Exploring the biocontrol potential of rocket (Eruca sativa) extracts and associated microorganisms against Verticillium wilt.

AIMS: This study aimed to assess the impact of rocket (Eruca sativa) extract on Verticillium wilt in eggplants, explore rhizospheric microorganisms for disease biocontrol, and evaluate selected strains' induced systemic resistance (ISR) potential while characterizing their genomic and biosynthetic profiles. METHODS AND RESULTS: Rocket extract application led to a significant reduction in Verticillium wilt symptoms in eggplants compared to controls. Isolated microorganisms from treated soil, including Paraburkholderia oxyphila EP1, Pseudomonas citronellolis EP2, Paraburkholderia eburnea EP3, and P. oxyphila EP4 and EP5, displayed efficacy against Verticillium dahliae, decreasing disease severity and incidence in planta. Notably, strains EP3 and EP4 triggered ISR in eggplants against V. dahliae. Genomic analysis unveiled shared biosynthetic gene clusters, such as ranthipeptide and non-ribosomal peptide synthetase-metallophore types, among the isolated strains. Additionally, metabolomic profiling of EP2 revealed the production of metabolites associated with amino acid metabolism, putative antibiotics, and phytohormones. CONCLUSIONS: The application of rocket extract resulted in a significant reduction in Verticillium wilt symptoms in eggplants, while the isolated microorganisms displayed efficacy against V. dahliae, inducing systemic resistance and revealing shared biosynthetic gene clusters, with metabolomic profiling highlighting potential disease-suppressing metabolites.

Biocontrol strategies against Botrytis cinerea in viticulture: evaluating the efficacy and mode of action of selected winemaking yeast strains.

Botrytis cinerea poses a recurring threat to viticulture, causing significant yield losses each year. The study explored the biocontrol capabilities of commercially used winemaking yeasts as a strategy to manage B. cinerea in grape berries. The winemaking yeast strains-Saccharomyces cerevisiae ES181, Saccharomyces pastorianus KBG6, S. cerevisiae BCS103, Lachancea thermotolerans Omega, and Torulaspora delbrueckii TD291-reduced B. cinerea growth and conidiation in vitro. Furthermore, they demonstrated a decreased disease severity and number of conidia in grape berries. Among these strains, S. cerevisiae BCS103 was the most effective, inducing the expression of the defense-related gene PR4 in berries. Its diffusible compounds and volatile organic compounds also reduced the expression of BcLTF2, a positive regulator of B. cinerea conidiogenesis. The examined winemaking yeast strains, especially S. cerevisiae BCS103, demonstrated effective inhibition of B. cinerea in vitro and in grape berries, influencing key defense genes and reducing BcLTF2 expression, offering potential solutions for disease management in viticulture. The study underscores the promise of commercially available winemaking yeast strains as eco-friendly tools against B. cinerea in viticulture. Leveraging their safety and existing use in winemaking offers a potential avenue for sustainable disease management.

The bacterial biocontrol agent Paenibacillus alvei K165 confers inherited resistance to Verticillium dahliae.

The biocontrol agent Paenibacillus alvei K165 was previously shown to protect Arabidopsis thaliana plants against Verticillium dahliae. Here we show that K165 also confers inherited immune resistance to V. dahliae. By performing a histone acetyltransferases mutant screen, ChIP assays, and transcriptomic experiments, we were able to show that histone acetylation significantly contributes to the K165 biocontrol activity and establishment of inheritable resistance to V. dahliae. K165 treatment primed the expression of immune-related marker genes and the cinnamyl alcohol dehydrogenase gene CAD3 through the function of histone acetyltransferases. Our results reveal that offspring of plants treated with K165 have primed immunity and enhanced lignification, both contributing towards the K165-mediated inherited immune resistance. Thus, our study paves the way for the use of biocontrol agents for the establishment of inheritable resistance to agronomically important pathogens.

The Innate Immune Signaling System as a Regulator of Disease Resistance and Induced Systemic Resistance Activity Against Verticillium dahliae.

In the last decades, the plant innate immune responses against pathogens have been extensively studied, while biocontrol interactions between soilborne fungal pathogens and their hosts have received much less attention. Treatment of Arabidopsis thaliana with the nonpathogenic bacterium Paenibacillus alvei K165 was shown previously to protect against Verticillium dahliae by triggering induced systemic resistance (ISR). In the present study, we evaluated the involvement of the innate immune response in the K165-mediated protection of Arabidopsis against V. dahliae. Tests with Arabidopsis mutants impaired in several regulators of the early steps of the innate immune responses, including fls2, efr-1, bak1-4, mpk3, mpk6, wrky22, and wrky29 showed that FLS2 and WRKY22 have a central role in the K165-triggered ISR, while EFR1, MPK3, and MPK6 are possible susceptibility factors for V. dahliae and bak1 shows a tolerance phenomenon. The resistance induced by strain K165 is dependent on both salicylate and jasmonate-dependent defense pathways, as evidenced by an increased transient accumulation of PR1 and PDF1.2 transcripts in the aerial parts of infected plants treated with strain K165.

Emerging Roles of Epigenetics in Grapevine and Winegrowing.

Epigenetics refers to dynamic chemical modifications to the genome that can perpetuate gene activity without changes in the DNA sequence. Epigenetic mechanisms play important roles in growth and development. They may also drive plant adaptation to adverse environmental conditions by buffering environmental variation. Grapevine is an important perennial fruit crop cultivated worldwide, but mostly in temperate zones with hot and dry summers. The decrease in rainfall and the rise in temperature due to climate change, along with the expansion of pests and diseases, constitute serious threats to the sustainability of winegrowing. Ongoing research shows that epigenetic modifications are key regulators of important grapevine developmental processes, including berry growth and ripening. Variations in epigenetic modifications driven by genotype-environment interplay may also lead to novel phenotypes in response to environmental cues, a phenomenon called phenotypic plasticity. Here, we summarize the recent advances in the emerging field of grapevine epigenetics. We primarily highlight the impact of epigenetics to grapevine stress responses and acquisition of stress tolerance. We further discuss how epigenetics may affect winegrowing and also shape the quality of wine.

A Case of Plant Vaccination: Enhancement of Plant Immunity against Verticillium dahliae by Necrotized Spores of the Pathogen.

The soil-borne fungus Verticillium dahliae is causing a devastating vascular disease in more than 200 species of dicotyledonous plants. The pathogen attacks susceptible plants through the roots, colonizes the plant vascular system, and causes the death of aerial tissues. In this study, we used Arabidopsis and eggplants to examine the plant protective and immunization effects of autoclaved V. dahliae spores against V. dahliae. We observed that the application of V. dahliae autoclaved spores in eggplants and Arabidopsis resulted in enhanced protection against V. dahliae, since the disease severity and pathogen colonization were lower in the plants treated with V. dahliae autoclaved spores when compared to controls. In addition, upregulation of the defense related genes PR1 and PDF1.2 in the Arabidopsis plants treated with the V. dahliae autoclaved spores was revealed. Furthermore, pathogenicity experiments in the Arabidopsis mutant cerk1, defective in chitin perception, revealed a loss of protection against V. dahliae in the cerk1 treated with the V. dahliae autoclaved spores. The participation of the chitin receptor CERK1 is evident in Arabidopsis immunization against V. dahliae using autoclaved spores of the pathogen.

Combined Transcriptomic and Metabolomic Analysis Reveals Insights into Resistance of Arabidopsis bam3 Mutant against the Phytopathogenic Fungus Fusarium oxysporum.

The wilt-inducing strains of Fusarium oxysporum are responsible for severe damage to many economically important plant species. The most cost-effective and environmentally safe method for the management of Fusarium wilt is the use of resistant cultivars when they are available. In the present study, the Arabidopsis genotype with disruptions in the ß-amylase 3 (BAM3) gene, which encodes the major hydrolytic enzyme that degrades starch to maltose, had significantly lower susceptibility to Fusarium oxysporum f. sp. raphani (For) compared to wild-type (wt) plants. It showed the lowest disease severity and contained reduced quantities of fungal DNA in the plant vascular tissues when analyzed with real-time PCR. Through metabolomic analysis using gas chromatography (GC)-mass spectrometry (MS) and gene-expression analysis by reverse-transcription quantitative PCR (RT-qPCR), we observed that defense responses of Arabidopsis bam3 mutants are associated with starch-degradation enzymes, the corresponding modification of the carbohydrate balance, and alterations in sugar (glucose, sucrose, trehalose, and myo-inositol) and auxin metabolism.

Towards Biological Control of Aspergillus carbonarius and Botrytis cinerea in Grapevine Berries and Transcriptomic Changes of Genes Encoding Pathogenesis-Related (PR) Proteins.

Grapevine bunch rot, caused by Botrytis cinerea and Aspergillus carbonarius, causes important economic losses every year in grape production. In the present study, we examined the plant protective activity of the biological control agents, Paenibacillus alvei K165, Blastobotrys sp. FP12 and Arthrobacter sp. FP15 against B. cinerea and A. carbonarius on grapes. The in vitro experiments showed that strain K165 significantly reduced the growth of both fungi, while FP15 restricted the growth of A. carbonarius and FP12 was ineffective. Following the in vitro experiments, we conducted in planta experiments on grape berries. It was shown that K165, FP12 and FP15 reduced A. carbonarius rot severity by 81%, 57% and 37%, respectively, compared to the control, whereas, in the case of B. cinerea, the only protective treatment was that with K165, which reduced rot by 75%. The transcriptomic analysis of the genes encoding the pathogenesis-related proteins PR2, PR3, PR4 and PR5 indicates the activation of multiple defense responses involved in the biocontrol activity of the examined biocontrol agents.

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

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.

Phenolic Responses of Resistant and Susceptible Olive Cultivars Induced by Defoliating and Nondefoliating Verticillium dahliae Pathotypes.

Verticillium wilt is the most serious olive disease worldwide. The olive-infecting Verticillium dahliae pathotypes have been classified as defoliating (D) and nondefoliating (ND), and the disease is mainly controlled in olive orchards by using resistant or tolerant cultivars. Limited information is available about the nature of resistance in most of the olive cultivars. In the present study, the phenolic responses of the susceptible to V. dahliae olive cv. Amfissis and the resistant cv. Koroneiki upon D and ND V. dahliae infection were monitored in relation to the fungal DNA levels in the vascular tissues with the purpose to explore the defense mechanisms of olive trees against V. dahliae. Quantitative polymerase chain reaction revealed that the decrease in symptom severity shown in Koroneiki trees was associated with significant reduction in the growth of both V. dahliae pathotypes in the vascular tissues compared with Amfissis. In Koroneiki trees, the levels of o-diphenols and verbascoside were positively associated with the DNA levels of the D and ND pathotypes. In addition, a positive association was observed between the levels of verbascoside and the fungal DNA level in Amfissis trees, whereas a negative association was revealed between the fungal DNA level and the total phenols and oleuropein content in both cultivars. The levels of verbascoside were clearly higher in Koroneiki trees compared with Amfissis trees, indicating for the first time in the literature the involvement of verbascoside in the defense mechanism of olive trees against V. dahliae.

The Ethylene Biosynthesis Genes ACS2 and ACS6 Modulate Disease Severity of Verticillium dahliae.

Verticillium dahliae is one of the most destructive soilborne plant pathogens since it has a broad host range and there is no chemical disease management. Therefore, there is a need to unravel the molecular interaction between the pathogen and the host plant. For this purpose, we examined the role of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs) of Arabidopsis thaliana upon V. dahliae infection. We observed that the acs2, acs6, and acs2/6 plants are partially resistant to V. dahliae, since the disease severity of the acs mutants was lower than the wild type (wt) Col-0 plants. Quantitative polymerase chain reaction analysis revealed that acs2, acs6, and acs2/6 plants had lower endophytic levels of V. dahliae than the wt. Therefore, the observed reduction of the disease severity in the acs mutants is rather associated with resistance than tolerance. It was also shown that ACS2 and ACS6 were upregulated upon V. dahliae infection in the root and the above ground tissues of the wt plants. Furthermore, the addition of 1-aminocyclopropane-1-carboxylic acid (ACC) and aminooxyacetic acid (AOA), the competitive inhibitor of ACS, in wt A. thaliana, before or after V. dahliae inoculation, revealed that both substances decreased Verticillium wilt symptoms compared to controls irrespectively of the application time. Therefore, our results suggest that the mechanism underpinning the partial resistance of acs2 and acs6 seem to be ethylene depended rather than ACC related, since the application of ACC in the wt led to decreased disease severity compared to control.

An integrated approach to improve plant protection against olive anthracnose caused by the Colletotrichum acutatum species complex.

The olive tree (Olea europaea L.) is the most important oil-producing crop of the Mediterranean basin. However, although plant protection measures are regularly applied, disease outbreaks represent an obstacle towards the further development of the sector. Therefore, there is an urge for the improvement of plant protection strategies based on information acquired by the implementation of advanced methodologies. Recently, heavy fungal infections of olive fruits have been recorded in major olive-producing areas of Greece causing devastating yield losses. Thus, initially, we have undertaken the task to identify their causal agent(s) and assess their pathogenicity and sensitivity to fungicides. The disease was identified as the olive anthracnose, and although Colletotrichum gloeosporioides and Colletotrichum acutatum species complexes are the two major causes, the obtained results confirmed that in Southern Greece the latter is the main causal agent. The obtained isolates were grouped into eight morphotypes based on their phenotypes, which differ in their sensitivities to fungicides and pathogenicity. The triazoles difenoconazole and tebuconazole were more toxic than the strobilurins being tested. Furthermore, a GC/EI/MS metabolomics model was developed for the robust chemotaxonomy of the isolates and the dissection of differences between their endo-metabolomes, which could explain the obtained phenotypes. The corresponding metabolites-biomarkers for the discrimination between morphotypes were discovered, with the most important ones being the amino acids L-tyrosine, L-phenylalanine, and L-proline, the disaccharide α,α-trehalose, and the phytotoxic pathogenesis-related metabolite hydroxyphenylacetate. These metabolites play important roles in fungal metabolism, pathogenesis, and stress responses. The study adds critical information that could be further exploited to combat olive anthracnose through its monitoring and the design of improved, customized plant protection strategies. Also, results suggest the necessity for the comprehensive mapping of the C. acutatum species complex morphotypes in order to avoid issues such as the development of fungicide-resistant genotypes.

Induction of resistance to Verticillium dahliae in Arabidopsis thaliana by the biocontrol agent K-165 and pathogenesis-related proteins gene expression.

The biocontrol bacterium Paenibacillus alvei K165 has the ability to protect Arabidopsis thaliana against Verticillium dahliae. A direct antagonistic action of strain K165 against V. dahliae was ruled out, making it likely that K165-mediated protection results from induced systemic resistance (ISR) in the host. K165-mediated protection was tested in various Arabidopsis mutants and transgenic plants impaired in defense signaling pathways, including NahG (transgenic line degrading salicylic acid [SA]), etr1-1 (insensitive to ethylene), jar1-1 (insensitive to jasmonate), npr1-1 (nonexpressing NPR1 protein), pad3-1 (phytoalexin deficient), pad4-1 (phytoalexin deficient), eds5/sid1 (enhanced disease susceptibility), and sid2 (SA-induction deficient). ISR was blocked in Arabidopsis mutants npr1-1, eds5/sid1, and sid2, indicating that components of the pathway from isochorismate and a functional NPR1 play a crucial role in the K165-mediated ISR. Furthermore, the concomitant activation and increased transient accumulation of the PR-1, PR-2, and PR-5 genes were observed in the treatment in which both the inducing bacterial strain and the challenging pathogen were present in the rhizosphere of the A. thaliana plants.

Reactive oxygen species and plant resistance to fungal pathogens.

Reactive oxygen species (ROS) have been studied for their role in plant development as well as in plant immunity. ROS were consistently observed to accumulate in the plant after the perception of pathogens and microbes and over the years, ROS were postulated to be an integral part of the defence response of the plant. In this article we will focus on recent findings about ROS involved in the interaction of plants with pathogenic fungi. We will describe the ways to detect ROS, their modes of action and their importance in relation to resistance to fungal pathogens. In addition we include some results from works focussing on the fungal interactor and from studies investigating roots during pathogen attack.

Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae.

Vascular wilts caused by Verticillium spp. are very difficult to control and, as a result, are the cause of severe yield losses in a wide range of economically important crops. The responses of Arabidopsis thaliana mutant plants impaired in known pathogen response pathways were used to explore the components in defence against Verticillium dahliae. Analysis of the mutant responses revealed enhanced resistance in etr1-1[ethylene (ET) receptor mutant] plants, but not in salicylic acid-, jasmonic acid- or other ET-deficient mutants, indicating a crucial role of ETR1 in defence against this pathogen. Quantitative polymerase chain reaction analysis revealed that the decrease in symptom severity shown in etr1-1 plants was associated with significant reductions in the growth of the pathogen in the vascular tissues of the plants, suggesting that impaired perception of ET via ETR1 results in increased disease resistance. Furthermore, the activation and increased accumulation of the PR-1, PR-2, PR-5, GSTF12, GSTU16, CHI-1, CHI-2 and Myb75 genes, observed in etr1-1 plants after V. dahliae inoculation, indicate that the outcome of the induced defence response of etr1-1 plants seems to be dependent on a set of defence genes activated on pathogen attack.