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

The bacterial biocontrol agent Paenibacillus alvei K165 confers inherited resistance against 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 against 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 against V. dahliae. K165 treatment primed the expression of immune-related marker genes and the cinnamyl alcohol dehydrogenases 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 that way for the use of biocontrol agents for the establishment of inheritable resistance against agronomically important pathogens

The role of epigenetic inheritance in the biological control of Vetricillium dahliae

Epigenetic modification caused by biotic or abiotic stress can severely affect the expression of genes related to plant’s defense responses. Epigenetic inheritance is the transfer of those modifications to the next generation via the germline. The aim of this study is to unravel the mechanism of the epigenetically inherited resistance against Vericillium dahliae that the biocontrol agent Paenibacillus alvei K165 confers to the offspring of Arabidopsis thaliana treated plants. The phenomenon was confirmed with endophytic quantification using Real time PCR. Metabolomic and transcriptomic analyses were performed in the above ground part of the plants treated with K165 and their offspring to study their response against the fungus Verticillium dahliae. The results of the transcriptomic analysis revealed a significant number of genes in the phenylpronanoid biosynthesis pathway that are affected by K165 in the plants treated with it as well as in their offspring. Also it seems that both the salicylic acid and the jasmonic acid/ethylene pathways are upregulated by K165. According to the results of the metabolomic analysis there is significant differentiation of the metabolic profiles of plants from all the treatments. The lipid metabolism also seems to be up-regulated in plants treated with K165 and their offspring.To confirm the transcriptomic analysis results the expression levels of a set of genes that are affected by K165 and in the resistant offspring were studied using Real time PCR. The selected genes have a basic role in plant defense, participating in the salicylic acid pathway, the jasmonic acid pathway and the phenylpropanoid biosynthesis pathway. It was revealed that although at 3 days post inoculation with Verticillium, salicylic acid pathway is up-regulated, later at 7 days it is the jasmonic acid/ethylene pathway that takes over since the expression of PDF1.2 is significantly elevated in the Κ165-treated plants. On the other hand in the resistant offspring upon infection with the fungus both pathways are induced at the same time. As far as the phenylpropanoid biosynthesis pathway is concerned it seems that an important number of genes is affected both in K165 treated plants and in their offspring but they are also affected by Verticillium. However the expression of CAD8 is significantly increased in the resistant offspring. Lignin concentration was measured using a UV spectrophotometer since it is the final product of phenylpropanoid biosynthesis pathway in plants. Indeed it was found significantly higher in K165 treated plants and in their offspring. Pathogenicity experiments were performed using Arabidopsis thaliana mutants to study the role of CAD1, CAD6 and CAD 8 genes in the observed phenomenon since they seem to be significantly affected in K165 treated plants and their offspring. The results confirmed the role of these genes in the inherited resistance caused by K165 since the bacterium failed to protect the mutants and their offspring. Immunoprecipitation experiments using specific antibodies against H3 acetylation and western blot analysis were performed in order to confirm the epigenetic nature of the described phenomenon by detecting inheritable epigenetic modifications caused by K165. The whole genome acetylation levels in the resistant offspring were found significantly increased. The acetylation levels of PR1, PR5, PDF1.2, CAD4 and CAD8 genes in the promoter and open reading frame areas were studied using Real time PCR. The results showed increased acetylation levels in the promoters of PR1, PDF1.2, CAD4 and CAD8 in the resistant offspring. Finaly the role of HAC and HAG acetyltransferase genes in the observed epigenetically inherited resistance was studied with pathogenicity experiments in Arabidopsis mutants. Once again the bacterium failed to protect the mutants and their offspring. Therefore the role of those genes in the inherited resistance caused by K165 was confirmed. The results suggest that the phenomenon is controlled epigenetically.Επιγενετικές τροποποιήσεις που προκαλούνται από βιοτικές ή αβιοτικές καταπονήσεις δύνανται να επηρεάσουν σημαντικά την έκφραση γονιδίων που σχετίζονται με την άμυνα των φυτών εναντίον παθογόνων μικροοργανισμών. Η επιγενετική κληρονομικότητα αναφέρεται στη διατήρηση ορισμένων εξ’ αυτών των τροποποιήσεων στην επόμενη γενεά. Το αντικείμενο της συγκεκριμένης διατριβής είναι η μελέτη του μηχανισμού μέσω του οποίου ένα ωφέλιμο για τα φυτά βακτήριο το Paenibacillus alvei K165 προσδίδει κληρονομούμενη ανθεκτικότητα εναντίον του φυτοπαθογόνου μύκητα Verticillium dahliae, μέσω της εφαρμογής του σε μητρικά φυτά Arabidopsis thaliana, το οποίο επιβεβαιώθηκε και με ενδοφυτική ποσοτικοποίηση του μύκητα. Πραγματοποιήθηκαν μεταγραφωμικές και μεταβολομικές αναλύσεις στο υπέργειο μέρος φυτών στα οποία εφαρμόστηκε το στέλεχος Κ165 καθώς και των απογόνων τους ώστε να μελετηθεί η ανθεκτικότητά τους εναντίον του μύκητα Verticillium dahliae. Τα αποτελέσματα της μεταγραφωμικής ανάλυσης κατέδειξαν έναν σημαντικό αριθμό γονιδίων με ρόλο στο μονοπάτι βιοσύνθεσης φαινυλοπροπανοειδών των οποίων η έκφραση επηρεάζεται σημαντικά τόσο στα ίδια τα φυτά που εφαρμόστηκε το στέλεχος Κ165 όσο και στους απογόνους τους. Επίσης φαίνεται πως τόσο το μονοπάτι του σαλικυλικού οξέος όσο και αυτό του ιασμονικού/αιθυλενίου ενεργοποιούνται μέσω της εφαρμογής του στελέχους Κ165. Οι μεταβολομικές αναλύσεις έδειξαν σαφή διαφοροποίηση των μεταβολομικών προφίλ των εφαρμογών που ελέχθησαν καθώς και αύξηση του μεταβολισμού των λιπιδίων στα φυτά που δέχθηκαν την εφαρμογή του στελέχους Κ165 και στους απόγονους τους. Για επιβεβαίωση των αποτελεσμάτων της μεταγραφομικής ανάλυσης πραγματοποιήθηκε μελέτη της έκφρασης ενός σημαντικού αριθμού γονιδίων των οποίων η έκφραση φάνηκε ότι επηρεάζεται τόσο στα ίδια τα φυτά που εφαρμόστηκε το στέλεχος Κ165 όσο και στους απογόνους τους. Τα γονίδια που μελετήθηκαν έχουν βασικό ρόλο στην άμυνα των φυτών και συμμετέχουν στα μονοπάτια του σαλικυλικού οξέος, του ιασμονικού/αιθυλενίου και της βιοσύνθεσης φαινυλοπροπανοειδών. Η μελέτη της έκφρασης των γονιδίων PR1, PR5, NPR1 και PDF1.2 έδειξε πως ενώ τις πρώτες ημέρες από την εφαρμογή του μύκητα υπάρχει ενεργοποίηση και του μονοπατιού του σαλικυλικού οξέος, αργότερα την σκυτάλη φαίνεται να αναλαμβάνει το μονοπάτι ιασμονικού/αιθυλενίου καθώς παρατηρείται σημαντική υπερέκφραση του γονιδίου PDF1.2 στα φυτά που εφαρμόστηκε το βακτήριο. Ωστόσο στους ανθεκτικούς απογόνους παρατηρείται παράλληλη ενεργοποίηση των δύο μονοπατιών. Όσον αφορά στο μονοπάτι βιοσύνθεσης φαινυλοπροπανοειδών φαίνεται πως πράγματι, ένας μεγάλος αριθμός γονιδίων επηρεάζεται σημαντικά στα φυτά που εφαρμόστηκε το στελέχος Κ165 και στους απογόνους αυτών. Ωστόσο, το γονίδιο CAD8 ξεχωρίζει μέσω της σημαντικής υπερέκφρασής του στους ανθεκτικούς απογόνους και το γονίδιο 4CL2 μέσω της μεγάλης υπερέκφρασής του στα φυτά που εφαρμόστηκε το στέλεχος Κ165 ενώ το γονίδιο CCR2 παρουσιάζει μεγάλη υπερέκφραση στους ανθεκτικούς απογόνους και ακόμα μεγαλύτερη στα ίδια τα φυτά στα οποία εφαρμόζεται το βακτήριο. Αντιθέτως ο μύκητας φαίνεται πως μειώνει την έκφραση του συγκεκριμένου γονιδίου. Μετρήθηκε επίσης η συγκέντρωση της λιγνίνης στο υπέργειο μέρος των φυτών με τη χρήση φασματοφωτομετρίας, καθώς πρόκειται για το τελικό προϊόν που παράγεται από την ενεργοποίηση του μονοπατιού βιοσύνθεσης φαινυλοπροπανοειδών στα φυτά. Οι μετρήσεις επιβεβαίωσαν την αυξημένη συσσώρευση λιγνίνης στα φυτά που δέχθηκαν την εφαρμογή του στελέχους Κ165 αλλά και στους απογόνους αυτών. Με τη πραγματοποίηση πειραμάτων παθογένειας σε μεταλλαγμένες σειρές Arabidopsis thaliana μελετήθηκε ο ρόλος των γονιδίων CAD1, CAD6, CAD8 των οποίων η έκφραση φαίνεται να επηρεάζεται σημαντικά στους ανθεκτικούς απογόνους. Τα αποτελέσματα των πειραμάτων παθογένειας επιβεβαίωσαν τον ρόλο των παραπάνω γονιδίων στη πρόκληση του φαινομένου καθώς το στέλεχος Κ165 απέτυχε να προστατέψει τα μεταλλαγμένα φυτά και τους απογόνους τους από τον μύκητα V. dahliae. Η ενδοφυτική ποσοτικοποίηση του μύκητα επίσης έδειξε ότι δεν υπάρχει μείωση της συγκέντρωσης του μύκητα στα φυτά που δέχθηκαν το στέλεχος Κ165 ή στους απογόνους αυτών. Πραγματοποιήθηκαν ακόμα πειράματα ανοσοκατακρίμνησης χρωματίνης και ανοσοαπoτυπώματος κατά western με σκοπό να επιβεβαιωθεί η επιγενετική φύση του φαινομένου μέσω του εντοπισμού κληρονομούμενων επιγενετικών τροποποιήσεων που προκαλεί το στέλεχος Κ165. Τα επίπεδα ακετυλίωσης συνολικά στο γονιδίωμα των ανθεκτικών απογόνων βρέθηκαν αυξημένα σε σχέση με τον μάρτυρα. Χρησιμοποιήθηκαν εξειδικευμένα αντισώματα για τον εντοπισμό ακετυλιώσεων στην ιστόνη 3 και μελετήθηκαν τα επίπεδα ακετυλίωσης των γονιδίων PR1, PR5, PDF1.2, CAD4 και CAD8 στις περιοχές των υποκινητών και στο ανοιχτό πλαίσιο ανάγνωσης με τη χρήση της μεθόδου Real time PCR. Τα επίπεδα ακετυλίωσης στους υποκινητές των γονιδίων PR1, PDF1.2, CAD4 και CAD8 των ανθεκτικών απογόνων βρέθηκαν ιδιαίτερα αυξημένα σε σχέση με τους μάρτυρες. Τέλος, μελετήθηκε ο ρόλος των γονιδίων που είναι υπεύθυνα για την ακετυλίωση των αμινοξέων της ιστόνης 3 στην πρόκληση της επιγενετικά κληρονομούμενης ανθεκτικότητας μέσω πραγματοποίησης πειραμάτων παθογένειας σε μεταλλαγμένες σειρές Arabidopsis thaliana. Τα αποτελέσματα επιβεβαίωσαν τον ρόλο των HAC και HAG ακετυλοτρανσφερασών στη πρόκληση της κληρονομούμενης ανθεκτικότητας από το στέλεχος Κ165 καθώς αυτό απέτυχε να μειώσει τα συμπτώματα που προκαλεί ο μύκητας στα μεταλλαγμένα φυτά που εφαρμόστηκε το βακτήριο και τους απογόνους αυτών. Η ενδοφυτική ποσοτικοποίηση του μύκητα επίσης έδειξε ότι δεν υπάρχει μείωση της συγκέντρωσης του μύκητα στα φυτά που δέχθηκαν το στέλεχος Κ165 ή στους απογόνους αυτών

Feeding the Microbes: A Strategy to Control Verticillium Wilt

Verticillium dahliae is one of the most devastating soilborne pathogens for horticulture production. The pathogen has a broad host range and currently there is no effective chemical disease management, therefore, novel sustainable integrated disease management strategies should be considered. In this respect, we questioned whether the soil application of common microbiological growth media can influence the plant—microbe interactions and, subsequently, offer protection against V. dahliae. Indeed, the addition of Nutrient Broth (NB) and Potato Dextrose (PD) in non-sterilized soil reduced Verticillium wilt symptoms in eggplants. The addition of NB in sterilized soil did not reduce the disease symptoms compared to controls, however the addition of PD enhanced plant protection against V. dahliae. Following the results of a split root bioassay in eggplants, the possibility that NB and PD triggered the plant defense mechanisms against V. dahliae was excluded, since NB and PD did not reduce wilt symptom. Therefore, PD may be used as an easy food source for V. dahliae, detouring the pathogen from the root system of plants, while NB may affect the soil microbiome by enhancing antagonism in rhizosphere, or antagonistic interaction between V. dahliae and rhizospheric microbiome. Subsequently, several bacterial strains were isolated from the NB-treated rhizosphere and examined for their biocontrol activity against V. dahliae. Among the examined strains, a Pseudomonas putida strain, Z13, significantly reduced Veticillium severity and incidence under greenhouse conditions

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

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

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

<i>Paenibacillus alvei</i> K165 and <i>Fusarium oxysporum</i> F2: Potential Biocontrol Agents against <i>Phaeomoniella chlamydospora</i> 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

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