Population diversity of Pseudomonas syringae subsp. savastanoi on olive and oleander

Twenty-one strains of Pseudomonas syringae subsp. savastanoi , isolated from knots on olive and oleander trees growing in close proximity to or in physical contact with one another, were evaluated for knot induction and for bacteriocin production. In addition, DNA preparations from the bacterial strains were tested for hybridization to probes containing the tryptophan monooxygenase (iaaM) and isopentenyl transferase (ipt) genes, which are involved in indole-3-acetic acid and cytokinin biosynthesis, respectively, in P.s. subsp. savastanoi.
The strains showed features characteristic of strains usually isolated from their respective host plants. For example, all 10 oleander strains were virulent both to oleander and olive, did not produce bacteriocins and harboured the iaaM gene on plasmids. In contrast, all 11 olive strains were virulent only to olive, 10 strains produced bacteriocins, and nine strains carried the iaaM gene on the chromosome. Two olive strains (OAll, OD21) harboured the gene coding for iaaM on plasmids. Furthermore, strain OD21 carried the iaaM gene on the same plasmid as the ipt gene. This is the first report both of a plasmid-borne iaaM in typical olive strains (virulent only to olive and bacteriocin producers), and of the presence of the ipt gene on the same plasmid. In olive and oleander strains the ipt gene was located either on plasmids or on the chromosome. These results suggest that under natural conditions the pathogen does not appear to spread from oleander to olive even when trees are growing in close proximity or in physical contact.
The location of the phytohormone genes on plasmids or on the chromosome is discussed in relationship to bacteriocin-production and knot-induction on the host species.     

In vitro analysis of the interaction of Pseudomonas savastanoi pvs. savastanoi and nerii with micropropagated olive plants

This study assessed the use of in vitro olive plants to evaluate the virulence of Pseudomonas savastanoi pv. savastanoi strains isolated from olive and P. savastanoi pv. nerii strains isolated from oleander knots. First, different olive isolates were inoculated into stem wounds and differences in knot formation and weight of overgrowths were observed for the selected strains. Tissue proliferation was clearly visible in all inoculated plants 30 days after inoculation. Virulence of P. savastanoi pv. nerii mutants with defects in regard to biosynthesis of indole-3-acetic acid and/or cytokinins was tested using this system. In agreement with data previously reported, all mutant strains multiplied in olive but induced attenuated symptoms. To analyze the virulence of P. savastanoi pv. savastanoi affected in their ability to grow in olive tissue, a trpE tryptophan auxotroph mutant was generated using a collection of signature tagged mutagenesis transposons. Virulence of this mutant was clearly reduced as evidenced by swelling of the olive tissue that evolved into attenuated knots. Furthermore, mixed infections with its parental strain revealed that the wild-type strain completely out-competed the trpE mutant. Results shown here demonstrate the usefulness of in vitro olive plants for the analysis of P. savastanoi pvs. savastanoi and nerii virulence. In addition, this system offers the possibility of quantifying virulence differences as weight of overgrowths. Moreover, we established the basis for the use of mixed infections in combination with signature tagged mutagenesis for high-throughput functional genomic analysis of this bacterial pathogen.     

Genetic characterization by fluorescent AFLP of Pseudomonas savastanoi pv. savastanoi strains isolated from different host species

The genetic diversity of 71 Pseudomonas savastanoi pv. savastanoi strains isolated from different host species and from diverse geographical regions was determined by fluorescent amplified fragment length polymorphism (f-AFLP) analysis. The study was carried out using three different selective primer combinations. Strains of P. syringae pv. syringae , P. syringae pv. phaseolicola , P. syringae pv. glycinea , P. syringae pv. tagetis and P. amygdali were also included as outgroups. Based on cluster analysis of f-AFLP data, all P. savastanoi pv. savastanoi strains showed a high degree of similarity, grouping in a cluster and forming a taxon clearly separate from outgroup strains. AFLP analyses failed to support placing strains of P. savastanoi pv. savastanoi , P. syringae pv. phaseolicola and P. syringae pv. glycinea in the same species. Strains of P. savastanoi pv. savastanoi formed subclusters that correlated with the host species. Strains identified within these subclusters were related to the geographical region where the strains were isolated. Strains of P. savastanoi pv. savastanoi from olive were divided into two subclusters. Strains from oleander were differentiated from those from ash and were divided into two additional subclusters, distinct from olive strains. Three strains isolated from jasmine showed a high level of similarity among them but, at a lower Dice similarity coefficient, were linked to a subcluster including olive strains. Finally, two strains isolated from privet were similar to strains from olive and were included in the same subcluster.     

Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

ABSTRACT The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.     

Heterogeneity of Pseudomonas savastanoi populations infecting Myrtus communis in Sardinia (Italy)

Genetic, phenotypic and host range diversity among Pseudomonas savastanoi isolates from Myrtus communis were investigated. Thirty‐one isolates from six Sardinian commercial myrtle orchards and three isolates from plants growing spontaneously on the island of Rhodes (Greece) were compared with reference strains of P. savastanoi from olive, oleander, ash and myrtle. Multilocus sequence analysis (MLSA) indicated the presence of a monomorphic population with a very low level of variability. Conversely, Biolog phenotypic fingerprinting and phytohormone production analyses showed a considerable metabolic diversity, as bacteria obtained from single infected tissue differed more than bacteria obtained from different orchards. When pathogenicity tests were carried out on myrtle plants, different types of symptoms were induced: knots, canker lesions with or without tissue proliferations and, occasionally, wilting of the inoculated twig, a symptom never reported before for P. savastanoi. Comparable symptoms were also observed in the natural environment both on spontaneous and cultivated plants. Moreover, the host range of the myrtle population was heterogeneous and not well defined. Some isolates showed a wide host range whilst others were pathogenic only to their natural host. Overall these findings suggest that the diversity of the P. savastanoi population from myrtle does not depend so much on the locality or the natural host and does not allow the Sardinian and Greek isolates, together with previously characterized myrtle strains, to be ascribed to a known pathovar of P. savastanoi, nor to propose their belonging, as a whole, to a new pathovar.     

Interaction between Pseudomonas savastanoi pv. savastanoi and Pantoea agglomerans in olive knots

Results of a survey of olive knot disease in central Italy in 2002 and 2003 showed that Pantoea agglomerans was found associated with the pathogen Pseudomonas savastanoi pv. savastanoi ( Ps. savastanoi ) in 70% of the olive knots examined. Pathogenicity tests in which these two bacteria were co-inoculated on the stems of 1-year-old olive plants at ratios of 1:1, 1:100 and 100:1 showed that the growth of P. agglomerans was apparently aided by the presence of an actively growing population of Ps. savastanoi . At the same time, however, a dominant population of P. agglomerans at the inoculation site tended to depress the growth of Ps. savastanoi , probably because of competition for space and nutrients between these bacteria and by means of antibiotic production by P. agglomerans. In some cases the association of P. agglomerans, which in culture was found to produce indole-3-acetic acid but not cytokinins, with Ps. savastanoi resulted in an increase in the size of knots. This boosting effect of P. agglomerans on proliferation was probably due to the release of IAA by this bacterium at the inoculation sites.     

Systemic spread of Pseudomonas savastanoi pv. savastanoi in olive explants

The manner in which the bacterium Pseudomonas savastanoi pv. savastanoi ( Pss ), the causal agent of knot disease, infects olive plants is erratic and has not been fully documented. To investigate the process of Pss invasion, olive explants were inoculated in vitro and examined visually and by light microscopy at 2-weekly intervals for 10 weeks. In all host genotypes tested, interaction with the pathogen resulted in: (i) a progressive collapse of the stem, originating at the inoculation site at the apex of the explant, and proceeding downwards; and (ii), localized outgrowths on the stem located at various distances from the inoculation site. Histological analysis revealed that the anatomy of the outgrowths closely resembled that of knots formed in vivo ; they showed that Ps. savastanoi also diffused within the olive explants through the xylem vessels, and that the olive host reacted to pathogen invasion, possibly by producing substances of polysaccharidic and/or phenolic nature.     

Induction of root-mat symptoms on cucumber plants by Rhizobium, but not by Ochrobactrum or Sinorhizobium, harbouring a cucumopine Ri plasmid

Ochrobactrum CSL 2573, Rhizobium CSL 2411 and Sinorhizobium CSL 2611 strains harbouring the Agrobacterium cucumopine Ri plasmid (pRi), previously were shown to induce root-mat symptoms in an in vitro cucumber cotyledon assay. In whole-plant, rockwool-grown cucumber host tests Rhizobium CSL 2411 was shown to be as efficient an inducer of root-mat symptoms as the virulent Agrobacterium radiobacter strain NCPPB 4042, which also harbours a cucumopine pRi. Conjugal transfer of pRi to ingressing, avirulent Agrobacterium isolates was observed within root tissues with symptoms. Ochrobactrum CSL 2573 and Sinorhizobium CSL 2611 were not able to induce root-mat symptoms on plants. Rhizobium CSL 2411 and Ochrobactrum CSL 2573 were reisolated from inoculated plants, but Sinorhizobium CSL 2611 was not detected or isolated from inoculated plants 68 days after inoculation. It was postulated that the differences in pathogenicity observed between the in vitro and in situ host tests were caused by a lack of proper attachment to inoculated root tissues by pRi-harbouring Ochrobactrum and Sinorhizobium in the whole-plant host tests.     

The colonization processes of Myrtus communis by strains of Pseudomonas savastanoi with a differential ability to produce phytohormones

The ability to produce indole-3-acetic acid (IAA) through the indole-3-acetamide (IAM) pathway as well as cytokinins is a common trait of Pseudomonas savastanoi populations causing disease on oleander and olive. These phytohormones are required for the induction and development of an outgrowth of plant cell tissue termed a knot. However, in myrtle orchards of Sardinia (Italy), strains of P. savastanoi unable to produce cytokinins were found coexisting with cytokinin-producing strains. Data presented here show that the ability to produce IAA through the IAM pathway is also a variable trait within this population, raising questions on the exact role of these plant growth substances in the disease process on myrtle. Three P. savastanoi strains were selected based on their differential ability to produce phytohormones in vitro, and their interaction with the host was investigated over a period of 8 months using histological methods. All strains successfully invaded the infected twigs, moving systemically (unhalted by host defences) upward and downward from the inoculation point, both by completely degrading the cell walls and by taking advantage of the xylem vessels and intercellular spaces. Moreover, all strains induced the development of cankers, which slowly evolved into typical knots only on the twigs inoculated with the phytohormone-producing strains. This study further demonstrates that cytokinins and IAA are essential for knot development; moreover, it ascertains that bacterial production of cytokinins is not necessary for host colonization and for the expression of pathogenicity (i.e. the ability to cause disease) of P. savastanoi on myrtle.     

Occurrence of unusual strains of Pseudomonas syringae subsp. savastanoi on olive in central Italy1

Bacteria forming levan colonies and not producing fluorescent pigments have been isolated from olive knots and the olive phylloplane in central Italy. By their pathogenicity to olive and their morphological, biochemical and physiological features, they clearly belong to Pseudomonas syringae subsp. savastanoi.     

Factors Affecting Pseudomonas savastanoi pv. savastanoi Plant Inoculations and Their Use for Evaluation of Olive Cultivar Susceptibility

ABSTRACT Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.     

Occurrence of specific reactions induced by Pseudomonas syringae pv. syringae on bean pods, lilac and pear plants

A study on the pathogenicity of 81 strains of Pseudomonas syringae pv. syringae (PSS) isolated from 16 different hosts was conducted on lilac plants, bean pods and pear seedlings, using artificial inoculation.
Only 55 among the 81 strains induced a necrotic lesion when inoculated on lilac leaves. On bean pods, all but one of the bean isolates, and only eight strains among the 52 strains isolated from other hosts, induced typical green water-soaked lesions. On pear leaves, only pear isolates incited a typical progressive necrotic reaction, the isolates from other origins inducing no symptoms or a weak reaction limited to the inoculation point. This study indicates that in addition to the large variability observed in aggressiveness of PSS strains, host specificity occurred on bean and pear.     

Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and increases antioxidative enzyme levels in Nicotiana spp

Responses of cytokinin overproducing transgenic Nicotiana plants to infections with compatible and incompatible Pseudomonas syringae pathovars were compared. Plants used were transformed with the ipt(isopentenyl transferase) gene that catalyzes the synthesis of cytokinin. In cytokinin overproducing lines that carry the ipt gene fused to the CaMV 35S (Nt+ipt), the wound-inducible proteinase inhibitor II (Ntx+ipt), or the light-inducible Rubisco small subunit protein (Npl+ipt) promoter, development of the hypersensitive response (HR) after infection with incompatible bacteria (P. syringae pv. tomato) was significantly inhibited as compared to the untransformed (Nt) controls. Over a 12 h period following inoculation, P. syrinage pv. tomato populations were slightly reduced in leaves of the cytokinin-overproducing Nt-ipt line compared with the Nt control. When the compatible P. syringae. pv. tabaci was used to infect the ipt transformed lines, slight or no significant differences in necrosis development were observed. Following infection, the titer of P. syringae pv. tabaci increased rapidly in both the transgenic and control lines but was higher in Nt+ipt plants. Leaf superoxide dismutase and catalase enzyme activities were about 60% higher in ipt leaf extracts than in the controls. This augmented antioxidant capacity likely decreased the amount of H(2)O(2) that may be associated with the higher tolerance of plants to pathogen-induced necrosis. In addition, the Nt+ipt lines had a significantly lower molar ratio of free sterols to phospholipids. The more stable membrane lipid composition and the higher antioxidant capacity likely contributed to the suppressed HR symptoms in the cytokinin overproducing Nt+ipt plants. In conclusion, the overproduction of cytokinins in tobacco appears to suppress the HR symptoms induced by incompatible bacteria.     

Comparison among Japanese isolates of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>, causal agent of olive knot disease

Olive knot disease in Japan was first reported in Shizuoka Prefecture in 2014, and the causal agent was identified as Pseudomonas savastanoi pv. savastanoi. Subsequently, olive trees having knots were also found in Aichi and Kanagawa Prefectures in 2015, and the isolates from knots were also suspected to be P. savastanoi pv. savastanoi through preliminary examinations. Therefore, the Aichi and Kanagawa isolates were identified through comparison of isolates from three prefectures. Phylogenic analysis based on 16S rDNA and housekeeping genes (gyrB, rpoD, gltA and gap1) revealed that the isolates belonged to the same cluster as the pathotype strain, ICMP4352^PT. The iaaM, H and L genes, which are involved in promotion of symptoms, and the ina gene coding the ice nucleation protein, were detected by PCR from all the isolates. In rep-PCR (ERIC and REP) analyses, the isolates yielded DNA fragment-banding patterns that were nearly identical to that of ICMP4352^PT, but slight variations in banding patterns were observed among them. In a pathogenicity test, the isolates formed distinct knots on olive and pink jasmine. Phenotypic properties of the isolates were almost identical to those of ICMP4352^PT, with the exception of d-sorbitol utilization. Consequently, Aichi and Kanagawa isolates from olive were identified as P. savastanoi pv. savastanoi, and several genetic diversities in terms of rep-PCR were found in the Japanese population of P. savastanoi pv. savastanoi, indicating their heterogeneity.     

Phenotypic variability in different strains of Pseudomonas syringae subsp. savastanoi isolated from different hosts

One hundred and sixty strains of Pseudomonas syringae subsp. savastanoi from Olea europaea, Olea europaea var. sylvestris, Nerium oleander, Fraxinus angustifolia and Retama sphaerocarpa, and four type strains of other pathovars were studied, investigating 102 phenotypic traits, among which we include biochemical characteristics, assimilation of different carbon sources, sensitivity or resistance to antibiotics and indoleacetic acid (IAA) production. Results were analysed with an affinity dendrogram via the Jaccard coefficient. They indicate an influence of environmental factors on the formation of the 15 phenons obtained, since isolated (knot) strains from the same species but different geographical areas are segregated. Segregation, also detected in strains from different hosts within the same area, added to the pathogenicity test helps to characterise these strains as different pathovars.     

中国猕猴桃细菌性花腐病菌的鉴定

 从福建、湖南和湖北猕猴桃病花上分离到能引起花腐病的32个细菌菌株,经细菌学和BiologGN测试板测定,可以看出中国的猕猴桃细菌性花腐病菌与新西兰的猕猴桃花腐病菌、丁香假单胞菌丁香致病变种Pseudomonas syringae pv.syringae和绿黄假单胞菌P.viridiflava相似,与萨氏假单胞菌P.savastanoi和猕猴桃溃疡病菌P.syringae pv.actinidiae有更多的不同,但是DNA/DNA同源性测定结果却显示出中国的菌株可分为2个类型:第1个类型与新西兰猕猴桃花腐病菌和萨氏假单胞菌有很高的同源性,第2类型与绿黄假单胞菌有很高的同源性,说明中国菌株分别属于这2个种。第1类型来自于福建和湖北,第2类型来自于湖南。     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Relationship of genetic structure of Pseudomonas savastanoi pv. savastanoi populations from Italian olive trees and patterns of host genetic diversity

A total of 360 Pseudomonas savastanoi pv. savastanoi isolates obtained from 11 Italian olive ( Olea europaea ) cultivars grown in different provinces were assessed with repetitive PCR using short interspersed elements of the bacterial genome as primers (ERIC, BOX and REP primer sets). The population structure of the isolates was determined by using three different hierarchical clustering algorithms: UPGMA, single-link and complete-link methods. REP primers were the most discriminatory. The various fingerprints obtained from the same cultivar and locality persisted over 2 years of knot sampling. Repetitive PCR and UPGMA analysis, using the three data sets combined, revealed 20 patterns with an overall similarity of 81%, with no grouping of the isolates. The resulting dendrogram shows a bush-like topology. Similar results were obtained with the other two clustering methods. In contrast, data obtained from the literature showed that the genetic structure of olive is characterized by bifurcated dendrograms and clear grouping of cultivars. Therefore it appears that the host plant and its pathogen did not cospeciate. The strict adaptation of the bacterium to olive would represent a case of association by colonization.     

<Emphasis Type="Italic">Pantoea ananatis</Emphasis> strains are differentiated into three groups based on reactions of tobacco and welsh onion and on genetic characteristics

Ninety-six strains of Pantoea ananatis were isolated from 14 plant species including melon, rice, tea and other crops of economic importance. They were classified into three groups (group I, II, III) based on a welsh onion stabbing assay, tobacco infiltration test, and polymerase chain reaction to detect indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). Group Ι strains were characterized as causing significant blight symptom on welsh onion and inducing a hypersensitive response (HR)-like reaction on tobacco leaves after 36–48 h and encompassed 20 isolates from foxtail millet, hydrangea, pineapple, river water and rice. These 20 isolates did not possess iaaM, iaaH, or etz genes. Group II, consisting of 34 melon isolates, harbored iaaM, iaaH and etz genes, but did not cause either blight on welsh onion or HR-like reaction on tobacco. Group III strains did not have the iaaM, iaaH, and etz genes, nor did they cause any reaction on welsh onion or tobacco. The 42 strains in group III were isolated from bamboo grass, Chinese silver grass, citrus, dogwood, melon, mugwort, silk tree, sweet corn, tea and welsh onion. Representative strains of the three groups were tested for pathogenicity on melon and rice. Group Ι strains caused palea browning on rice but not internal fruit rot on melon. On the contrary, group II strains did not cause disease on rice but caused internal fruit rot on melon. Group III strains were not pathogenic on rice or melon. These results suggested that the host range of P. ananatis may be predicted by the reactions of welsh onion and tobacco and detection of iaaM, iaaH and etz genes. These tools may serve as rapid tests to identify the pathogenicity groups of P. ananatis.     

Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.