Search

Essential oils (EOs) and oleoresins are complex mixtures mainly made up of terpenes, synthesized by a wide variety of plants. Individual terpenes may show broad-spectrum activity against different plant pathogens, and their combination into EO and oleoresin mixtures enhances plant chemical defense. The interest in EOs has significantly increased due to the trend of using natural products as herbicides, insecticidal and antimicrobial agents. In addition, the use of plant mixtures is an emerging approach to face the problem of antimicrobial resistance in agriculture. This chapter reports guidelines about plant sample collection for the production of EOs and provides protocols to test their activity as antimicrobial agents against bacteria and fungi. It also describes a solvent-free method for the inclusion of EOs into β-cyclodextrins. This type of formulate is prepared to turn liquid EOs into easily manageable water-soluble powders, and to control the release of volatile compounds, aiming to increase EOs' applications in agriculture.

Some antagonistic bacteria contribute to the management of plant diseases by stimulating the host natural defense and/or by providing direct biocontrol of pathogens. The objective of this study was to evaluate the biocontrol performances and the ability to induce a systemic response to crown gall of four strains belonging to Bacillus and Pseudomonas genera. Evaluation of in vitro antagonistic ability showed that the four bacteria had a different spectrum of activity against the three tested strains of Agrobacterium tumefaciens. A delay in time of appearance and a significant reduction of the tumor size were observed in tomato plants obtained by bio-primed seeds and stem inoculated with A. tumefaciens. Seed priming with antagonistic strains stimulated some systemic defense mechanisms in tomato plants that can be related to the reduction of disease symptoms induced by A. tumefaciens. Both antagonists and A. tumefaciens strains induced variations in phenol content and peroxidase activity while polyphenol oxidase activity was mainly affected by the single A. tumefaciens strain. The response was different in relation to the various combinations of antagonist/pathogen, however Pseudomonas brassicacearum EPR3 strain was the most effective strain.

Pollution affects most of the urban and forest environments at different levels causing well-known effects on human and plant health. The influence that pollutants exert on plant-associated microbiota might direct plant health and, in some cases, also the removal of pollutants by plants. With the advent of nanotechnologies, an increasing amount of engineered nanoparticles are being introduced into the environment, and consequently, their impact on plant-associated microorganisms needs to be investigated. In this context, silver nanoparticles (Ag-NPs) were experimentally supplied at leaf and root level of poplar plants to assess Ag-NPs effects on plant microbiota. Leaf Ag-NP treatment increased bacteria and fungi evenness and determined a significant reduction in both microbial groups, while root Ag-NP treatment reduced the bacterial and fungal biodiversity. Bioinformatics functional analysis showed that Ag-NP treatment reduced the aerobic and stimulated facultative anaerobic and oxidative stress-tolerant bacteria. Our study offers new insights into the effects of Ag-NPs on both phyllosphere and rhizosphere poplar-associated microbiota and may represent a first attempt to understand the behavior of microbial communities of a tree species growing in a polluted environment.

The Pseudomonas fluorescens complex contains at least eight phylogenetic groups and each of these includes several bacterial species sharing ecological and physiological traits. Pseudomonas chlororaphis classified in a separate group is represented by three different subspecies that show distinctive traits exploitable for phytostimulation and biocontrol of phytopathogens. The high level of microbial competitiveness in soil as well as the effectiveness in controlling several plant pathogens and pests can be related to the P. chlororaphis ability to implement different stimulating and toxic mechanisms in its interaction with plants and the other micro- and macroorganisms. Pseudomonas chlororaphis strains produce antibiotics, such as phenazines, pyrrolnitrine, 2-hexyl, 5-propyl resorcinol and hydrogen cyanide, siderophores such as pyoverdine and achromobactine and a complex blend of volatile organic compounds (VOCs) that effectively contribute to the control of several plant pathogens, nematodes and insects. Phenazines and some VOCs are also involved in the induction of systemic resistance in plants. This complex set of beneficial strategies explains the high increasing interest in P. chlororaphis for commercial and biotechnological applications. The aim of this review is to highlight the role of the different mechanisms involved in the biocontrol activity of P. chlororaphis strains.

International audience; Samples of pear shoots, blossoms and leaves showing typical fire blight symptoms were collected from diseased pear trees during spring and summer over a period of 3 years from orchards situated in the Mitidja region, a main pear producing area in Northern Algeria. From all collected plant samples, thirty-seven putative Erwinia amylovora strains were isolated and identified by biochemical, serological and molecular tests. Molecular typing of these strains was performed using six variable number of tandem repeats sequences (VNTRs). Minimal spanning trees showed that four different haplotypes were present within the bacterial population analyzed. The efficacy of twenty potential bacterial antagonists was evaluated in vitro against two E. amylovora pathogenic strains. Four antagonists (Bacillus amyloliquefaciens, Bacillus methylotrophicus and two Pseudomonas brassicaceaurum) were selected to perform a biocontrol assay on immature pear fruits. Results showed that the two Pseudomonas strains were the most effective for preventive treatment on pear fruits, leading to a necrosis reduction of up to 90%. No curative effect was observed. The four selected antagonists were characterized for features linked to biocontrol activity, such as biofilm, siderophores and biosurfactant-production, motility, and effect of environmental conditions on bacterial growth.

Thirty-five bacterial strains were selected on the basis of colony morphology from larvae and pupae of Rhynchophorous (Olivier) ferrugineus collected from infested Phoenix canariensis Chabaud trees growing in different Italian locations. 16S rDNA sequencing showed that bacteria isolated from larvae were mostly Gram positive and belonged to Bacillus , Brevibacillus and Paenibacillus genera. Bacteria isolated from pupae were affiliated to Bacillus and five different Gram negative genera ( Alcaligenes , Morganella , Myroides , Providencia and Serratia ). Some strains produced lytic enzymes like chitinase, lecithinase, lipase and protease in vitro. The enthomopathogenic strains belonging to Bacillus thuringiensis, Providencia rettgeri and Serratia marcescens species may be considered of interest as potential biocontrol agents of R. ferrugineus .

The massive use of traditional pesticides represents a worldwide problem that can no longer be postponed, due to the serious impact that these agrochemicals display on the environment. Possible strategies to lower their use include the development of natural antimicrobial agents and the improvement of their delivery efficacy. In this study, nanovectors composed of lipids extracted from the marine microalga Nannochloropsis sp. were fabricated, structurally characterized, and loaded with thymol. These nanovectors were tested as antibacterial agents against the plant pathogen Xanthomonas campestris pv. vesicatoria. The mean size of the nanoparticles was below 150 nm, with a polydispersity index ranging from 0.12 and 0.33 and a slightly negative surface charge (-4.74 to -1.19 mV). Their “backbone” was mainly made of neutral lipids, representing 76% of the lipid fraction. Concerning the loading ratio, more than 70% of thymol was uploaded into the carriers. In vitro tests showed marked bactericidal effect of the thymol-delivering nanovectors at the concentration of 250 ppm. Similar results were obtained from standard liposomes made of purified soybean phosphatidylcholine, thus encouraging further investigation on the potentialities of this algae-derived nanoformulation as novel drug delivery system.

Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC–MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.

Nell'ambito del progetto, il CNR-IBIMET è coinvolto nelle azioni della Fase 5 - Integrated Pest Management (IPM) ed impiego di bio-pesticidi con il Dipartimento di Scienze delle Produzioni AgroAlimentari e dell'Ambiente (DISPAA), l'Istituto per la Protezione Sostenibile delle Piante (CNR - IPSP), l'Istituto di Bioscienze e Biorisorse (CNR - IBBR), la Società Agricola Romiti Vivai e GIEFFE Vivai. Le attività della Fase 5 sono state suddivise facendo una distinzione tra il lavoro richiesto per la realizzazione degli strumenti di supporto "Uso razionale dei fitofarmaci" e quello necessario per la "Gestione delle fitopatie e delle piante infestanti". Ambedue le azioni sono tese all'individuazione di tecnologie innovative per un aumento delle conoscenze e una razionalizzazione degli interventi di lotta fitosanitaria. La prima si basa su strumenti di monitoraggio e sulla modellistica agrometeorologica, mentre la seconda mira ad individuare e promuovere l'impiego di prodotti a minor impatto ambientale, come biopesticidi e bioerbicidi.

Pseudomonas chlororaphis subsp. aureofaciens encompasses bacterial strains that effectively control phytopathogenic fungi through the production of the natural antibiotics named phenazines. In this work, the involvement of phenazine production in the interaction between the biological control agent P. chlororaphis subsp. aureofaciens M71 and the fungus Seiridium cardinale, a serious cypress pathogen, was investigated. Field trials were carried out to assess the role of phenazines in the control of S. cardinale in vivo. Results showed that P. chlororaphis subsp. aureofaciens M71 and 30-84, both able to produce phenazine-1-carboxylic acid (PCA), drastically reduced the canker development incited by S. cardinale. Conversely, strain M71b, a natural gacA mutant of P. chlororaphis subsp. aureofaciens M71, showed a decrease in PCA production and a reduction in controlling S. cardinale. These results were enforced by the reduction of canker size higher than 94% registered when 6 ?g of pure PCA was directly applied on each cypress wound. Furthermore, PCA was detected in cypress plant tissues only when P. chlororaphis subsp. aureofaciens M71 was interacting with S. cardinale for 30 days. All these data support that the biological control of S. cardinale achieved by the application of P. chlororaphis subsp. aureofaciens M71 relies mainly on the ability of the bacterial strain to produce PCA in planta.

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was tested for its efficacy in controlling L. Seiridium cardinale, the fungus responsible for bark canker of common cypress (Cupressus sempervirens).The bacterium was able to completely inhibit the mycelial growth and conidium germination of the fungus in vitro and prevented canker induction in field trials. Strain M71 produced two phenazine compounds, phenazine-1-carboxyilic acid and 2-hydroxyphenazine. They were extracted from the bacterial culture, purified, identified and tested for their activity against S. cardinale and three other fungi responsible for cypress canker, viz. Diplodia cupressi, Seiridium cupressi and Seiridium unicorne. Phenazine- 1-carboxylic acid was the sole compound active against the four fungi. The application in vivo of this phenazine molecule against S. cardinale reduced canker size indicating that the compound is directly involved in the control of the fungal pathogen by P. chlororaphis subsp. aureofaciens strain M71. Furthermore, the antagonist showed an interesting capacity for epiphytic fitness since it was able to establish itself on the crown of cypress plants and survive on it for more than three months

Nine Italian peach nurseries, which use Agrobacterium rhizogenes strain K84 to protect plants from crown gall, were monitored for three years with the aim of determining whether transconjugant populations may arise following plasmid exchanges between K84 and autochtonous soil agrobacteria. Six hundred and seventy-eight Agrobacterium isolates were obtained from 120 tumours developed on apricot and peach rootstocks that had been treated in pre-planting with the antagonist. Agrobacteria were characterized for pathogenicity, biovar, opine catabolism and agrocin 84 sensitivity. Colony hybridization was used for screening the isolates harbouring plasmids pTi and/or pAgK84. Analysis of plasmid content and Southern blotting were performed on putative transconjugant agrobacteria found in tumours collected from one nursery where a biological control breakdown was observed. The RFLP analysis of 16S + IGS regions showed that pAgK84 was transferred from the antagonist to virulent and avirulent soil agrobacteria belonging to different ribotypes. Pathogenic transconjugants, inoculated on GF677 rootstocks, were not controlled in vivo by K84 and stably maintained pTi and pAgK84 in the bacterial cells for at least one year. At the end of a biocontrol trial, new transconjugant tumorigenic agrobacteria originated by the transfer of pAgK84 to the pathogen. Virulent and avirulent transconjugants may represent a real threat for biological control by K84 strain since all of them produced agrocin and were insensitive to it. Survival in soil of these populations could make the future application of K84 ineffective.

Physiological, biochemical and molecular characteristics of Agrobacterium tumefaciens strains isolated in Italy from different host plants were analysed. Diseased plants were collected from several nurseries located in nine different regions. Out of 1293 strains isolated from 12 fruit tree and six ornamental plant species, a group of 120 strains was chosen as representative of the whole collection. The majority of the strains were biovar 2 (82.5%), agrocin 84 sensitive, and were isolated from stone fruit trees. Most of the strains identified as biovar 1 were isolated from ornamental plants and were insensitive to A. radiobacter antagonistic strain K84. Some strains that were isolated from Euonymus spp, Prunus GF 677 and Pyrus communis (pear) OHF tumours could not be allocated to any of the three Agrobacterium biovars. PCR-restriction fragment length polymorphism of the rrs gene plus the intergenic spacer was used for strain fingerprinting and characterisation. Results showed a wide genetic variability within the biovar 1 strains and homogeneity within the biovar 2 group. Biovar 2 strains from Sardinia were highly variable and differed from the biovar 2 strains isolated from the other regions of Italy.

Ficus benjamina plants, galled both at epi- and hypogeous parts, were observed in Italy and in The Netherlands, and these were the first records of the appearance of weeping fig crown gall in Europe. A total of 241 Agrobacterium isolates was obtained from 41 tumours and studied for their morphological, physiological and phytopathological characters. Two main groups of agrobacteria were distinguished by their colony morphology and through classical biovarietal tests that allowed allocation of 86 isolates into biovar 1 and 155 into an intermediate biovar rather different from any of the three biovars defined for agrobacteria. Most of the isolates were unable to utilize mannopine, nopaline or octopine as C and N sources; only 62 strains utilized nopaline. However, when nonopine-utilizing strains were inoculated into F. benjamina, only nopaline was detected in the developing tumours. BIOLOG ML 1™ system analysis applied to 50 representative strains allowed identification of the biovar 1 isolates as Agrobacterium tumefaciens and most of the intermediate biovar isolates as the newly proposed species Agrobacterium fici. Analysis of sensitivity to a set of 14 antibiotics confirmed the allocation of the 50 strains into two well defined main clusters matching the BIOLOG identification. Out of 141 tumorigenic isolates, 66 were sensitive in vitro to agrocine 84, but four of these strains showed scarce or no sensitivity to the antagonist A. radiobacter K84 when tested in fig plants. The two types of agrobacteria could usually be isolated from the same tumours. Tumorigenic strains were able to induce tumours in six herbaceous plant species, in eight to 10 out of 12 woody plants and in six to eight out of nine Ficus species, indicating a wide host-range Ti plasmid. Agrobacteria were able to survive and move in the vascular system of galled ficus plants and to induce tumour growth in stem-cutting propagated plants. Moreover, agrobacteria were detected in many healthy F. benjamina plants as part of the endophytic microflora. These findings suggest potential for spread of the disease through latently infected plant propagation material produced as cuttings or by tissue culture.

The volatile constituents of the flowerheads of Centaurea nicaeensis All., C. parlatoris Helder and C. solstitialis L. ssp. schouwii (DC.) Dostal were extracted by hydrodistillation and analysed by GC and GC-MS. Altogether 113 components were identified. Fatty acids and hydrocarbons were the most abundant components in the oils. Caryophyllene and caryophyllene oxide were the main compounds of the sesquiterpene fraction. The study on the biological activity of the oils shows no significant activity.

Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phenazine compounds as main secondary metabolites. These metabolites were identified as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this study, the spectrum of the activity of PCA and 2-OH P was evaluated against a group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA was active against most of the tested plant pathogens, while 2-OH P slightly inhibited a few fungal species. Furthermore, four semisynthesised derivatives of PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their antifungal activity against 11 phytopathogenic species. Results showed that the carboxyl group is a structural feature important for the antifungal activity of PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1- carboxamide, the two more lipophilic and reversible PCA derivatives remained substantially unaltered compared with PCA.

In the Sidi M'djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR– RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.

Agrobacteriumlarrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically. The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria. The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS. The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A.larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: Download : Download full-size image All sugars are α- d -pyranoses if not stated otherwise, Kdo is 3-deoxy- d -manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA.