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.     

Genetic and phenotypic diversity of Mediterranean populations of the olive knot pathogen,Pseudomonas savastanoi pv. savastanoi

Pseudomonas savastanoi pv. savastanoi (Psav) is a member of P. syringae sensu lato, and causes olive knot disease, a disease first reported over 2000 years ago. Analysing 124 isolates of Psav from 15 countries by rep‐PCR, the population genetic structure of Psav was investigated. A total of 113 distinct fingerprints were detected. Cluster analysis revealed the existence of two clusters and four subclusters. These clusters were associated with the geographic origin of isolates, which in turn correspond to historic human migration events and trade routes across the Mediterranean Sea. In contrast, multilocus sequence typing (MLST) of 2788 bp of the gapA, gltA, gyrB and rpoD genes found only one variable site among 77 representative isolates. Virulence variation was observed within the Psav population, with the most virulent strains generating knots that had a weight that was 10‐fold greater than those generated by the least virulent strains. Taken together, these data suggest that today's Psav population is the result of clonal expansion of a single strain, that moderate migration of the pathogen occurred between countries, and that changes in virulence arose during its evolution.     

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.     

Dissemination of Pseudomonas savastanoi pv. savastanoi populations and subsequent appearance of olive knot disease

Pseudomonas savastanoi pv. savastanoi (Psv) is the causal agent of olive knot disease. The bacterium survives epiphytically and gains ingress through new wounds where infections and colonization result in knot formation. The natural spread of the bacterium and the subsequent appearance of the disease in olive orchards is poorly understood. The aim of this study was to monitor Psv epiphytic populations in inoculated plants with knots versus non‐inoculated healthy trees within the same orchard over four years. Additionally, disease severity was measured in both inoculated and non‐inoculated control trees. Epiphytic Psv populations moved from inoculated to non‐inoculated trees, although average Psv populations were higher in inoculated trees. Olive knot severity increased over the course of the study in all treatments and cultivars, with all plants reaching a high level of disease by the end of the study. However, the delay in the onset of disease was longer in non‐inoculated than in inoculated trees. Molecular typing of Psv isolates recovered from non‐inoculated control trees confirmed that they were similar to the inoculated strain. These data demonstrate that Psv can move over short distances in olive orchards through dissemination of epiphytic bacteria and suggest a relationship between the presence of epiphytic Psv and the number of knots on trees.     

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.     

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.     

Pseudomonas syringae pv. actinidiae: chemical control,resistance mechanisms and possible alternatives

Pseudomonas syringae pv. actinidiae (Psa) is a Gram‐negative bacterium that causes the bacterial canker of both green (Actinidia deliciosa) and yellow (Actinidia chinensis) fleshed kiwifruit. Since the emergence of an economically devastating Psa outbreak in Japan in the 1980s, the disease took a contagious turn causing severe economic loss to kiwifruit industries in Italy, South Korea, Spain, New Zealand and other countries. Research shows that the pathogenic strains isolated from different infected orchards vary in their virulence characteristics and have distinct genes coding for the production of different toxins. The global Psa outbreak has activated research around the world on developing efficient strategies to contain the pandemic and minimize loss to the kiwifruit industry. Chemical and biological control options, orchard management and breeding programmes are being employed in this global effort. Synergy between different disease control strategies has been recognized as important. Phytotoxicity, resistance development and regulatory measures in certain countries restrict the use of copper compounds and antibiotics, which are otherwise the mainstay chemicals against bacterial plant diseases. Therefore, because of the limitations of existing chemicals, it is important to develop novel chemical controls against Psa. Antimicrobial peptides, which are attractive alternatives to conventional antibiotics, have found promising applications in plant disease control and could contribute to expanding the chemical control tool box against Psa. This review summarizes all chemical compounds trialled so far against Psa and provides thoughts on the development of antimicrobial peptides as potential solutions for the future.     

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.     

Application of Pseudomonas fluorescens isolates to wheat as potential biological control agents against take-all

Two isolates of Pseudomonas fluorescens (2–79 and 13–79) from the USA were evaluated in the UK as biological control agents against Gaeumannomyces graminis var. tritici , the cause of take-all in wheat. Biological control agents were applied as seed coatings in carboxymethyl cellulose (CMC) to seven wheat trials sown in 1987 and 1988 on fen peat and clay soils, and as peat-based and microgranule formulations in one of these trials. In a trial of spring wheat on fen peat, all treatments with biological control agents reduced the percentage take-all infection of crown roots and seminal roots, but the effects of only one isolate were statistically significant ( P <0·05). Effects of biological control agents on infection rates in five other trials were not significant. In the trial in which application methods were compared, peat-based inoculum initially appeared most effective but none of the treatments reduced take-all significantly throughout the season. Application of biological control agents was associated with yield increases in several trials; these were not consistently associated with effects on take-all. These results suggest that the isolates of P. fluorescens have potential to reduce take-all and increase yields of wheat in the UK, but the beneficial effects are inconsistent. There is a need to develop isolates which reliably control severe take-all in a variety of soil types.     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.     

Use of green leaves from olive trees as soil amendment for the control of Meloidogyne1

Plant extracts have been used against nematodes mainly in the third-world countries. The possibility of using leaves of olive trees for reducing populations of nematodes (Meloidogyne spp.) in the soil was investigated in the present work. The root-knot incidence in tomato roots was reduced as well as the presence of nematodes in the soil. The methanol extract of the leaves inhibited hatching of the eggs almost completely.     

上海地区大豆细菌性疫病发生危害与防治

大豆细菌性疫病又称大豆细菌性斑点病[Pseu-domonas syringaepv.glycinea(Coerper)Young,Dye&Wilkie],是我国和世界大豆产区主要病害,导致大豆产量损失和品质下降。据报道,在适宜发病条件下此病可使大豆减产18%~22%,一般减产20%左右,最高达50%以上。近年来,随着上海地区菜用大豆种植面积的不断扩大,发生了大豆细菌性疫病。为防治该病进行了田间流行调查和发生特点观测。1病害发生情况2002~2004年,对上海郊区的大豆种植地进行了调查,根据大豆细菌性疫病危害程度进行病级标准划分。0级:无症;1级:复叶上有1~5个病斑;2级:复叶上有6~10个病斑;3…     

Field evaluation of treatments for the control of the bacterial apical necrosis of mango (Mangifera indica) caused by Pseudomonas syringae pv. syringae

Bacterial apical necrosis is a critical disease in the main production area of mango in Europe. It is caused by Pseudomonas syringae pv. syringae, and produces necrotic lesions on mango buds and leaves, causing severe yield losses due to a decrease of flowering and fruit set. A field study to evaluate control treatments against bacterial apical necrosis was carried out during three seasons on mango trees cv. Tommy Atkins in Huelva (Spain). Experimental treatments included Bordeaux mixture, fosetyl-Al, acibenzolar-s-methyl, gibberelic acid, silicon gel, a mixture between acibenzolar-S-methyl and Bordeaux mixture, and combined applications of fosetyl-Al with Bordeaux mixture or silicon gel. The treatments which caused a consistent reduction in bacterial apical necrosis symptoms at similar levels to the conventional treatment with Bordeaux mixture, were the plant resistance activator acibenzolar-S-methyl and the phosphonate derivative fosetyl-Al applied singly or in combination with other compounds, which could be alternative treatments. These treatments showed a significant decrease in the necrotic buds and/or leaves numbers; however, minor differences in P. syringae-like population levels were observed. The analysis of the inhibitory and bactericidal concentrations of cupric compounds against P. syringae strains isolated from mango tissues suggests that the commercial copper-based treatments with Bordeaux mixture used in the management of mango crops do not work in a bactericidal mode of action.     

Comparative genomics‐informed design of two LAMP assays for detection of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae and discrimination of isolates belonging to the pandemic biovar 3

The aim of this study was to develop a rapid, sensitive and reliable field‐based assay for detection of the quarantine pathogen Pseudomonas syringae pv. actinidiae (Psa), the causal agent of the most destructive and economically important bacterial disease of kiwifruit. A comparative genomic approach was used on the publicly available Psa genomic data to select unique target regions for the development of two loop‐mediated isothermal amplification (LAMP) assays able to detect Psa and to discriminate strains belonging to the highly virulent and globally spreading Psa biovar 3. Both LAMP assays showed specificity in accordance with their target and were able to detect reliably 125 CFU per reaction in less than 30 min. The developed assays were able to detect the presence of Psa in naturally infected kiwifruit material with and without symptoms, thus increasing the potential of the LAMP assays for phytosanitary use.     

春雷霉素和噻霉酮对番茄斑疹病菌联合毒力及防病效果

通过平板菌落计数法测定了春雷霉素和噻霉酮对引起番茄斑疹病的丁香假单胞杆菌番茄致病变种(Pseudomonas syringae pv.tomato)的毒力及两种药剂混配的联合毒力。结果表明:春雷霉素和噻霉酮对番茄斑疹病菌具有抑制作用,EC50值分别为5.32μg/mL和2.66μg/mL;联合毒力的测定结果表明:春雷霉素和噻霉酮质量比1∶1的配比增效作用最强。田间试验结果表明:春雷霉素、噻霉酮及春雷霉素与噻霉酮(1∶1)混剂,田间防治效果均达到80%以上,混剂防治效果显著高于单剂。     

Effects of para-aminobenzoic acid on bacterial speck symptom development and Pseudomonas syringae pv. tomato populations in tomato leaves

Para-aminobenzoic (PABA) is reported to induce resistance against a range of plant pathogens in different crops in a salicylic acid-dependent manner. However, factors affecting its efficacy are not well understood. Foliar PABA applications on tomato seedlings reduced lesion incidence caused by Pseudomonas syringae pv. tomato (Pst) in a dose-dependent manner in distal leaves up to 18 mM under controlled environment conditions, but only three out of six commercial processing tomato cultivars tested showed a response to PABA. Leaves in direct contact with 9 and 18 mM PABA of both PABA-responsive and PABA-nonresponsive cultivars showed phytotoxicity. In a PABA-responsive cultivar, one, two and three PABA applications were equally effective at reducing lesion incidence in distal leaves, but the duration of control only persisted for approximately 7 days. Although PABA application reduced lesion incidence in distal leaves, the Pst population in leaves was unaffected. Lesions on PABA-treated plants were larger than nontreated plants, and thus the proportion of leaf surface area with lesions was unaffected by PABA treatment. In in vitro assays, 18 and 72 mM PABA produced zones of inhibition against Pst 15 and 50% larger than the ethanol control, demonstrating direct antimicrobial effects of PABA. PABA application did not affect symptom development in a mixed infection of Pst or Xanthomonas spp. in one field experiment with a PABA-responsive cultivar. Further research is needed to understand why PABA was unsuccessful in the field before it is to be used as a practical disease management tool for foliar bacterial diseases of tomato.

     

Pathogenesis and control of bacterial speck,Pseudomonas syringae pv. tomato,on tomato*

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, was observed to cause severe symptoms, especially on protected tomato crops, during the winter season in the coastal area of Lebanon. This study was conducted to investigate the aetiology and pathogenesis of the bacterium involved and the efficacy of different chemicals for the control of the disease. Biochemical, physiological and pathological tests verified the identity of the bacterium involved as P. s. tomato. Periodic histological sectioning of inoculated tomato leaves showed that bacterial cells resided and multiplied in depressions and around trichome bases for 24 h before penetration through stomata and trichome basal cells. The bacteria invaded intercellular spaces and caused cell lignification, collapse and shrinkage, 48 h after inoculation. Necrotic lesions filled with bacterial masses and collapsed lignified cells were readily observable at and after 72 h. No detectable histological changes were observed in the yellow halo region surrounding the necrotic leaf specks. A thermostable toxin was produced by the pathogen and is involved in chlorotic symptom expression. An antibiotic mixture of streptomycin + oxytetracy‐cline was most effective in controlling infections followed by copper oxychloride + mancozeb, tribasic copper sulphate + sulphur, copper oxychloride and copper oxychloride + zineb.