Forewarned is forearmed : harmonized approaches for early detection of potentially invasive pests and pathogens in sentinel plantings

This work was supported by COST Action Global Warning (FP1401). DLM and YB contribution was also supported by the Russian Foundation for Basic Research (Grant No. 17-04-01486). MG was supported by Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant III43002. MKA was supported by the Ministry of Science and Higher Education of the Republic of Poland. NK was supported by Le Studium foundation (France) and RFBR (Grant No. 19-04-01029). RE, IF and MK contribution was also supported by CABI with core financial support from its member countries (see http://www.cabi.org/about-cabi/who-we-work-with/key-donors/ for details). IF contribution was further supported through a grant from the Swiss State Secretariat for Science, Education and Research (Grant C15.0081, awarded to RE).Peer reviewedPublisher PD

The Top 10 oomycete pathogens in molecular plant pathology

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.Keywords: microbiology, diversity, genomics, oomycetes plant patholog

Development of molecular diagnostic methods for detections pathogenic genes of fusarium and study of interaction with plants

Cotton fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (FOV), is considered as a major threat for commercial cotton production worldwide due to its devastating effect on the vascular system of cotton plants and its ability to survive in water, seeds or soil for very long periods. Therefore accurate characterization and sensitive detection in potential contamination sources of these pathogenic FOV isolates are prerequisites for the application of a prevention program and for the developing of key strategies in disease management. Polymerase chain reaction (PCR) amplification of the repetitive regions of the ribosomal intergenic spacer regions (IGS) and digestion with three restriction enzymes (AluI, HaeIIIII, RsaI) resulted in three unique restriction profiles (IGS haplotypes 1,2,3) for Fusarium oxysporum f. sp. vasinfectum isolates, capable of distinguishing them from other formae speciales of Fusarium oxysporum. In addition, a portion of the IGS region flanking the 5’ end was sequenced and single nucleotide polymorphisms (SNPs) were revealed. Using these data there were developed two specific Real Time PCR-based assays for the absolute quantification of genomic DNA from these isolates in soil substrates and infected cotton tissues. Standard curves showed a linear relation (r2=0.99) between log values of fungal genomic DNA and Real Time PCR threshold cycles. The detection sensitivity when inoculums were added to sterile soils was in some cases lower than 104 conidia·g-1 soil tested. Furthermore in order to identify possible involvement of salicylic acid (SA)- or jasmonate (JA)- dependent signal transduction pathways in defense mechanisms against the aforementioned fungus, we examined the effect of treatment of cotton plants (resistant and susceptible cultivars) with chemical inducers such as BION (a chemical analogue of SA) and methyl jasmonate (MeJA) before challenge with a pathogenic isolate of this fungus. The cotton pathogenesis-related (PR) genes examined were PR10 and chitinase (PR3) for which plylogenetic analysis showed low degree of homology with other similar function genes. Expression changes of these genes in roots and hypocotyls (of a susceptible variety, LACTA and of a resistant one, EMERALD) by Real Time PCR provided useful data of defense reactions on this type of plant-pathogen interaction. It was observed a high induction of expression of chitinase gene in hypocotyls and roots of the susceptible variety after treatment with MeJA, while in PR10 was rapidly induced in both tissues of the same variety after treatment with BION. Finally, in contrast to the hypocotyls of susceptible variety, the hypocotyls of the resistant variety over expressed both genes after simple infection with Fusarium oxysporum f. sp. vasinfectum isolate.Η φουζαρίωση του βάμβακος που προκαλείται από τον μύκητα Fusarium oxysporum f. sp. vasinfectum (FOV) θεωρείται κύρια απειλή για την καλλιέργεια βάμβακος παγκοσμίως εξαιτίας των καταστρεπτικών συνεπειών που προκαλεί μέσω του αποικισμού του παθογόνου στις ηθμαγγειώδεις δεσμίδες των φυτών και της ικανότητας του να επιβιώνει για μεγάλες χρονικές περιόδους στο νερό, τους σπόρους και στο έδαφος. Επομένως, είναι αναγκαίος ο χαρακτηρισμός και η αξιόπιστη μοριακή ανίχνευση αυτών των απομονώσεων του FOV σε πιθανές πηγές μόλυνσης καθώς αποτελούν απαραίτητες προϋποθέσεις για την εφαρμογή προγραμμάτων φυτοπροστασίας. Η ενίσχυση με αλυσιδωτή αντίδραση της πολυμεράσης (PCR) των επαναλαμβανόμενων τμημάτων των ριβοσωμικών ενδογονιδιακών περιοχών (IGS) και κοπή με τρία περιοριστικά ένζυμα (AluI, HaeIIIII, RsaI) είχε ως αποτέλεσμα τη δημιουργία τριών ευδιάκριτων προτύπων (IGS απλότυποι 1,2,3) για αυτές τις απομονώσεις του Fusarium oxysporum f. sp. vasinfectum, ικανές να τις διαχωρίζουν από άλλες ειδικές μορφές (formae speciales) του Fusarium oxysporum. Επιπρόσθετα, πραγματοποιήθηκε αλληλούχιση των τμημάτων που συνορεύουν με το 5΄ άκρο των περιοχών αυτών και εντοπίστηκαν πολυμορφισμοί απλού νουκλεοτιδίου (SNPs). Χρησιμοποιώντας αυτά τα δεδομένα αναπτύχθηκαν δυο εξειδικευμένες δοκιμές PCR πραγματικού χρόνου για την απόλυτη ποσοτικοποίηση του γενωμικού DNA αυτών των απομονώσεων σε διάφορα εδάφη και φυτικούς ιστούς επιμολυσμένων φυτών. Τα πρότυπα διαγράμματα αναφοράς κατέδειξαν γραμμικές συσχετίσεις (r2=0.99) μεταξύ του λογάριθμου της ποσότητας του γενωμικού DNA και των κύκλων ανίχνευσης της PCR πραγματικού χρόνου. Η ευαισθησία ανίχνευσης όταν μολύνθηκαν τα εδάφη ήταν σε κάποιες περιπτώσεις λιγότερο από 104 κονίδια·g-1 εδάφους. Επιπλέον, με σκοπό να αναγνωριστεί η πιθανή ανάμειξη των εξαρτώμενων από το σαλικυλικό (SA) ή το ιασμονικό οξύ (JA) βιοχημικών οδών μεταγωγής σήματος στην ενεργοποίηση των αντιδράσεων άμυνας έναντι αυτών των απομονώσεων, εξετάστηκε η επίδραση της επέμβασης σε φυτά βαμβακιού (ευπαθούς και ανθεκτικής ποικιλίας) χημικών επαγωγέων όπως το ΒΙΟΝ (ένα ανάλογο του SA) και του μεθυλεστέρα του ιασμονικού οξέος (MeJA) πριν την μυκητολογική πρoσβολή με μια μολυσματική απομόνωση του μύκητα αυτού. Τα γονίδια του βάμβακος που εξετάστηκαν και σχετίζονται με την παθογένεση (PR γονίδια) ήταν αυτά του PR-10 (δράση ριβονουκλεάσης) και της χητινάσης (PR-3) και για τα οποία η φυλογενετική ανάλυση έδειξε χαμηλό βαθμό ομολογίας με άλλες παρόμοιες πρωτεΐνες. Η μελέτη των επαγόμενων αλλαγών σχετικής έκφρασης αυτών των δυο γονιδίων με PCR πραγματικού χρόνου στις ρίζες και στα υποκότυλα (μιας ευπαθούς ποικιλίας, της LACTA και μιας ανθεκτικής, της EMERALD) παρέχει πολύτιμες πληροφορίες για τις αντιδράσεις άμυνας σε αυτού του είδους τις αλληλεπιδράσεις φυτού-παθογόνου. Σημαντική επαγωγή της έκφρασης της χητινάσης στα υποκότυλα και τις ρίζες της ευπαθούς ποικιλίας παρατηρήθηκε ύστερα από επέμβαση με MeJA, ενώ τα επίπεδα των μεταγράφων του PR10 αυξήθηκαν πολύ πιο ραγδαία και στους δυο ιστούς της ίδιας ποικιλίας ύστερα από επέμβαση με ΒΙΟΝ. Σε αντίθεση με τα υποκότυλα της ευπαθούς ποικιλίας στην ανθεκτική τα επίπεδα έκφρασης και των δυο γονιδίων αυξήθηκαν ύστερα από μόλυνση με τη μολυσματική απομόνωση

Detection of Venturia inaequalis Isolates with Multiple Resistance in Greece

The excessive use of fungicides against Venturia inaequalis, the causal agent of apple scab, has led to the emergence of resistant populations to multiple fungicides over the years. In Greece, there is no available information on fungicide resistance, despite the fact that control failures have been reported on certain areas. An amount of 418 single-spore isolates were collected from three major apple production areas and tested for their sensitivity to eight commonly used fungicides from unrelated chemical groups. The isolates were tested on malt extract agar media enriched with the discriminatory dose of each fungicide using the point inoculation method. To define the discriminatory dose for assessing the levels of resistance, EC50 values on both spore germination and mycelial growth assays were previously determined. Isolates exhibiting high resistance to trifloxystrobin (92% in total) and difenoconazole (3%); and moderate resistance to cyprodinil (75%), dodine (28%), difenoconazole (36%), boscalid (5%), and fludioxonil (7%) were found for the first time in Greece. A small percentage of the isolates were also found less sensitive to captan (8%) and dithianon (6%). Two isolates showed various levels of resistance to all eight fungicides. Despite the occurrence of strains with multiple resistances to many fungicides, we concluded that this practical resistance in the field arose mainly due to the poor control of apple scab with trifloxystrobin and difenoconazole

Differential triggering of the phenylpropanoid biosynthetic pathway key genes transcription upon cold stress and viral infection in tomato leaves

Plants develop a plethora of defense strategies during their acclimation and interactions with various environmental stresses. Secondary metabolites play a pivotal role in the processes during stress acclimation, therefore deciphering their relevant responses exchange the interpretation of the underlying molecular mechanisms that may contribute to improved adaptability and efficacy. In the current study, tomato plants were exposed to short-term cold stress (5◦C for 16 h) or inoculated (20 d) with either Cucumber Mosaic Virus (CMV) or Potato Virus Y (PVY). Responses were recorded via the assessments of leaf total phenolic (TP) content, total flavonoid (TF) levels, and phenylalanine ammonia-lyase (PAL) enzyme activity. The transcription of the gene families regulating the core phenylpropanoid biosynthetic pathway (PBP) at an early (PAL, cinnamic acid 4-hydroxylase, 4-coumarate-CoA ligase) or late (chalcone synthase and flavonol synthase) stage was also evaluated. The results showed that cold stress stimulated an increase in TP and TF contents, while PAL enzyme activity was also elevated compared to viral infection. Besides genes transcription of the enzymes involved in the core PBP was mostly induced by cold stress, whereas transcription of the genes regulating flavonoid biosynthesis was mainly triggered by viral infection. In conclusion, abiotic and biotic stressors induced differential regulation of the core PBP and flavonoid biosynthetic metabolism. Taking the above into consideration, our results highlight the complexity of tomato responses to diverse stimuli allowing for better elucidation of stress tolerance mechanisms at this crop

Compatible Consortium of Endophytic <i>Bacillus halotolerans</i> Strains Cal.l.30 and Cal.f.4 Promotes Plant Growth and Induces Systemic Resistance against <i>Botrytis cinerea</i>

Evaluating microbial-based alternatives to conventional fungicides and biofertilizers enables us to gain a deeper understanding of the biocontrol and plant growth-promoting activities. Two genetically distinct Bacillus halotolerans strains (Cal.l.30, Cal.f.4) were evaluated for the levels of their compatibility. They were applied individually or in combination under in vitro and greenhouse conditions, using seed bio-priming and soil drenching as inoculum delivery systems, for their plant growth-promoting effect. Our data indicate that application of Cal.l.30 and Cal.f.4 as single strains and as a mixture significantly enhanced growth parameters of Arabidopsis and tomato plants. We investigated whether seed and an additional soil treatment with these strains could induce the expression of defense-related genes in leaves of young tomato seedling plants. These treatments mediated a long lasting, bacterial-mediated, systemic-induced resistance as evidenced by the high levels of expression of RP3, ACO1 and ERF1 genes in the leaves of young tomato seedlings. Furthermore, we presented data showing that seed and soil treatment with B. halotolerans strains resulted in an effective inhibition of Botrytis cinerea attack and development on tomato leaves. Our findings highlighted the potential of B. halotolerans strains as they combine both direct antifungal activity against plant pathogens and the ability to prime plant innate immunity and enhance plant growth

Phenotypic and molecular characterization of apple (Malus × domestica Borkh) genetic resources in Greece

The phenotypic and genetic analysis of apple (Malus × domestica Borkh) genotypes is essential for breeding species. Information on the morphology and genetic structure of apple offers significant help for germplasm maintenance and selection of suitable material to breed superior cultivars. This study shows the results of an investigation on the morphology and the genetic diversity for 19 apple cultivars, which are preserved in an ex situ collection in Naoussa, Central Macedonia, Greece. Information was recorded over a 5-year period for 47 traits describing plant morphology and phenotype, as well as leaf and fruit quality. Data were analyzed using the principal component analysis (PCA) method. The Euclidean distance metric and the Ward’s agglomeration method were used in an unsupervised hierarchical cluster analysis of all cultivars. The cultivars were grouped into four main clusters, suggesting that the characterized apple collection has a high potential for specific breeding goals. Furthermore, the cultivars were genotyped using seven microsatellite primers. Moderate levels of polymorphism were detected, and 38 distinctive alleles (5.4 alleles per primer pair) were identified. Both multivariate clustering approach (phenotypic data) and the genetic distance clustering approach (genetic data) grouped the apple cultivars according to their type. Hence, these data could be used for protection or patenting processes of existing or new apple cultivars carried out by the EU-Community Plant Variety Office

Global Transcriptome Analysis of the Peach (<i>Prunus persica</i>) in the Interaction System of Fruit–Chitosan–<i>Monilinia fructicola</i>

The peach (Prunus persica L.) is one of the most important stone-fruit crops worldwide. Nevertheless, successful peach fruit production is seriously reduced by losses due to Monilinia fructicola the causal agent of brown rot. Chitosan has a broad spectrum of antimicrobial properties and may also act as an elicitor that activate defense responses in plants. As little is known about the elicitation potential of chitosan in peach fruits and its impact at their transcriptional-level profiles, the aim of this study was to uncover using RNA-seq the induced responses regulated by the action of chitosan in fruit–chitosan–M. fructicola interaction. Samples were obtained from fruits treated with chitosan or inoculated with M. fructicola, as well from fruits pre-treated with chitosan and thereafter inoculated with the fungus. Chitosan was found to delay the postharvest decay of fruits, and expression profiles showed that its defense-priming effects were mainly evident after the pathogen challenge, driven particularly by modulations of differentially expressed genes (DEGs) related to cell-wall modifications, pathogen perception, and signal transduction, preventing the spread of fungus. In contrast, as the compatible interaction of fruits with M. fructicola was challenged, a shift towards defense responses was triggered with a delay, which was insufficient to limit fungal expansion, whereas DEGs involved in particular processes have facilitated early pathogen colonization. Physiological indicators of peach fruits were also measured. Additionally, expression profiles of particular M. fructicola genes highlight the direct antimicrobial activity of chitosan against the fungus. Overall, the results clarify the possible mechanisms of chitosan-mediated tolerance to M. fructicola and set new foundations for the potential employment of chitosan in the control of brown rot in peaches

Phenotypic and molecular characterization of apple (Malus × domestica Borkh) genetic resources in Greece

ABSTRACT: The phenotypic and genetic analysis of apple (Malus × domestica Borkh) genotypes is essential for breeding species. Information on the morphology and genetic structure of apple offers significant help for germplasm maintenance and selection of suitable material to breed superior cultivars. This study shows the results of an investigation on the morphology and the genetic diversity for 19 apple cultivars, which are preserved in an ex situ collection in Naoussa, Central Macedonia, Greece. Information was recorded over a 5-year period for 47 traits describing plant morphology and phenotype, as well as leaf and fruit quality. Data were analyzed using the principal component analysis (PCA) method. The Euclidean distance metric and the Ward’s agglomeration method were used in an unsupervised hierarchical cluster analysis of all cultivars. The cultivars were grouped into four main clusters, suggesting that the characterized apple collection has a high potential for specific breeding goals. Furthermore, the cultivars were genotyped using seven microsatellite primers. Moderate levels of polymorphism were detected, and 38 distinctive alleles (5.4 alleles per primer pair) were identified. Both multivariate clustering approach (phenotypic data) and the genetic distance clustering approach (genetic data) grouped the apple cultivars according to their type. Hence, these data could be used for protection or patenting processes of existing or new apple cultivars carried out by the EU-Community Plant Variety Office