The type III effector PthG of <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> pv. <Emphasis Type="Italic">gypsophilae</Emphasis> modifies host plant responses to auxin,cytokinin and light

Pantoea agglomerans pvs. gypsophilae and betae are related gall-forming bacteria. While P. agglomerans pv. beta initiates gall formation on both beet and gypsophila, the gypsophila pathovar causes gall formation only on gypsophila. PthG is a type III effector determining host range of these pathogens, initiating the hypersensitivity response in beet, but is a virulence factor in gypsophila. The role of PthG and its mode of action in pathogenicity remain unclear. Transgenic Nicotiana tabacum plants expressing pthG were created. PthG over-expression was often lethal, and surviving pthG-bearing lines showed morphological and developmental abnormalities such as leaf deformation and abnormal vascular branching, dwarf stature, loss of apical dominance, seedling apical meristem loss, rapid germination, reduced fertility, plants which cease growth for several weeks later producing a new lateral shoot, and loss of endophyte resistance (bearing unusual saprophyte populations). Some transformants required light for seed germination and showed rapid seedling greening. In in vitro assays PthG expression modified responses to auxin and cytokinin, inhibiting root and shoot production but not callus formation. In vitro differentiation responses to light were modified by PthG expression. This effector may interfere in the plant auxin signalling pathways resulting in higher observed auxin and ethylene levels, and subsequent blockage of root and shoot development. Apparently PthG tunes the host response to high hormone levels, changing the developmental response. Since shoot and root development are delayed, we hypothesize that callus/gall formation is supported by this activity. However, interference by PthG with hormone and light signalling does not explain all the responses observed in pthG-bearing lines.     

Detection of Erwinia herbicola pv. gypsophilae in gypsophila plants by PCR

Three PCR primer pairs, based on the cytokinins (etz) or IAA biosynthetic genes, were used for detecting Erwinia herbicola pv. gypsophilae in Gypsophila paniculata plants. The primers were specific to all gall-forming E. herbicola strains and distinguished them from saprophytic strains associated with gypsophila plants or from other gall-forming bacteria. In pure culture of the pathogen, less than one bacterial cell was detected with nested PCR using the etz primers - an increase of 100-fold in sensitivity as compared with single-round PCR. In the presence of plant extract a reduction of tenfold in sensitivity was observed by nested PCR. When cells were grown on a semi-selective medium prior to PCR (Bio-PCR), five cells from pure culture of the pathogen were detected. The bacteria could be detected by nested-PCR or Bio-PCR in symptomless gypsophila cuttings after 7 days. The Bio-PCR procedure described in this study can be used to establish disease-free nuclear stock of mother plants of gypsophila.     

The bionomics ofLiriomyza Trifolii on Gypsophila and Bean Leaves

A study was carried out to determine the distribution of feeding and oviposition punctures made byLiriomyza trifolii Burgess on gypsophila(Gypsophila paniculata ) and bean(Phaseolus vulgaris) leaves, and the development times of the immature stages on these plants. The absolute number of punctures per leaf area was much greater on bean leaves than on gypsophila leaves. The number of mines per leaf was also higher on beans than on gypsophila, but the ratio of mines/punctures was higher on gypsophila. Gypsophila leaves were punctured on both sides, but most of the punctures were on the distal third of the upper leaf side. On beans the punctures were dispersed uniformly on the upper side of the leaf. The larval period (±SE) at temperatures of 20, 25 and 30°C was 9.9±0.2, 4.4±0.1 and 3.7±0.1, and 5.5±O. l, 3.7±0.1 and 2.4±0.1 days, on gypsophila and bean, respectively. The pupal period at 17, 20, 25 and 30°C lasted 19.9±0.2, 14.7±0.1, 10.4±0.1 and 7.8±0.1 days, respectively.     

A strain of Erwinia herbicola pathogenic on Gypsophila paniculata

A yellow pigmented, rod-shaped, Gram negative, peritrichously flagellated, fermentative bacterium was isolated from galls on the stems of the ornamental plant,Gypsophila paniculata. The galls appeared to be similar to those described on the plant by Brown (1934). The isolate proved pathogenic when inoculated into healthy stems; it was identified as a strain ofErwinia herbicola (Lohnis) Dye. It is suggested that the organism should be namedE. herbicola f. sp.gypsophilae.Samenvatting Een geel gepigmenteerde, staafvormige, Gram-negatieve, fermentatieve bacterie met petritriche flagellen werd geïsoleerd uit gallen op de stengels van de sierplantGypsophila paniculata. De ziektesymptomen kwamen overeen met de door Brown in 1934 van deze plant beschreven gallen. Het isolaat bleek na inoculatie in gezonde stengels pathogeen te zijn en werd geïdentificeerd als een stam vanErwinia herbicola. Voorgesteld wordt dit organism aan te duiden alsE. herbicola f. sp.gypsophilae.     

Hostplants ofAgrobacterium gypsophilae

Symptoms obtained after either natural or artificial infection on roses, carnations and cacti byAgrobacterium gypsophilae are described, as well as a modification of the isolation method for this pathogen.     

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.     

A hypersensitive response was induced by virulent bacteria in transgenic tobacco plants overexpressing a plant ferredoxin-like protein (PFLP)

The hypersensitive response (HR) displayed by resistance plants against invading pathogens is a prominent feature of an incompatible plant pathogen interaction. It has been shown that tobacco cell cultures transgenic for a plant ferredoxin-like protein (PFLP) that functions as an electron acceptor of Photosystem I increased harpin-mediate HR. In this work we report increased bacterial disease resistance of pflp transgenic tobacco. Compared to the controls, four distinctive characteristics were found in the pflp-transgenics after inoculation with virulent bacterial cells Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci: (i) instead of typical disease symptoms, an HR-like necrosis was observed; (ii) the proliferation of the virulent pathogen was highly retarded; (iii) the expression of hsr203j, an HR marker gene, was apparently induced; (iv)H₂O₂ accumulation was induced immediately. Together, those results demonstrate that enhanced production of PFLP in the transgenic plant conditions the induction of a hypersensitive response during compatible pathogen attack.     

Interaction between silicon amendment,bacterial wilt development and phenotype of Ralstonia solanacearum in tomato genotypes

Silicon amendment significantly reduced bacterial wilt incidence expressed as area under disease progress curve for tomato genotypes L390 (susceptible) by 26.8% and King Kong2 (moderately resistant) by 56.1% compared to non-treated plants grown in hydroponic culture. However, wilt incidence in silicon-treated plants of genotype L390 reached 100% at 13 days post-inoculation (dpi), while in genotype King Kong2, plant death was retarded by 6 days, with 20% reduction of final wilt incidence. Bacterial numbers were significantly lower in silicon-treated compared to non-treated plants in King Kong2 at 2 dpi in midstems and in all organs at 5 dpi, and in Hawaii 7998 (resistant) in all organs at 2 dpi. Differences between genotypes were obvious on midstem level (5 dpi), where bacterial populations were generally significantly lower compared to roots. Increased tolerance was observed in genotypes L390 and King Kong2 with silicon treatment.Silicon accumulated in roots and was low in stems and leaves. Inoculation with Ralstonia solanacearum did not significantly affect silicon uptake and distribution. Negative correlations between root silicon content and bacterial numbers of midstems in genotypes Hawaii 7998 and King Kong2 suggested an induced resistance. Indications for an influence of host genotype and silicon treatment on the phenotypic conversion of R. solanacearum strain To-udk2-sb from fluidal to non-fluidal colonies in planta were observed.This is the first report on the effect of silicon on a bacterial disease and in a silicon-non-accumulator plant.     

Compost enhances plant resistance against the bacterial wilt pathogen Ralstonia solanacearum via up-regulation of ascorbate-glutathione redox cycle

The interactions between the pathogen Ralstonia solanacearum and potato Solanum tuberosum plants were studied to investigate the reactive oxygen species metabolic system and ascorbate (ASC)-glutathione (GSH) redox cycle in response to compost application. Single potato eyepieces were germinated and grown in pots containing sandy soil with or without compost at a rate of 7.5 g kg^?1 soil. Non-compost- and compost-treated plants (CTP) were inoculated with R. solanacearum 25 days after planting and then analyzed after 10 days, unless otherwise stated. The present results revealed that pathogen infection caused a remarkable decrease in plant growth related parameters and productivity and an increase in disease incidence. However, under these conditions compost had substantially improved plant growth and decreased disease incidence and bacterial population. R. solanacearum resulted in significant enhancement in the activities of NADPH oxidase, lipoxygenase, the production rate of superoxide and hydroxyl radicals, levels of hydrogen peroxide, membrane lipid peroxidation, and protein oxidation indicating the induction of oxidative stress in potato roots. However, the pathogen-mediated enhancement in indices of oxidative stress was considerably decreased by compost application, which enhanced the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) in infected potato plants, implying a better ROS-scavenging activity. Data also indicated that there were general increases in ASC and GSH content in infected compost treated plants, but non-compost treated ones significantly had lower levels of such redox metabolites. In addition, significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulphide) were generally found in CTP than in non-compost treated-ones. The obtained results suggest that compost provides effective protection against the Ralstonia bacterial pathogen via up-regulation of the capacity of the ASC-GSH cycle and modulation of the cellular redox status, thereby eliminating ROS damage and sustaining membrane stability.     

Evaluation of a DNA probe for the detection of Erwinia herbicola strains pathogenic on Gypsophila paniculata

A 7.5-kb DNA fragment carrying genes for indole acetic acid biosynthesis in Erwinia herbicola pv. gypsophilae was used as a DNA probe to detect gall-forming E. herbicola strains. A quick colony hybridization procedure was developed to detect E. herbicola pv. gypsophilae in cuttings of gypsophila, and was compared with ELISA and pathogenicity tests. The sensitivity of the colony hybridization procedure was sufficient to detect less than 100 colony-forming units after enrichment culture. In mixed cultures, with saprophytes associated with the gypsophila plant, about 10⁴ CFU/ml of the pathogen were detectable. The colony hybridization is specific to gall-forming E. herbicola strains, and is relatively easy to perform. The advantages of using the colony hybridization procedure for practical purposes, compared with ELISA and pathogenicity tests, are discussed.     

Molecular diagnostic procedures for production of pathogen-free propagation material

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.     

Promotion of growth, health and stress tolerance of Styrian oil pumpkins by bacterial endophytes

Substantial yield losses of Styrian oil pumpkin caused by the fungus Didymella bryoniae and bacterial pathogens were recently reported. Here we applied bacterial endophytes with a broad antagonistic activity to pumpkin plants by seed priming. Effects of the bacterial inoculants with and without chemical seed treatments on plant growth and health were evaluated during three different field trials in two consecutive years (2010 and 2011). Biological seed treatments strongly supported the germination of pumpkin seeds. In 2010, the germination of the biologically treated seeds was comparable to the rate following a chemical treatment; whilst in 2011 effects of biological seed treatments were more obvious, including an increased emergence rate up to 109?% by Serratia plymuthica S13. Furthermore, tolerance against desiccation stress was observed for Serratia as well as for Lysobacter gummosus L101 treatment. The biological treatment showed different effects against fungal diseases: no effect on fruit rot was observed, whereas powdery mildew could be significantly suppressed by Paenibacillus polymyxa PB71 and L. gummosus L101 in 2010. In addition, both strains led to reproducible increases in harvest yields. In this study, we found bacterial endophytes suitable as inoculants for plant growth promotion, biocontrol, as well as for enhancing stress tolerance in Styrian oil pumpkins.     

A paucity of bacterial root diseases: Streptomyces succeeds where others fail

There are relatively few bacterial diseases of roots, in comparison to those of aerial plant tissues. Numerous species and pathovars of Pseudomonas,Erwinia and Xanthomonas are important pathogens of leaf and stem tissue on dozens of plant families but these bacterial genera only infrequently attack roots or other underground plant structures. In contrast, there is a growing list of Streptomyces species that are very effective root pathogens. These filamentous, Gram-positive bacteria can cause scab, rot and gall diseases of plant roots and other underground plant structures. The best known pathogenic Streptomyces species is S. scabiei. Horizontal transfer of pathogenicity genes among diverse scab-causing streptomycetes appears to explain the emergence of several new plant pathogens over the last half century. It is proposed that the ability to penetrate plant tissue is essential for successful root infection as there are few natural openings in roots. In contrast, leaves have many natural openings that allow bacteria access to the interior tissues. Thaxtomin, a phytotoxin produced by many plant pathogenic streptomycetes, appears to aid penetration of developing plant tissues by inhibiting primary cell wall development.     

Induction of a viable but not culturable (VBNC) state in some Pseudomonas syringae pathovars upon exposure to oxidation of an apoplastic phenolic,acetosyringone

Acetosyringone is a phenolic metabolite often found in plant apoplasts. Its oxidation by hydrogen peroxide and peroxidase results in a prolonged increase in the redox potential of the reaction mixture, similar to redox increases observed in tobacco suspension cells upon treatment with incompatible bacteria. Since high redox potentials, being oxidative, are generally detrimental to bacteria, the effect of acetosyringone oxidation on bacterial viability was examined. Pseudomonas syringae pv. syringae was added to reaction mixtures containing acetosyringone, hydrogen peroxide and peroxidase and samples were removed to determine viability by dilution plating. Initial studies were done with low bacterial concentrations, 10⁵ CFU ml⁻¹, to ensure that scavenging of H₂O₂ was negligible and did not interfere with the reaction mixture. No colonies were formed by bacteria that had been added to reaction mixtures with acetosyringone ranging from 25 to 100 μΜ. Examination of the bacteria by microscopy and flow cytometry, using fluorescent stains that indicate bacterial membrane integrity, suggested that these bacteria had maintained their membrane integrity. In addition they were able to respire based on oxygen uptake. When bacteria were added to on-going reaction mixtures at a time point after the prolonged redox response, the CFU ml⁻¹ increased indicating that a stable reaction product was not responsible for the non-culturability bioactive effect. Other bacterial isolates, P. s. pv. tabaci and Pseudomonas fluorescens, were less susceptible to the bioactive effect of the acetosyringone oxidation. Other phenolics were tested and had lesser degrees of bioactivity and in some cases reduced the bioactivity of acetosyringone oxidation. The ‘viable but non-culturable’ (VBNC) state of the bacteria in this study is compared to that described for other medical and plant pathogens.     

Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley,oat and wheat

When barley, wheat or oat leaf epidermal cells were attacked by their appropriate forma specialis (f.sp.) of Blumeria graminis DC. Speer (f.sp. hordei, tritici and avenae, respectively), many attempted penetrations succeeded, functional haustoria were formed and very few plant cells died. When attacked by either of the two possible inappropriate ff.spp., penetration attempts failed in association with papilla deposition by epidermal cells, attacked cells died, or if visible haustoria were formed the plant cell died very soon afterwards. Double inoculation experiments were performed where each cereal species was first attacked by its appropriate f.sp., as inducer, and later by the different ff.spp. as challenger. Infection by the appropriate inducer profoundly affected cellular responses to challenger attack. Suppression of defensive responses was dramatic within epidermal cells containing the inducer haustorium, evident to some extent in adjacent cells, but undetectable at two cells distance. Suppression of penetration resistance allowed most challenger attacks, even by inappropriate ff.spp., to form a haustorium. Furthermore, death of penetrated epidermal cells was also suppressed so that haustoria of the inappropriate ff.spp. functioned to support colony development. In oat, delayed epidermal cell death prevented full colony development by inappropriate ff.spp., but in barley and wheat, no cell death was apparent by four days after inoculation and colonies of the inappropriate ff.spp. produced extensive hyphae, secondary haustoria and conidial chains.     

Narrow sense heritability estimates of bacterial leaf spot resistance in pseudo F2 (F1) population of mulberry (Morus spp.)

Bacterial leaf spot incited by Xanthomonas campestris pv. mori is a devastating foliar disease of mulberry reported globally. Host plant resistance is the most sustainable and economic control measure but so far unexplored. Highly heterozygous plant behaviour and scant genetic information of bacterial leaf spot resistance limits a targetted breeding approach in mulberry. In the present research eight pseudo-F₂(F₁)full-sib progenies derived from selected resistant and susceptible sources were evaluated symptomatically for bacterial leaf spot resistance under natural disease occurrence in 2008 and 2009. Significant variation for bacterial leaf spot resistance was observed in the parents and progenies. Broad sense heritability estimate (0.9) indicates that selection of resistant genotypes can be useful for exploitation in future advanced breeding programs for mulberry. High narrow sense heritability estimates (0.76)[2008] and (0.79)[2009] suggest additive gene effects for the disease resistant trait. The continuous frequency distribution of diseases severity across the progenies indicates that bacterial leaf spot resistance in mulberry may be inherited quantitatively.