Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum

Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.

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Application methods and modes of action of Pantoea agglomerans and Paenibacillus sp. to control the grapevine trunk disease-pathogen, Neofusicoccum parvum

OENO One ◽

10.20870/oeno-one.2021.55.2.4530 ◽

2021 ◽

Vol 55 (3) ◽

Author(s):

Rana Haidar ◽

Yacoub Amira ◽

Jean Roudet ◽

Fermaud Marc ◽

Rey Patrice

Keyword(s):

Soil Surface ◽

Pantoea Agglomerans ◽

In Planta ◽

Bacterial Strains ◽

Neofusicoccum Parvum ◽

Modes Of Action ◽

Grapevine Trunk Diseases ◽

Paenibacillus Sp ◽

Application Methods

Despite an increasing number of studies being carried out on the biocontrol of grapevine trunk diseases (GTDs), no commercial bacterial products have yet been developed to control GTDs. Knowledge of the precise modes of action (MOA) and the different application methods (AM) for biocontrol agents is crucial if they are to be successful in the field. In light of this, the present study aimed at selecting the most appropriate AM for eight bacterial strains with high potential for controlling Neofusicoccum parvum. These strains were applied on one-year-old grapevine in pots (grown from cuttings) using three methods: co-inoculation at stem level, preventive inoculation at stem level and preventive inoculation at the soil surface. The inhibitory activity of the bacterial strain against N. parvum was significantly dependent on the AM. Application of bacterial strains to stems, especially in a preventive way, was much more efficient than inoculation in the soil. When performing preventive inoculation on stems, the inhibition of N. parvumwood necrosis reached 50 and 65 % for Pantoea agglomerans (S1) and Paenibacillus sp. (S19) respectively. To decipher the underlying processes linked to fungal inhibition, the way in which several MOA affected the antagonistic capacity of these two strains was studied via in vitro and in planta assays. While P. agglomerans (S1) inhibited N. parvum by the secretion of antifungal volatile compounds, Paenibacillus sp. (S19) mainly inhibited this pathogen by antibiosis. In addition, both bacterial strains induced systemic defenses in grapevine. However, this affect tended to be higher at 15-dpi after inoculation with P. agglomerans (S1) than after innoculation with Paenibacillus sp. (S19) (three defense genes repressed versus five respectively). Finally, P. agglomerans (S1) and Paenibacillus sp. (S19) were shown to be potential biocontrol candidates for fighting N. parvum in grapevine, due to the combination of direct control via their antifungal activity and indirect control via their ability to activate the grapevine defense system.

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Characterization of an antifungal soil bacterium and its antagonistic activities againstFusariumspecies

Canadian Journal of Microbiology ◽

10.1139/w03-033 ◽

2003 ◽

Vol 49 (4) ◽

pp. 253-262 ◽

Cited By ~ 31

Author(s):

Yiu-Kwok Chan ◽

Wayne A McCormick ◽

Keith A Seifert

Keyword(s):

Bacillus Subtilis ◽

Culture Filtrate ◽

Fusarium Species ◽

Hyphal Growth ◽

Antagonistic Activity ◽

Plant Diseases ◽

Ear Rot ◽

In Planta ◽

Head Blight

Bacteria were isolated from a cultivated soil and screened for antagonistic activity against Fusarium graminearum, a predominant agent of ear rot and head blight in cereal crops. Based on its in vitro effectiveness, isolate D1/2 was selected for characterization and identified as a strain of Bacillus subtilis by phenotypic tests and comparative analysis of its 16S ribosomal RNA gene (rDNA) sequence. It inhibited the mycelial growth of a collection of common fungal phytopathogens, including eight Fusarium species, three other ascomycetes, and one basidiomycete. The cell-free culture filtrate of D1/2 at different dilutions was active against macroconidium germination and hyphal growth of F. graminearum, depending on the initial macroconidium density. It induced the formation of swollen hyphal cells in liquid cultures of this fungus grown from macroconidia. A bioassay also demonstrated that D1/2 offered in planta protection against the damping-off disease in alfalfa seedlings caused by F. graminearum, while the type strain of B. subtilis was ineffective. Hence, B. subtilis D1/2 or its culture filtrate has potential application in controlling plant diseases caused by Fusarium.Key words: antifungal activity, Bacillus subtilis, biological control, biopesticide, Fusarium species.

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Antifungal Agents Based on Chitosan Oligomers, ε-polylysine and Streptomyces spp. Secondary Metabolites against Three Botryosphaeriaceae Species

Antibiotics ◽

10.3390/antibiotics8030099 ◽

2019 ◽

Vol 8 (3) ◽

pp. 99 ◽

Cited By ~ 2

Author(s):

Laura Buzón-Durán ◽

Jesús Martín-Gil ◽

Eduardo Pérez-Lebeña ◽

David Ruano-Rosa ◽

José L. Revuelta ◽

...

Keyword(s):

Cost Effective ◽

Botryosphaeria Dothidea ◽

Diplodia Seriata ◽

Neofusicoccum Parvum ◽

Grapevine Trunk Diseases ◽

Streptomyces Spp ◽

Effective Option ◽

Trunk Diseases ◽

Botryosphaeriaceae Species

Grapevine trunk diseases (GTDs) are a major threat to the wine and grape industry. The aim of the study was to investigate the antifungal activity against Neofusicoccum parvum, Diplodia seriata, and Botryosphaeria dothidea of ε-polylysine, chitosan oligomers, their conjugates, Streptomyces rochei and S. lavendofoliae culture filtrates, and their binary mixtures with chitosan oligomers. In vitro mycelial growth inhibition tests suggest that the efficacy of these treatments, in particular those based on ε-polylysine and ε-polylysine:chitosan oligomers 1:1 w/w conjugate, against the three Botryosphaeriaceae species would be comparable to or higher than that of conventional synthetic fungicides. In the case of ε-polylysine, EC90 values as low as 227, 26.9, and 22.5 µg·mL−1 were obtained for N. parvum, D. seriata, and B. dothidea, respectively. Although the efficacy of the conjugate was slightly lower, with EC90 values of 507.5, 580.2, and 497.4 µg·mL−1, respectively, it may represent a more cost-effective option to the utilization of pure ε-polylysine. The proposed treatments may offer a viable and sustainable alternative for controlling GTDs.

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Wood Metabolomic Responses of Wild and Cultivated Grapevine to Infection with Neofusicoccum parvum, a Trunk Disease Pathogen

Metabolites ◽

10.3390/metabo10060232 ◽

2020 ◽

Vol 10 (6) ◽

pp. 232 ◽

Cited By ~ 2

Author(s):

Clément Labois ◽

Kim Wilhelm ◽

Hélène Laloue ◽

Céline Tarnus ◽

Christophe Bertsch ◽

...

Keyword(s):

Vitis Vinifera ◽

Strong Increase ◽

Primary Metabolism ◽

Alcohol Content ◽

Neofusicoccum Parvum ◽

Specialized Metabolites ◽

Grapevine Trunk Diseases ◽

Botryosphaeria Dieback ◽

Trunk Diseases ◽

Resveratrol Oligomer

Grapevine trunk diseases (GTDs), which are associated with complex of xylem-inhabiting fungi, represent one of the major threats to vineyard sustainability currently. Botryosphaeria dieback, one of the major GTDs, is associated with wood colonization by Botryosphaeriaceae fungi, especially Neofusicoccum parvum. We used GC-MS and HPLC-MS to compare the wood metabolomic responses of the susceptible Vitis vinifera subsp. vinifera (V. v. subsp. vinifera) and the tolerant Vitis vinifera subsp. sylvestris (V. v. subsp. sylvestris) after artificial inoculation with Neofusicoccum parvum (N. parvum). N. parvum inoculation triggered major changes in both primary and specialized metabolites in the wood. In both subspecies, infection resulted in a strong decrease in sugars (fructose, glucose, sucrose), whereas sugar alcohol content (mannitol and arabitol) was enhanced. Concerning amino acids, N. parvum early infection triggered a decrease in aspartic acid, serine, and asparagine, and a strong increase in alanine and β-alanine. A trend for more intense primary metabolism alteration was observed in V. v. subsp. sylvestris compared to V. v. subsp. vinifera. N. parvum infection also triggered major changes in stilbene and flavonoid compounds. The content in resveratrol and several resveratrol oligomers increased in the wood of both subspecies after infection. Interestingly, we found a higher induction of resveratrol oligomer (putative E-miyabenol C, vitisin C, hopeaphenol, ampelopsin C) contents after wood inoculation in V. v. subsp. sylvestris.

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Activity of Anthracenediones and Flavoring Phenols in Hydromethanolic Extracts of Rubia tinctorum against Grapevine Phytopathogenic Fungi

Plants ◽

10.3390/plants10081527 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1527

Author(s):

Natalia Langa-Lomba ◽

Eva Sánchez-Hernández ◽

Laura Buzón-Durán ◽

Vicente González-García ◽

José Casanova-Gascón ◽

...

Keyword(s):

Fungal Species ◽

Phytopathogenic Fungi ◽

Ex Situ ◽

Antifungal Compound ◽

Promising Alternative ◽

Grapevine Trunk Diseases ◽

Rubia Tinctorum ◽

Botryosphaeria Dieback ◽

Trunk Diseases

In this work, the chemical composition of Rubia tinctorum root hydromethanolic extract was analyzed by GC–MS, and over 50 constituents were identified. The main phytochemicals were alizarin-related anthraquinones and flavoring phenol compounds. The antifungal activity of this extract, alone and in combination with chitosan oligomers (COS) or with stevioside, was evaluated against the pathogenic taxa Diplodia seriata, Dothiorella viticola and Neofusicoccum parvum, responsible for the so-called Botryosphaeria dieback of grapevine. In vitro mycelial growth inhibition tests showed remarkable activity for the pure extract, with EC50 and EC90 values as low as 66 and 88 μg·mL−1, respectively. Nonetheless, enhanced activity was attained upon the formation of conjugate complexes with COS or with stevioside, with synergy factors of up to 5.4 and 3.3, respectively, resulting in EC50 and EC90 values as low as 22 and 56 μg·mL−1, respectively. The conjugate with the best performance (COS–R. tinctorum extract) was then assayed ex situ on autoclaved grapevine wood against D. seriata, confirming its antifungal behavior on this plant material. Finally, the same conjugate was evaluated in greenhouse assays on grafted grapevine plants artificially inoculated with the three aforementioned fungal species, resulting in a significant reduction in the infection rate in all cases. This natural antifungal compound represents a promising alternative for developing sustainable control methods against grapevine trunk diseases.

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Combining an HA + Cu (II) Site-Targeted Copper-Based Product with a Pruning Wound Protection Program to Prevent Infection with Lasiodiplodia spp. in Grapevine

Plants ◽

10.3390/plants10112376 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2376

Author(s):

Pedro Reis ◽

Ana Gaspar ◽

Artur Alves ◽

Florence Fontaine ◽

Cecília Rego

Keyword(s):

Grapevine Trunk Diseases ◽

Pruning Wound ◽

Wound Protection ◽

Protection Strategies ◽

Botryosphaeria Dieback ◽

Trunk Diseases ◽

The Impact ◽

Fungistatic Effect

The genus Lasiodiplodia has been reported from several grape growing regions and is considered as one of the fastest wood colonizers, causing Botryosphaeria dieback. The aim of this study was to (i) evaluate the efficacy of Esquive®, a biocontrol agent, on vineyard pruning wound protection, applied single or, in a combined protection strategy with a new site-targeted copper-based treatment (LC2017), and (ii) compare their efficacy with chemical protection provided by the commercially available product, Tessior®. For two seasons, protectants were applied onto pruning wounds, while LC2017 was applied throughout the season according to the manufacturer’s instructions. Pruning wounds of two different cultivars were inoculated with three isolates of Lasiodiplodia spp. Efficacy of the wound protectants, varied between both years of the assay and according to the cultivar studied but were able to control the pathogen to some extent. The application of LC2017 did not show clear evidence of improving the control obtained by the sole application of the other products tested. Nevertheless, LC2017 showed a fungistatic effect against Lasiodiplodia spp., in vitro, and has previously shown an elicitor effect against grapevine trunk diseases. Therefore, this combination of two protection strategies may constitute a promising long-term approach to mitigate the impact of Botryosphaeria dieback.

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Endophytic colonisation of perennial ryegrass by Trichoderma atroviride

New Zealand Plant Protection ◽

10.30843/nzpp.2015.68.5850 ◽

2015 ◽

Vol 68 ◽

pp. 444-444

Author(s):

A. Daryaei ◽

R.E. Falloon ◽

E.E. Jones ◽

D.R.W. Kandula ◽

H. Ghazalibiglar ◽

...

Keyword(s):

Plant Growth ◽

Perennial Ryegrass ◽

Field Experiments ◽

Trichoderma Atroviride ◽

Growth Promoters ◽

Trichoderma Spp ◽

Beneficial Effects ◽

Reproductive Structures ◽

Plant Growth Promoters

Trichoderma spp are widely used as biocontrol agents and plant growth promoters Endophytic colonisation of perennial ryegrass (Lolium perenne) by T atroviride strains was studied in laboratory greenhouse and field experiments Four strains of T atroviride were inoculated into in vitro agar cultures or potting mix with ryegrass seeds for colonisation studies The strains were also produced as prill or granule formulations for application in the field experiment Microscopy was used to observe fungal structures in plant tissue segments from pot or fieldgrown plants Segments were also surface sterilised and placed onto Trichoderma selective media Fungal colonies recovered were verified as T atroviride by sequencing the tef1 gene No endophytic colonisation occurred in any noninoculated plants and no Trichoderma endophytic colonisation was observed in fieldgrown plants However microscopy revealed fungal hyphae and reproductive structures characteristic of Trichoderma in root and stem sheath tissues of inoculated plants from in vitro cultures and pots These results were verified by sequencing the tef1 gene This study has demonstrated endophytic colonisation of ryegrass by T atroviride strains which may be related to beneficial effects on plant growth and disease control

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Avaliação da compatibilidade in vitro de microrganismos (Bacillus subtilis, Pseudomonas fluorescens e Trichoderma atroviride) com fungicidas comerciais, para a produção sustentável de amendoim

South American Sciences ISSN 2675-7222 ◽

10.52755/sas.v2iedesp2.112 ◽

2021 ◽

Vol 2 (edesp2) ◽

pp. e21112

Author(s):

Dean Fernandez ◽

Matias Torassa ◽

Maria Alejandra Pérez

Keyword(s):

Bacillus Subtilis ◽

Pseudomonas Fluorescens ◽

Trichoderma Atroviride

A aplicação de fungicidas na semente de amendoim é uma necessidade na semeadura, porém a aplicação combinada com produtos biológicos pode reduzir os efeitos do uso de agroquímicos, favorecendo a qualidade do grão como alimento. O objetivo deste trabalho foi avaliar a compatibilidade in vitro de microrganismos (Bacillus subtilis, Pseudomonas fluorescens e Trichoderma atroviride) com fungicidas comerciais para a produção sustentável de amendoim. Os testes de compatibilidade foram realizados no Laboratório do Centro de Transferência de Qualidade Agroalimentar (FCA UNC). Foram avaliados três combinações de fungicidas comerciais disponíveis no setor de amendoim (F1, F2, F3), em diferentes doses (0, 30, 50, 100 e 1000 mg.l-1). Em placas de petri com meio APG com cada fungicida, foram inseridos B. subtilis, P. fluorescens e T. atroviride, que foram mantidos em câmaras com condições controladas. Sete dias após a semeadura, o crescimento bacteriano foi avaliado de acordo com a escala de compatibilidade e para Trichoderma foi calculada a porcentagem de inibição do crescimento radial (PICR). O delineamento foi completamente aleatório; para cada biológico, foram avaliadas três repetições para cada fungicida e dose. Todo o teste foi repetido três vezes. Os dados foram submetidos à análise de variância e os valores médios do PICR foram comparados por Tukey (p<0,05). Os resultados mostraram um efeito diferencial dos fungicidas sobre os biológicos. F1 (Tiabendazol + Fludioxonil + Metalaxil-M + Azoxistrobina) pode ser combinado com P. fluorescens em todas as doses e B. subtilis em doses não superiores a 50 mg.l-1. F2 (Ipconazol + Metalaxil) e F3 (Ipconazol + Metalaxil M + Carboxina) podem ser aplicados com P. fluorescens mesmo em altas concentrações. Apenas F3 pode ser combinado com T. atroviride até 100 mg.l-1. A combinação correta de fungicidas e produtos biológicos contribuirá efetivamente para a sustentabilidade do sistema de produção de amendoim.

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Control of the fungi Lasiodiplodia theobromae, the causal agent of dieback, in cv. syrah grapevines

Acta Scientiarum Agronomy ◽

10.4025/actasciagron.v43i1.44785 ◽

2020 ◽

Vol 43 ◽

pp. e44785

Author(s):

Carine Rusin ◽

Fabio Rossi Cavalcanti ◽

Patrícia Carla Giloni de Lima ◽

Cacilda Márcia Duarte Rios Faria ◽

Marcus André Kurtz Almança ◽

...

Keyword(s):

Bacillus Subtilis ◽

Plant Extracts ◽

Trichoderma Harzianum ◽

Garlic Extract ◽

Limiting Factors ◽

Lasiodiplodia Theobromae ◽

Grapevine Trunk Diseases ◽

Wound Protection ◽

Trunk Diseases

Grapevine trunk diseases are among the most important limiting factors of worldwide viticulture. In this context, we aimed to verify the effect of chemical fungicides, biological agents and plant extracts on the control of Lasiodiplodia theobromae in pruning wounds and their physiological effects in cv. Syrah grapevines. Plant extracts (clove, cinnamon, garlic, rosemary and lemongrass), commercial fungicides (difeconazole, tebuconazole, mancozeb, sulfur, pyraclostrobin, fosetyl-Al, and azoxystrobin), chitosan, Trichoderma harzianum and Bacillus subtilis were used for the in vitro trials. The protection of pruning wounds in the potted vines was studied using fosetyl-Al, tebuconazole, Trichoderma harzianum, Bacillus subtilis, garlic extract and clove extract. The experiments were carried out through two vegetative cycles: 2015/2016 and 2016/2017. The length of wood discoloration, pathogen re-isolation percentage, fresh mass of the pruning material, peroxidase activity, fluorescence, chlorophyll index, phenological stages, shoot length and leaf area were evaluated. The treatments with clove, garlic extract, tebuconazole, pyraclostrobin, mancozeb, fosetyl-Al and B. subtilis reduced mycelial growth by more than 90%. In the grapevines, the use of T. harzianum decreased the re-isolation of L. theobromae, but no differences were verified for the other plant evaluations. We concluded that the use of T. harzianum would be a potential option for wound protection without altering the physiological aspects of cv. Syrah grapevines.

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Arabidopsis thaliana Seedlings Influence Bacillus subtilis Spore Formation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-18-0278-r ◽

2019 ◽

Vol 32 (9) ◽

pp. 1188-1195 ◽

Cited By ~ 4

Author(s):

Vincent Charron-Lamoureux ◽

Pascale B. Beauregard

Keyword(s):

Arabidopsis Thaliana ◽

Bacillus Subtilis ◽

Systemic Resistance ◽

Soil System ◽

Beneficial Effects ◽

Bacillus Subtilis Spore ◽

Bacillus Spp ◽

Plant Growth Promoting ◽

Growth Promoting

Bacillus subtilis is a Gram-positive plant-growth-promoting rhizobacterium exerting many beneficial effects on plant health. Because they secrete antimicrobial compounds and elicit induced systemic resistance, B. subtilis and phylogenetically related species are of particular interest as antifungals in organic agriculture. These bacteria are also known for their capacity to differentiate phenotypically into endospores able to withstand many environmental stresses. However, although B. subtilis is often inoculated on plants as spores, dynamics of germination and sporulation on roots remain unexplored. Using a hydroponic culture system and a soil system for Arabidopsis thaliana, we observed that B. subtilis spores germinate rapidly on contact with plants. However, the vegetative cells are abundant on roots for only a few days before reversing back to spores. We observed that the germinant receptor GerK and sporulation kinases KinA and KinB identified in vitro control sporulation dynamics on plants. Surprisingly, when plants are inoculated with B. subtilis, free-living cells sporulate more rapidly than plant-associated cells. However, direct contact between plant and bacteria is required for the induction of sporulation in the surrounding B. subtilis. This study has fundamental implications for our understanding of interactions between Bacillus spp. and plants, and particularly for a more efficient usage of B. subtilis as a biofertilizer or biofungicide.

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