scholarly journals Profiling grapevine trunk pathogens in planta: A case for community-targeted DNA metabarcoding

2018 ◽  
Author(s):  
Abraham Morales-Cruz ◽  
Rosa Figueroa-Balderas ◽  
Jadran F. García ◽  
Eric Tran ◽  
Philippe E. Rolshausen ◽  
...  
Keyword(s):  
Microbial Ecology ◽  
Time Course ◽  
Universal Primers ◽  
In Planta ◽  
Universal Primer ◽  
Grapevine Trunk Diseases ◽  
Field Samples ◽  
Dna Metabarcoding ◽  
Trunk Diseases ◽  
Mock Communities

ABSTRACTDNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the Fungal Kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over- or under-estimated with universal primers. Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

The compositional turnover of grapevine-associated plant pathogenic fungal communities are greater among intraindividual microhabitats and terroirs than among healthy and Esca-diseased plants

2021 ◽  
Author(s):  
Adrienn Geiger ◽  
Zoltán Karácsony ◽  
Richárd Golen ◽  
Kálmán Zoltán Váczy ◽  
József Geml
Keyword(s):  
Pathogenic Fungi ◽  
Wine Industry ◽  
Fungal Communities ◽  
Opportunistic Pathogens ◽  
Grapevine Trunk Diseases ◽  
Esca Disease ◽  
Dna Metabarcoding ◽  
Trunk Diseases ◽  
Fungal Dna ◽  
Key Questions

Grapevine trunk diseases (GTD) are a major threat to the wine industry, causing yield loss and dieback of grapevines. While the increasing damage caused by GTDs in recent decades have spurred several studies on grapevine-associated pathogenic fungi, key questions about the emergence and severity of GTDs remain unanswered, including possible differences in plant pathogenic fungal communities in asymptomatic and symptomatic grapevines. We generated fungal DNA metabarcoding data from soil, bark, and perennial wood samples from asymptomatic and symptomatic grapevines sampled in three terroirs. We observed larger compositional differences in plant pathogenic fungi among different plants parts within grapevine plants than among individual grapevines. This is driven by the dominance of GTD-associated fungi in perennial wood and non-GTD pathogens in soil, as well as by the lack of significant differences among asymptomatic and Esca symptomatic grapevines. These results suggest that fungi generally associated with Esca disease belong to the core grapevine microbiome and likely are commensal endophytes and/or latent saprotrophs, some of which can act as opportunistic pathogens on stressed plants. In addition, we found significant compositional differences among sampling sites, particularly in soil, which suggest a certain influence of local edaphic and mesclimatic factors on plant pathogenic fungal communities. Furthermore, the observed differences among terroirs in plant pathogenic fungal communities in grapevine woody parts indicate that environmental factors likely are important for the development of Esca disease and further studies are needed to investigate the abiotic conditions on fungal compositional dynamics in Esca-affected plants.

scholarly journals Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk-disease complex

2017 ◽  
Author(s):  
Abraham Morales-Cruz ◽  
Gabrielle Allenbeck ◽  
Rosa Figueroa-Balderas ◽  
Vanessa E. Ashworth ◽  
Daniel P. Lawrence ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transcriptional Profiling ◽  
Underlying Disease ◽  
In Planta ◽  
Dna And Rna ◽  
Trunk Disease ◽  
Specificity And Sensitivity ◽  
Genome Wide ◽  
Field Samples ◽  
Trunk Diseases

SummaryGrapevines, like other perennial crops, are affected by so-called ‘trunk diseases’, which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e., metatranscriptomics) approach that can monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for mapping and quantifying DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally-infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.

scholarly journals Biocontrol potential of grapevine endophytes against grapevine trunk pathogens

2020 ◽  
Author(s):  
Isidora Silva-Valderrama ◽  
Diana Toapanta ◽  
Maria de los Angeles Miccono ◽  
Mauricio Lolas ◽  
Gonzalo A. Díaz ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Dual Culture ◽  
In Planta ◽  
Clonostachys Rosea ◽  
Phaeomoniella Chlamydospora ◽  
Grapevine Trunk Diseases ◽  
Trunk Diseases ◽  
Media Types ◽  
Wood Extract ◽  
Biocontrol Potential

AbstractGrapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several species of fungi, including Neofusicoccum parvum, Diplodia seriata, and Phaeomoniella chlamydospora. In this study, we report the isolation of 169 endophytic and 209 epiphytic fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Clonostachys rosea, Trichoderma sp., Purpureocillium lilacium, Epiccocum nigrum, Cladosporium sp., and Chaetomium sp. were evaluated for their potential of biocontrol activity against fungal trunk pathogens. Tests were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. C. rosea showed 98.2% inhibition of all pathogens in presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp.. Overall these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

scholarly journals Biocontrol Potential of Grapevine Endophytic and Rhizospheric Fungi Against Trunk Pathogens

2021 ◽  
Vol 11 ◽  
Author(s):  
Isidora Silva-Valderrama ◽  
Diana Toapanta ◽  
Maria de los Angeles Miccono ◽  
Mauricio Lolas ◽  
Gonzalo A. Díaz ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Dual Culture ◽  
In Planta ◽  
Clonostachys Rosea ◽  
Phaeomoniella Chlamydospora ◽  
Purpureocillium Lilacinum ◽  
Grapevine Trunk Diseases ◽  
Trunk Diseases ◽  
Wood Extract ◽  
Biocontrol Potential

Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

scholarly journals In planta Activity of the Novel Copper Product HA + Cu(II) Based on a Biocompatible Drug Delivery System on Vine Physiology and Trials for the Control of Botryosphaeria Dieback

2021 ◽  
Vol 12 ◽  
Author(s):  
Vincenzo Mondello ◽  
Olivier Fernandez ◽  
Jean-François Guise ◽  
Patricia Trotel-Aziz ◽  
Florence Fontaine
Keyword(s):  
Drug Delivery ◽  
Plant Defense ◽  
Plant Protection ◽  
Plant Protection Products ◽  
Plasmopara Viticola ◽  
In Planta ◽  
Grapevine Trunk Diseases ◽  
Botryosphaeria Dieback ◽  
Trunk Diseases ◽  
Disease Impact

The growing concerns on human and environment health are forcing the plant protection industry toward the formulation of more eco-sustainable plant protection products (PPP), both efficient and innovative in their approach to disease control. A large number of these innovative formulations now rely on a combination of pathogens antagonistic properties and stimulation of natural plant defense to pathogens. The formulation HA + Cu(II), in which copper is delivered to the plants by the drug-delivery molecule hydroxyapatite (HA), was found efficient against the grapevine pathogens Plasmopara viticola and Phaeoacremonium minimum and able to induce the host-plant defense system. We investigated the HA + Cu(II) impacts on grapevine physiology, both in uninfected and when infected by the Botryosphaeria dieback agents Diplodia seriata and Neofusicoccum parvum. This study of plant physiology and disease impact were addressed to evaluate both the HA + Cu(II) potential as a plant defense elicitor and its possible and future use as PPP in vineyard. Our results showed that HA + Cu(II) induced several key-defense genes without negatively affecting plant growth and photosynthetic activity. In addition, fungistatic effect on the two Botryosphaeriaceae at the in planta tested concentrations is reported. Altogether, our results obtained under controlled conditions fully support the potential of HA + Cu(II) as a promising PPP toward grapevine trunk diseases in vineyard.

scholarly journals Paenibacillus alvei K165 and Fusarium oxysporum F2: Potential Biocontrol Agents against Phaeomoniella chlamydospora in Grapevines

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 207
Author(s):  
Fedon-Ioannis Gkikas ◽  
Alexandros Tako ◽  
Danai Gkizi ◽  
Christina Lagogianni ◽  
Emmanouil A. Markakis ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Biocontrol Agents ◽  
In Planta ◽  
Dna Amount ◽  
Phaeomoniella Chlamydospora ◽  
Grapevine Trunk Diseases ◽  
Paenibacillus Alvei ◽  
Trunk Diseases ◽  
Associated Species

In the last two decades grapevine trunk diseases (GTDs) have emerged as the most significant threat for grapevine sustainability worldwide. The tracheomycotic fungus Phaeomoniella chlamydospora (Pch) is the predominant GTD-associated species and cannot be controlled with available chemicals. In the present study, we evaluated the effectiveness of two microbial strains (Paenibacillus alvei K165 and Fusarium oxysporum F2) against Pch in grapevine. In vitro bioassays, performed in a growth culture medium simulating the xylem environment, indicated that F2 decreased Pch growth and sporulation, whereas K165 did not have any effect on Pch growth. In planta experiments revealed that root-drench and stem-puncture application of K165 and F2 reduced the endophytic relative DNA amount of Pch by 90% and 82%, respectively, compared to controls. However, wood discoloration, the typical symptom of Pch infection, was not reduced in the F2 treated grapevines. Nevertheless, the F2 treated grapevines harbored higher lignin levels compared to mocks, as it was also done by K165. Therefore, F2 and K165 have the potential to be used as biocontrol agents against Pch in grapevines.

scholarly journals Production of Phytotoxic Metabolites by Botryosphaeriaceae in Naturally Infected and Artificially Inoculated Grapevines

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 802
Author(s):  
Pierluigi Reveglia ◽  
Regina Billones-Baaijens ◽  
Jennifer Millera Millera Niem ◽  
Marco Masi ◽  
Alessio Cimmino ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Protocatechuic Acid ◽  
Physiological Factors ◽  
Diplodia Seriata ◽  
Grapevine Trunk Diseases ◽  
Botryosphaeria Dieback ◽  
Trunk Diseases ◽  
One Year ◽  
Pathogen Dna

Grapevine trunk diseases (GTDs) are considered a serious problem to viticulture worldwide. Several GTD fungal pathogens produce phytotoxic metabolites (PMs) that were hypothesized to migrate to the foliage where they cause distinct symptoms. The role of PMs in the expression of Botryosphaeria dieback (BD) symptoms in naturally infected and artificially inoculated wood using molecular and analytical chemistry techniques was investigated. Wood samples from field vines naturally infected with BD and one-year-old vines inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera were analysed by cultural isolations, quantitative PCR (qPCR) and targeted LC-MS/MS to detect three PMs: (R)-mellein, protocatechuic acid and spencertoxin. (R)-mellein was detected in symptomatic naturally infected wood and vines artificially inoculated with D. seriata but was absent in all non-symptomatic wood. The amount of (R)-mellein detected was correlated with the amount of pathogen DNA detected by qPCR. Protocatechuic acid and spencertoxin were absent in all inoculated wood samples. (R)-mellein may be produced by the pathogen during infection to break down the wood, however it was not translocated into other parts of the vine. The foliar symptoms previously reported in vineyards may be due to a combination of PMs produced and climatic and physiological factors that require further investigation.

scholarly journals Assessment of Conjugate Complexes of Chitosan and Urtica dioica or Equisetum arvense Extracts for the Control of Grapevine Trunk Pathogens

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 976
Author(s):  
Natalia Langa-Lomba ◽  
Laura Buzón-Durán ◽  
Pablo Martín-Ramos ◽  
José Casanova-Gascón ◽  
Jesús Martín-Gil ◽  
...  
Keyword(s):  
Urtica Dioica ◽  
Gas Chromatography Mass Spectrometry ◽  
Equisetum Arvense ◽  
Grapevine Trunk Diseases ◽  
Trunk Diseases ◽  
Synergistic Behavior ◽  
Reported Data ◽  
Basic Substances

In the work presented herein, we analyze the efficacy of three basic substances that comply with European Regulation (EC) No 1107/2009, namely chitosan, horsetail (Equisetum arvense L.) and nettle (Urtica dioica L.), for the control of grapevine trunk diseases (GTDs) in organic farming. The E. arvense and U. dioica aqueous extracts, prepared according to SANCO/12386/2013 and SANTE/11809/2016, have been studied by gas chromatography–mass spectrometry (GC-MS), identifying their main active constituents. The three basic substances, either alone or in combination (forming conjugate complexes), have been tested in vitro against eight Botryosphaeriaceae species, and in vivo, in grafted plants artificially inoculated with Neofusicoccum parvum and Diplodia seriata. A clear synergistic behavior between chitosan and the two plant extracts has been observed in the mycelial growth inhibition tests (resulting in EC90 values as low as 208 μg·mL−1 for some of the isolates), and statistically significant differences have been found in terms of vascular necroses lengths between treated and non-treated plants, providing further evidence of aforementioned synergism in the case of D. seriata. The reported data supports the possibility of extending the applications of these three basic substances in Viticulture beyond the treatment of mildew.

scholarly journals Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1042-1048 ◽  
Author(s):  
C. L. Trout ◽  
J. B. Ristaino ◽  
M. Madritch ◽  
T. Wangsomboondee
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Direct Pcr ◽  
Field Samples ◽  
Pcr Products ◽  
Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

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