Synergistic interaction between the type III secretion system of the endophytic bacterium Pantoea agglomerans DAPP-PG 734 and the virulence of the causal agent of olive knot Pseudomonas savastanoi pv. savastanoi DAPP-PG 722

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.     

From the root to the stem: interaction between the biocontrol root endophyte Pseudomonas fluorescens PICF7 and the pathogen Pseudomonas savastanoi NCPPB 3335 in olive knots

Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi, is one of the most important biotic constraints for olive cultivation. Pseudomonas fluorescens PICF7, a natural colonizer of olive roots and effective biological control agent (BCA) against Verticillium wilt of olive, was examined as potential BCA against olive knot disease. Bioassays using in vitro-propagated olive plants were carried out to assess whether strain PICF7 controlled knot development either when co-inoculated with the pathogen in stems or when the BCA (in roots) and the pathogen (in stems) were spatially separated. Results showed that PICF7 was able to establish and persist in stem tissues upon artificial inoculation. While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumours, and sharply altered the localization of the pathogen in the hyperplasic tissue, which may pose epidemiological consequences. Confocal laser scanning microscopy combined with fluorescent tagging of bacteria revealed that when PICF7 was absent the pathogen tended to be localized at the knot surface. However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumours. This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue.     

Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.     

Reduction of Olive Knot Disease by a Bacteriocin from Pseudomonas syringae pv. ciccaronei

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10⁵ and 10⁸ CFU ml⁻¹, respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml⁻¹ at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml⁻¹) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.     

Development of a versatile tool for the simultaneous differential detection of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pathovars by End Point and Real-Time PCR

Background  

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta.     

Detection of Pseudomonas savastanoi pv. savastanoi in Olive Plants by Enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

A Na+/Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca²⁺ levels is observed, suggesting that Ca²⁺ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca²⁺ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na⁺/Ca²⁺ exchanger critical for virulence. Using the fluorescent Ca²⁺ probe Fura 2-AM, we demonstrate that Ca²⁺ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca²⁺ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca²⁺ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca²⁺ entry is a Na⁺/Ca²⁺ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na⁺/Ca²⁺ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca²⁺ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca²⁺ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca²⁺ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.     

Scanning Electron Microscopy of Olive and Oleander Leaves Colonized by Pseudomonas syringae subsp. Savastanoi

Leaves of olive and oleander were sprayed with suspensions of their homologous strains (PVBa230 and ITM519, respectively) of Pseudomonas syringae subsp. savastanoi and examined by SEM. It was found that both strains multiplied on the lower surface of the leaves of both species. Preferred sites for survival and multiplication were the shields of peltate hairs on olive and the stomatal pits on oleander. The findings suggest that, at least in olive leaves, some bacteria entered the leaf tissue through the stornata but that this was not important since cells which entered the host did not cause any disease symptoms. Cells of both strains were agglomerated and cells of ITM519 were further attached to the surface of the leaf hairs on oleander by fibrillar material. Inoculated leaves did not show any disease symptoms except at leaf abscission scars on olive plants where leaves had been excised prior to inoculation. It is suggested that on both olive and oleander preexisting wounds are necessary for symptoms to develop.     

The Grouping of Strains of Pseudomonas syringae subsp. savastanoi by DNA Restriction Fingerprinting

A selected group of strains of Pseudomonas syringae subsp. savastanoi from olive, oleander and ash were compared with pathogenicity tests and with DNA restriction fingerprinting using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The strains from each host were distinguishable by their pathogenicity to the same host and to the other two plant species. A division into the same groups was obtained with unweighted pair-group method with averages (UPGMA) clustering of the data from genomic fingerprinting, even though high overall similarity between the strains also indicated that they formed a single, well characterized taxon. It seems clear that the subspecies savastanoi of P. syringae comprises at least 3 groups of strains that differ in their precise host range, in the nature of the symptoms induced on the individual hosts, and in their genomic profile.     

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star‐shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.     

PsasM2I,a Type II Restriction–Modification System in Pseudomonas savastanoi pv. savastanoi: Differential Distribution of Carrier Strains in the Environment and the Evolutionary History of Homologous RM Systems in the Pseudomonas syringae Complex

A type II restriction–modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.     

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(--dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l^–1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, -NAA or IBA+-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l^–1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl^–1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.     

Effects of Drip Irrigation on Population of Verticillium dahliae in Olive Orchards

Verticillium wilt of olive (Olea europaea L.), caused by Verticillium dahliae, is nowadays the most serious olive disease in Spain. The disease increments are being observed particularly in young olive plantations, favoured by several factors including inadequate cultural practices and crop production intensification, such as irrigation. Thus, three olive orchards affected by Verticillium wilt, with disease incidence ranging 30–50%, were selected to determine if the drip irrigation could favour the increase of pathogen in soil. Pathogen in soil was quantified in wet zones around the drippers and in dry zones out of them. Inoculum density in all experiments was higher in wet than in dry areas. After 4 months of watering, soil pathogen population increased considerably in wet and dry areas but inoculum density remained higher in the wet soil.     

Micropropagation of a Local Olive Cultivar for Germplasm Preservation

In vitro shoot culture was applied to an Italian local cultivar Nebbiara of olive (Olea europaea L.) to preserve its endangered germplasm. This cultivar showed a notable difficulty for the in vitro establishment due to heavy pathogen contamination. Mercury chloride and sodium hypochloride in the sterilisation step and antibiotics in culture media allowed to overcome the problem. Proliferation of shoot apical bud on olive culture medium with 36 g dm^–3 mannitol and 4.56 M zeatin appeared very satisfactory. All the explants tested rooted during a subculture (1 month) preceeded by a 5-d long dark pre-treatment.     

Effect of Fruiting on Leaf Gas Exchange in Olive (Olea Europaea L.)

The effect on traits of photosynthesis and water relations of assimilate demand was studied in olive tree that has strong alternate bearing. The diurnal and seasonal leaf gas exchanges, area dry mass, and saccharide and chlorophyll (Chl) contents were measured by comparing shoots with fruit of "on-trees" (heavy fruit load) with shoots without fruit on both "on-trees" and "off-trees" (light fruit load). In spite of large seasonal and diurnal differences, leaf net photosynthetic rate (P _N), stomatal conductance (g _s), sub-stomatal CO₂ concentration (C ₁), transpiration rate (E), and respiration rate (R _D) were not significantly influenced by fruit load or by the presence or absence of fruit on the shoot. An only exception was at the beginning of July when the one-year-old leaves on shoots with fruit had slightly higher P _N and E than leaves on shoots without fruit. Water content, Chl and saccharide contents, and area dry mass of the leaf were not substantially influenced by the presence/absence of fruit on the shoot or fruit load. Hence the sink demand, associated with fruit growth, did not improve leaf photosynthetic efficiency in olive. This revised version was published online in August 2006 with corrections to the Cover Date.     

The genetic characterization of Pseudomonas syringae pv. tagetis based on the 16S–23S rDNA intergenic spacer regions

Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548 bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526 bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.     

Arthrinium phaeospermum,Phoma cladoniicola and Ulocladium consortiale,New Olive Pathogens in Italy

In recent years, leaf necrosis and twig dieback in the olive crop have been detected in Sicily (Italy). In this article, we identify the predominant fungal species associated with symptomatic leaves and twigs, using morphological features and DNA sequencing of the internal transcribed spacer (ITS) region, as Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale. The pathogenicity of these four species was tested on olive plants cv. Biancolilla. All species were pathogenic on leaves, but only U. consortiale produced cortical lesions on twigs, thus suggesting its main role in the Olea europaea twig dieback. To our knowledge, this is the first report of A. phaeospermum, P. cladoniicola and U. consortiale as olive pathogens.