G143A mutation in P. ampelicida.

A Novel Fuzzy C-Means based Chameleon Swarm Algorithm for Segmentation and Progressive Neural Architecture Search for Plant Disease Classification

This study proposed a novel framework for plant leaf disease identification. The proposed model consists of four steps including pre-processing, segmentation, feature extraction, and classification. At first, the unwanted noise and overfitting are removed, and also image contrast level is enhanced. Secondly, the Fuzzy C-Means (FCM) based Chameleon Swarm Algorithm (CSA) named as (FCM-CSA) is used for plant leaf diseased part segmentation. In the third stage, the feature extraction is performed using a fast GLCM feature extraction model. Finally, the Progressive Neural Architecture Search (PNAS) is used for plant leaf disease identification. The experimental investigations are carried out using MATLAB software with the Mendeley database. From this dataset, we have used Apple Cedar Apple Rust (ACAR), Cherry Powdery Mildew (CPM), Corn Common Rust (CCCR), Apple Healthy (AH), Grape Black Rot (GBR), Pepper Bell Bacterial Spot (PBBS), Potato Late Blight (PLB) and Tomato Leaf Mold (TLM) disease images. Different measures such as precision, recall, sensitivity, specificity, and accuracy results are used to validate the performance of the proposed model.

The effect of black rot on grape berry composition

AbstractGuignardia bidwellii, indigenous to North America, is a significant pathogen of grapes long known in Hungary, infecting only the growing green parts of the vine (leaves, petioles, shoots, and bunches). In the absence of adequate plant protection and extreme weather conditions such as a predominantly humid, warm year, black rot of grapes can be expected. The pathogen can cause high yield losses due to grape rot and reduce wine quality if the infection is severe.The evolution of certain biogenic amine compounds were investigated under the influence of grape black rot. The results obtained showed that they were present in low concentrations from an oenological point of view. Polyphenol composition was consistent with the literature, blackening affected mainly the concentration of catechin. Black rot fungus does not produce β-glucosidase enzyme. In terms of resveratrol content, black rot has no particular effect. However, likeBotrytis cinerea, it produces glycerol and, proportionally, gluconic acid in lower concentrations.It can be concluded that black rot of grapes does not cause health problems when introduced into wine processing.

Detección por PCR de Guignardia bidwellii, agente causal de la pudrición negra de la vid

<em>Guignardia bidwellii</em> (anamorfo: <em>Phyllosticta</em> <em>ampelicida</em>) es el agente etiológico de la pudrición negra de la vid, enfermedad de importancia económica en Europa y cuarentenaria para México. La identificación del estado anamorfo mediante caracteres morfológicos resulta complicada debido a la similitud entre especies del mismo género, por ejemplo, con el endófito cosmopolita <em>G. endophyllicola</em> (<em>P. capitalensis</em>), por lo que es necesario contar con herramientas de diagnóstico rápidas y específicas. Con este propósito se desarrolló un marcador molecular basado en los polimorfismos de un solo nucleótido (SNP) en la región ITS del ADNr, donde se diseñaron los oligos Bidwell y Ampel2 que mediante amplificación PCR generan un fragmento de 173 pb específico a <em>G. bidwellii f. euvitis</em>. La validación del método demostró que no hay amplificación cruzada con otros hongos fitopatógenos ni con el genoma de la vid. La técnica fue sensible al detectar hasta 30 pg µL-1 de ADN a partir de cultivos monospóricos y en mezcla con tejido vegetal. Se presenta este procedimiento de diagnóstico como una opción rápida y específica para el monitoreo y detección de <em>G. bidwellii f. euvitis</em> en apoyo a las estrategias de prevención, manejo, cuarentena y erradicación de la enfermedad.

Antifungal activity of Bignoniaceae plants on Aspergillus carbonarius and Aspergillus niger

Twenty four extracts from Bignoniaceae plants of northwest Argentina were tested for antifungal activity against Aspergillus species responsible of the grape black rot. Stems and leaves of Amphilophium cynanchoides, Macfadyena cynanchoides, Tecoma stans and Jacaranda mimosifolia were separately extracted with solvents of increasing polarity to obtain the dichloromethane (fCH2Cl2), ethyl acetate (fEtOAc) and methanol extracts (fMeOH). The fCH2Cl2 from stem of M. cynanchoides had the lowest IC50 (1.0–1.2 mg/mL) and MID values (0.6–1.2 mg) and the highest ID values (5.0–6.8 mm) on A. niger and A. carbonarius. The main contributors of the antifungal activity of fCH2Cl2 were identified as lapachol (MIC = 0.25–1.00 mg/ml) and 1-hydroxy-4-methylanthraquinone (MIC = 0.0625–0.125 mg/mL). These compounds synergized the antifungal activity of sodium metabisulfite and showed an additive effect in mixtures with propiconazol. They might be used as additives of commercial antifungals to protect grapes against A. niger and A. carbonarius.

Production of Pycnidia and Conidia by Guignardia bidwellii, the Causal Agent of Grape Black Rot, as Affected by Temperature and Humidity.

Black rot, caused by the fungus Guignardia bidwellii, is a polycyclic disease affecting grape leaves and berries. In environmentally controlled experiments and in a 3-year field study, the effects of temperature and relative humidity (RH) were assessed on the following growth parameters of G. bidwellii: (i) formation of pycnidia and cirri in grape leaf lesions, (ii) production and germination of conidia, and (iii) length of the period between lesion appearance and pycnidia production. Pycnidia were produced between 5 and 35°C and at 90 to 100% RH but more pycnidia were produced between 20 and 30°C. No pycnidia were produced at RH < 90%. The first pycnidia were produced in approximately 2 days after lesion appearance at ≥20°C and in 8 days at 5°C; pycnidia continued to be produced on the same lesion for 5 to 16 days after lesion appearance, depending on the temperature. Models were developed to describe the effect of temperature and RH on pycnidia production, accounting for 95 and 97% of variability, respectively. Cirri were extruded only between 15 and 35°C and mainly at 100% RH. Field experiments confirmed that pycnidia are produced for several days on a leaf lesion and that the length of the period between lesion appearance and pycnidia production depends on temperature. Overall, the findings showed that production of conidia requires high humidity; under field conditions, some hours at high humidity, which usually occur at nighttime, rather than constant high humidity may be sufficient.

Composition and evaluation of a novel web-based decision support system for grape black rot control

Black rot on grapevine is a fungal disease caused by Phyllosticta ampelicida (syn. Guignardia bidwellii) affecting grape leaves as well as clusters. A novel black rot decision support system termed VitiMeteo Black rot was assembled based on existing sub-models and incorporated into the established VitiMeteo forecast and decision support platform. Based on local weather data and a 5-day weather forecast, VitiMeteo Black rot simulates the relative susceptibility of grape clusters, the occurrence and severity of infection events as well as the duration of incubation periods. Data sets obtained in extended international (14 case studies; eight monitoring locations; 11 cultivars; seven countries in Europe and North America) field monitoring campaigns in 2012 and 2013 were used to evaluate the model predictions of newly expressed symptoms on leaves. In the case of the Vitis vinifera cultivars, on average 26.3 disease assessments took place per season. On average, 9.9 predictions were classified as true positive, 8.0 as true negative, 5.2 as false positive and 3.2 as false negative. Model precision, sensitivity and accuracy were on average 64, 77 and 67 %. Potential reasons for false positive and false negative predictions are discussed. VitiMeteo Black rot is freely available for several locations in Germany, Luxembourg and Austria on the internet via the VitiMeteo platform and might be expanded to other regions in the future.

Microsatellite markers for population studies of the ascomycete Phyllosticta ampelicida , the pathogen causing grape black rot

Grape black rot, caused by the homothallic ascomycete Phyllosticta ampelicida , is a disease originating from North America and is widespread in Europe. To investigate population structure and epidemics of this pathogen, we developed 11 microsatellite markers. A multiplex PCR assay was used to amplify genomic DNA from environmental samples including mummified berries and foliar lesions, and from fungal cultures. Environmental samples were collected from five countries (Switzerland, France, Germany, Luxembourg and the USA), and consisted of 64 different genotypes. Five additional genotypes were identified from pure cultures isolated in Switzerland and Germany. The allele rarefaction approach indicated that French vineyards in the region of Bordeaux displayed the greatest mean allelic richness, probably related to the fact that France is the country where the disease was first reported outside North America, in 1885. Our results also suggest the absence of links between the species/cultivar of Vitis hosts and the infecting P. ampelicida genotypes. This is the first report of development of microsatellite markers and their deployment for population studies of P. ampelicida .

Use of systems analysis to develop plant disease models based on literature data: grape black-rot as a case-study

The available knowledge on black-rot of grape was retrieved from literature, analyzed, and synthesized to develop a mechanistic model of the life cycle of the pathogen (Guignardia bidwelii) based on the systems analysis. Three life-cycle compartments were defined: (i) production and maturation of inoculum in overwintered sources (i.e., ascospores from pseudothecia and conidia from pycnidia in berry mummies and cane lesions); (ii) infection caused by ascospores and conidia; and (iii) disease onset and production of secondary inoculum. An analysis of published, quantitative information was conducted to develop a mechanistic model driven by weather and vine phenology; equations were developed for ascospore and conidial maturation in overwintered fruiting bodies, spore release and survival, infection occurrence and severity, incubation and latency periods, onset of lesions, production of pycnidia, and infectious periods. The model was then evaluated for its ability to represent the real system and its usefulness for understanding black-rot epidemics by using three typical epidemics. Finally, weaknesses in our knowledge are discussed. Additional research is needed concerning the influence of wetness duration and temperature on infection by ascospores, production dynamics of pycnidia and conidia in black-rot lesions, and the dynamics of conidia exudation from pycnidia.

Epidemiology, identification and disease management of grape black rot and potentially useful metabolites of black rot pathogens for industrial applications – a review

AbstractPhyllosticta ampelicida (teleomorph: Guignardia bidwellii) is the fungal plant pathogen that causes black rot on grapevine. It is able to infect all green, expanding grape tissues. Black rot poses a threat to both yield and wine quality; a severe attack can virtually cause a complete crop loss. The fungus is native to North America, and was spread to Europe at the end of the 19th century. With the beginning of the 21st century, an increasing importance of the disease was observed in several European winegrowing regions. Successful black rot control strategies combine sanitary measures, cultural techniques, growing cultivars with reduced susceptibility and the use of effective fungicides. Berries are most susceptible to infections between flowering and bunch closure and consequently, fungicide applications against black rot need to focus on this period. In this paper, forecast models and decision support systems that help to achieve satisfactory control with a minimum of fungicide input are presented. Black rot in grapevine was reported to be well controlled in field experiments by a broad range of fungicide classes including quinone outside inhibitors (98 ± 3% efficacy), demethylation inhibitors (98 ± 3% efficacy) and dithiocarbamates (92 ± 7% efficacy). Average efficacies of other fungicide classes tested so far ranged from 32 to 69%; meta data on the efficacy of fungicides in numerous field trials are given. At present, black rot causes major problems primarily if vineyard management and fungicide use were reduced or abandoned. Organically managed vineyards are highly affected. Hence, site and cultivar selection as well as cultural measures are of the highest importance especially in organic viticulture. Recent studies showed that some strains formerly classified as G. bidwellii in fact belong to a distinct species (P. parthenocissi). Black rot pathogens produce some phytotoxic secondary metabolites, such as phenguignardic acid, guignardic acid, alaguignardic acid and the guignardianones A, E and F, which could stimulate the development of new herbicides of natural origin.

Assignment of Configuration in a Series of Dioxolanone‐Type Secondary Metabolites from Guignardia bidwellii – A Comparison of VCD and ECD Spectroscopy

AbstractThe absolute configurations of a series of phytotoxic dioxolanone‐type secondary metabolites isolated from culture filtrates of the grape black rot fungus Guignardia bidwellii were determined by vibrational circular dichroism (VCD) spectroscopy in the mid‐IR frequency range. Comparison of the recorded data with DFT calculations showed good agreement between experiment and theory for VCD, whereas electronic circular dichroism (ECD) data for the compounds matched poorly with the predicted spectra obtained by time‐dependent DFT (TDDFT) calculations at the same level of theory.

Total Synthesis of (+)‐Phenguignardic Acid, a Phytotoxic Metabolite of Guignardia bidwellii

Abstract(+)‐Phenguignardic acid, a phytotoxic metabolite of the grape black rot fungus Guignardia bidwellii was synthesized, as well as its enantiomer, in eight steps from (R)‐phenyllactic acid and 3‐phenylprop‐2‐yn‐1‐ol. The formation of the carboxylate at a late stage, to avoid enolization of the precursor used in the central acetalization step, proved to be crucial. The synthesis of the natural product allowed the unabiguous assignment of its hitherto unknown absolute configuration.

Typification and phylogenetic study of Phyllosticta ampelicida and P. vaccinii

Phyllosticta ampelicida, the causal agent of black rot of grape, and P. vaccinii, the pathogen of early rot of cranberry, are neotypified and epitypified respectively by strains from American Type Culture Collection (ATCC). Morphological characteristics of these cultures are described and compared with type specimens and the original descriptions. Based on phylogeny inferred from DNA sequences of internal transcribed spacer (ITS), actin (ACT), translation elongation factor 1-alpha (TEF1) and glyceraldehyde-3-phosphate dehydrogenase (GPDH), traditionally defined P. ampelicida (teleomorph Guignardia bidwellii) is revealed to be a species complex including P. ampelicida sensu stricto, typified in this study, and two additional species, one of which is described as new P. parthenocissi. Our results did not support an anamorph/teleomorph connection between P. vaccinii and G. vaccinii in that they represent different species. Multilocus sequence data from the neotype and epitype were generated and deposited in GenBank.

Phytotoxic dioxolanone-type secondary metabolites from Guignardia bidwellii

Phenguignardic acid was recently described as a phytotoxic secondary metabolite from submerged cultures of the grape black rot fungus Guignardia bidwellii. Since the production rate of this natural product in submerged culture is very low, fermentation optimisation was carried out. The optimisation of cultivation conditions led to the identification of seven secondary metabolites, structurally related to guignardic acid, a known secondary metabolite from Guignardia species containing a dioxolanone moiety. All metabolites presented here have not been described to date and are presumably biosynthesised via deamination products of amino acids, such as phenylalanine, valine, tyrosine, and alanine. Four of the seven compounds showed phytotoxic activity. Based on the structures determined by NMR spectroscopy a preliminary structure activity relationship indicated a free carboxyl group as presumably required for the phytotoxic activity.

Phenguignardic Acid and Guignardic Acid, Phytotoxic Secondary Metabolites from Guignardia bidwellii

Bioactivity-guided isolation led to the identification of phenguignardic acid (2), a new phytotoxic secondary metabolite from submerged cultures of grape black rot fungus, Guignardia bidwellii. The compound is structurally related to guignardic acid (1), a dioxolanone moiety-containing metabolite isolated previously from Guignardia species. However, in contrast to guignardic acid, which is presumably synthesized from deamination products of valine and phenylalanine, the biochemical precursor for the biosynthesis of the new phytotoxin appears to be exclusively phenylalanine. Guignardic acid was also found in extracts of cultures from Guignardia bidwellii. The phytotoxic activities of both compounds were assessed in plant assays using either detached vine leaves or intact plants. Antimicrobial and cytotoxic activities of phenguignardic acid were determined.

A Cumulative Degree-Day-Based Model to Calculate the Duration of the Incubation Period of Guignardia bidwellii.

The duration of the incubation period of Guignardia bidwellii on leaves and clusters of Vitis vinifera strongly correlates to temperature. To describe this relationship mathematically, a new, cumulative degreeday- based model was developed. According to this model, first symptoms on leaves appear after reaching a threshold of 175 cumulative degree-days (calculated as the sum of average daily temperatures between 6 and 24°C starting on the day after the infection). On clusters, the duration of the incubation period is additionally affected by their respective developmental stages. For 'Riesling', the duration of the incubation period on clusters corresponds to the duration on leaves until reaching the phenological stage "berries beginning to touch" but extends continuously with ongoing phenological development. Therefore, a correction factor recognizing cluster phenology was derived to calculate the cumulative degree-day thresholds for the occurrence of first symptoms on clusters after reaching "majority of berries touching". Hence, this present model allows the estimation of fungal development and forecasts the appearance of new symptoms on leaves as well as on clusters, enabling growers to more precisely schedule curative as well as protective fungicide applications against grape black rot.

Molecular, proteomic and morphological characterization of the ascomycete Guignardia bidwellii, agent of grape black rot: a polyphasic approach to fungal identification

Guignardia bidwellii is the etiological agent of grape black rot, a disease affecting Vitis and other Vitaceae that can cause heavy crop losses in vineyards. Its identification is based mainly on morphological characters and the symptoms on plants but, due to their variability, they may be difficult to interpret to reliably distinguish the pathogen to species. To date, despite the economic importance of G. bidwellii, no molecular investigations have been carried out on Vitis isolates and few sequence data are available for cultures derived from ornamental host plants. We analyzed samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins by morphological, molecular and proteomic techniques, including ITS1-ITS2 regions and calmodulin gene sequencing, as well as matrix-assisted laser desorption/ionization analysis by time-of-flight mass spectrometry (MALDI-TOF MS). This polyphasic approach allowed assessing the phylogenetic relationships among the different isolates and suggested the existence of two distinct species. The advantages of a polyphasic approach for the identification of G. bidwellii are highlighted.

Protective and curative grape black rot control potential of pyraclostrobin and myclobutanil

Strobilurin or triazole type fungicides have proven to be very effective in strategies to control grape black rot (causal agent: Guignardia bidwellii) due to their combined protective and curative activities. However, differences in their long-lasting protective capacity or in the limitation of the time frame for curative treatments have not yet been clearly described regarding this pathogen. To address these issues, trials with artificial inoculation were conducted using one representative of each group, pyraclostrobin and myclobutanil on leaves of potted vines as well as on berries of field grown vines. It could be demonstrated that pyraclostrobin possesses excellent pre-infection as well as post-infection activities on both, leaves and berries. Post-bloom, pyraclostrobin was able to completely inhibit infections on berries up to three weeks after application. Full curative control of developing black rot infections was achieved for up to 85% of the total incubation period. Myclobutanil turned out to be of limited protective activity. If applied the day prior to inoculation, the efficacy level reached around 90% but decreased continuously with increasing temporal distance between application and inoculation. However, curative applications of myclobutanil in the first half of the incubation period completely prevented symptom development on leaves as well as on berries. Due to their combined reach-back and forward activity to control black rot, both, pyraclostrobin as well as myclobutanil can be recommended in practical viticulture, especially in the most susceptible period between flowering and berries beginning to touch. Pyraclostrobin offers an exceptional protective and long-lasting curative potential, whereas myclobutanil is most effective in post-infection use.

Simulating the susceptibility of clusters to grape black rot infections depending on their phenological development

In the years 2006–2008, clusters of Vitis vinifera (cv. Riesling) were inoculated with a conidia suspension of Guignardia bidwellii, the causal agent of grape black rot, at weekly intervals from one week before until eight weeks after bloom. Berries’ susceptibility turned out to be strongly linked to the speed of the phenological development in the respective season. In view of this, a degree day-based model simulating the relative susceptibility as a function of the phenological development was established. Infections on inflorescences were already possible prior to bloom. The period of highest susceptibility for direct infection on berries lasted from fruit set to the beginning of bunch closure. Successful infections took place until the end of bunch closure. Ontogenic resistance occurred clearly before veraison. Consequently, control measures should focus on the period between directly prior to bloom and bunch closure. Thus, the time span of black rot control coincides with the period in which vineyards need to be protected against the two major grape pathogens, Erysiphe necator and Plasmopara viticola. Applications of fungicides aimed at powdery and downy mildew control, also effective against G. bidwellii (e.g. strobilurins, triazoles and dithiocarbamates), offer a simultaneous control strategy for these three fungal diseases.

Field validation of dosaviña, a decision support system to determine the optimal volume rate for pesticide application in vineyards

Dose expression and volume rate are key questions for spray application in orchard and vineyard plantations. Crop characteristics seem to be one of the most important factors to determine the optimal value, but parameters as the leaf area, canopy volume, and canopy density are difficult to quantify. dosaviña, a decision support system, has been developed as a tool to calculate the optimal volume rate for spray applications in vineyards. The method is based on achieving the optimal coverage (impacts cm −2) according to the characteristics of the crop canopy, and includes the establishment of an efficiency factor that depends on aspects such as the type of sprayer used, nozzle type and size, operational parameters, and weather conditions. Field tests, in order to evaluate the interest in and functionality of the dosaviña computer program, were arranged for different wine grape varieties in New York State (USA) and Lleida (Spain), with a wide spectrum of wine varieties ( Baco, Riesling, Vignoles, Merlot, and Cabernet Sauvignon), sprayers, and working conditions. Deposition, uniformity, and control of different diseases ( powdery mildew, Botrytis bunch rot, and grape black rot) were evaluated at different crop stages, by comparing the volume rate applied according to dosaviña with the conventional rate most generally used by farmers. The volume rate was reduced by an average 39.9% for dosaviña, with similar or even higher values of deposition and uniformity. The resulting reduction in pesticide use (average 53%) did not present any difference in disease control for the selected varieties. Methodologies based on coverage and leaf area, as prescribed by dosaviña, seem to be an effective alternative approach to determine the most adequate volume rate according to the canopy structure, with the consequent diminution of pesticide use and the increase of environmentally friendly practices.