Blight Bacterium Research Articles

Indole Alkaloid Production by the Halo Blight Bacterium Treated with the Phytoalexin Genistein.

When Pseudomonas savastanoi pv. phaseolicola, the bacterium that causes halo blight, induces hypersensitive immunity in common bean leaves, salicylic acid and phytoalexins accumulate at the site of infection. Both salicylic acid and the phytoalexin resveratrol exert antibiotic activities and toxicities in vitro, adversely disrupting the P. savastanoi pv. phaseolicola proteome and metabolism and stalling replication and motility. These efficacious properties likely contribute to the cessation of bacterial spread in beans. Genistein is an isoflavonoid phytoalexin that also accumulates during bean immunity, so we tested its antibiotic potential in vitro. Quantitative proteomics revealed that genistein did not induce proteomic changes in P. savastanoi pv. phaseolicola in the same way that salicylic acid or resveratrol did. Rather, a dioxygenase that could function to metabolize genistein was among the most highly induced enzymes. Indeed, high-throughput metabolomics provided direct evidence for genistein catabolism. Metabolomics also revealed that genistein induced the bacterium to produce indole compounds, several of which had structural similarity to auxin. Additional mass spectrometry analyses proved that the bacterium produced an isomer of the auxin indole-3-acetic acid but not indole-3-acetic acid proper. These results reveal that P. savastanoi pv. phaseolicola can tolerate bean genistein and that the bacterium likely responds to bean-produced genistein during infection, using it as a signal to increase pathogenicity, possibly by altering host cell physiology or metabolism through the production of potential auxin mimics.

Label-Free Surface-Enhanced Raman Scattering Detection of Fire Blight Pathogen Using a Pathogen-Specific Bacteriophage.

Fire blight is one of the most devastating plant diseases, causing severe social and economic problems. Herein, we report a novel method based on label-free surface-enhanced Raman scattering (SERS) combined with an Erwinia amylovora-specific bacteriophage that allows detecting efficiently fire blight bacteria E. amylovora for the first time. To achieve the highest SERS signals for E. amylovora, we synthesized and compared plasmonic nanoparticles (PNPs) with different sizes, i.e., bimetallic gold core-silver shell nanoparticles (Au@AgNPs) and monometallic gold nanoparticles (AuNPs) and utilized the coffee-ring effect for the self-assembly of PNPs and enrichment of fire blight bacteria. Furthermore, we investigated the changes in the SERS spectra of E. amylovora after incubation with an E. amylovora-specific bacteriophage, and we found considerable differences in the SERS signals as a function of the bacteriophage incubation time. The results indicate that our bacteriophage-based label-free SERS analysis can specifically detect E. amylovora without the need for peak assignment on the SERS spectra but simply by monitoring the changes in the SERS signals over time. Therefore, our facile method holds great potential for the label-free detection of pathogenic bacteria and the investigation of viral-bacterial interactions.

Rice ferredoxin OsFd4 contributes to oxidative stress tolerance but compromises defense against blight bacteria

Ferredoxins (Fds) in plastids are the most upstream stromal electron receptors shuttling electrons to downstream metabolic systems and function in various physiological processes of dicots, but their roles in monocots’ response to stresses are still unclear. In this study, the functions of OsFd4, the major non-photosynthetic type Fd in rice, were characterized under oxidative stress and Xanthomonas oryzae pv. oryzae (Xoo) infection. OsFd4-knockout mutants displayed no defects in key agronomic traits and blast resistance, but were more sensitive to hydrogen peroxide (H2O2) treatment than the wild type. Transient expression of OsFd4 alleviated H2O2-induced rice cell death, suggesting that OsFd4 contributes to rice tolerance to exogenous oxidative stress. Deletion of OsFd4 enhanced rice immune responses against Xoo. OsFd4 formed a complex in vivo with itself and OsFd1, the major photosynthetic Fd in rice, and OsFd1 transcripts were increased in leaf and root tissues of the OsFd4-knockout mutants. These results indicate that OsFd4 is involved in regulating rice defense against stresses and interplays with OsFd1.

On-site applicable diagnostic fluorescent probe for fire blight bacteria

Fire blight is a representative plant infection that contaminates edible plants and causes socio-economic problems in agricultural and livestock industries globally. It is caused by the pathogen Erwinia amylovora (E.amylovora) creates lethal plant necrosis and spreads rapidly across plant organs. We newly disclose the fluorogenic probe B-1 for real-time on-site detection of fire blight bacteria for the first time. B-1 exhibited no emission signals but manifested bright emission properties in the presence of fire blight bacteria. Based on these features, fluorescence imaging of the fire blight bacteria and its real-time detection from the infected host plant tissues were conducted. The detection limit against E.amylovora was 102CFU/mL, which had excellent sensitivity. The fluorogenic probe-based on-site diagnostic technology was supplemented by introducing a new portable UV device. This work holds enormous potential to be a new advanced tool for detecting fire blight in agricultural and livestock industries.

Evaluation of Bacterial Perpetuation Assays and Plant Biomolecules Antimicrobial Activity against Cotton Blight Bacterium Xanthomonas citri subsp. malvacearum; An Alternative Source for Food Production and Protection.

Cotton (Gossypium hirsutum) is a global cash crop which has gained importance in earning foreign exchange for each country. Bacterial blight caused by Xanthomonascitri subsp. malvacearum (Xcm) has been a seriousdisease in Pakistan’s cotton belt on multiple occasions. Bacterium was isolated and identified through various biochemical and diagnostic tests wherehypersensitivity reaction, Gram staining, KOH (potassium hydroxide), catalase, starch hydrolysis, lecithinase and Tween 80 hydrolysis tests confirmed bacterium as Gram-negative and plant pathogenic. Xcm perpetuation assays wereevaluated on various cotton varieties under glasshouse conditions in completely randomized design by three different methods, wherein the scratch method proved to be the best upon CIM-496 and showed 83.33% disease incidence as compared with the other two methods, where Bt-3701 responded with 53.33% incidence via the spray gun method, and 50% with the water splash method on CIM-616, as compared with the control. Similarly, for disease severity percentage, Bt-3701 was pragmatic with 47.21% through scratch method, whereas, in the spray gun method, 45.51% disease severity was noted upon Bt-802, and 31.27% was calculated on Cyto-179 through the water splash method. Owing to the unique antibacterial properties of aqueous plant extracts, the poison food technique showed Aloe vera, Mentha piperita, Syzygiumcumini and Azadirachta indica with 17.77, 29.33, 18.33 and 20.22 bacterial colonies counted on nutrient agarmedium petri plate, respectively, as compared with the control. Measurement of the inhibition zone by disk diffusion technique showed Mentha piperita, Syzygiumcumini, Citrus limon, Moringa oleifera and Syzygium aromaticum to present the most promising results by calculating the maximum diameter of the inhibition zone, viz., 8.58, 8.55, 8.52, 8.49 and 8.41 (mm), respectively, at the highest tested concentration (75 ppm, parts per million) compared with the control. It is probable that the decoction’s interaction with the pathogen population on the host plant will need to be considered in future experiments. However, at this moment, more research into the effective management of cotton bacterial blight by plant extracts in terms of concentration determination and development of biopesticides will provide future avenues to avoid environmental pollution.

Loss function of SL (sekiguchi lesion) in the rice cultivar Minghui 86 leads to enhanced resistance to (hemi)biotrophic pathogens

BackgroundSerotonin, originally identified as a neurotransmitter in mammals, functions as an antioxidant to scavenge cellular ROS in plants. In rice, the conversion of tryptamine to serotonin is catalyzed by SL (sekiguchi lesion), a member of cytochrome P450 monooxygenase family. The sl mutant, originated from rice cultivar Sekiguchi-asahi, exhibits spontaneous lesions, whereas its immune responses to pathogens have not been clearly characterized.ResultsHere we identified three allelic mutants of SL in an indica rice restore line Minghui 86 (MH86), named as sl-MH-1, − 2 and − 3, all of which present the typical lesions under normal growth condition. Compared with those in MH86, the serotonin content in sl-MH-1 is dramatically decreased, whereas the levels of tryptamine and L-trytophan are significantly increased. The sl-MH-1 mutant accumulates high H2O2 level at its lesion sites and is more sensitive to exogenous H2O2 treatment than the wild type. When treated with the reductant vitamin C (Vc), the lesion formation on sl-MH-1 leaves could be efficiently suppressed. In addition, sl-MH-1 displayed more resistant to both the blast fungus and blight bacteria, Pyricularia oryzae (P. oryzae, teleomorph: Magnaporthe oryzae) and Xanthomonas oryzae pv. Oryzae (Xoo), respectively. The pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses, like reactive oxygen species (ROS) burst and callose deposition, were enhanced in sl-MH-1. Moreover, loss function of SL resulted in higher resting levels of the defense hormones, salicylic acid and jasmonic acid. The RNA-seq analysis indicated that after P. oryzae infection, transcription of the genes involved in reduction-oxidation regulation was the most markedly changed in sl-MH-1, compared with MH86.ConclusionsOur results indicate that SL, involving in the final step of serotonin biosynthesis, negatively regulates rice resistance against (hemi)biotrophic pathogens via compromising the PTI responses and defense hormones accumulation.

HpaXpm, a novel harpin of Xanthomonas phaseoli pv. manihotis, acts as an elicitor with high thermal stability, reduces disease, and promotes plant growth

BackgroundHarpins are proteins secreted by the type III secretion system of Gram-negative bacteria during pathogen–plant interactions that can act as elicitors, stimulating defense and plant growth in many types of non-host plants. Harpin-treated plants have higher resistance, quality and yields and, therefore, harpin proteins may potentially have many valuable agricultural applications. Harpins are characterized by high thermal stability at 100 °C. However, it is unknown whether harpins are still active at temperatures above 100 °C or whether different temperatures affect the activity of the harpin protein in different ways. The mechanism responsible for the heat stability of harpins is also unknown.ResultsWe identified a novel harpin, HpaXpm, from the cassava blight bacteria Xanthomonas phaseoli pv. manihotis HNHK. The predicted secondary structure and 3-D structure indicated that the HpaXpm protein has two β-strand domains and two major α-helical domains located at the N- and C-terminal regions, respectively. A phylogenetic tree generated using the maximum likelihood method grouped HpaXpm in clade I of the Hpa1 group along with harpins produced by other Xanthomonas spp. (i.e., HpaG-Xag, HpaG-Xcm, Hpa1-Xac, and Hpa1Xm). Phenotypic assays showed that HpaXpm induced the hypersensitive response (HR), defense responses, and growth promotion in non-host plants more effectively than Hp1Xoo (X. oryzae pv. oryzae). Quantitative real-time PCR analysis indicated that HpaXpm proteins subjected to heat treatments at 100 °C, 150 °C, or 200 °C were still able to stimulate the expression of function-related genes (i.e., the HR marker genes Hin1 and Hsr203J, the defense-related gene NPR1, and the plant growth enhancement-related gene NtEXP6); however, the ability of heat-treated HpaXpm to induce HR was different at different temperatures.ConclusionsThese findings add a new member to the harpin family. HpaXpm is heat-stable up to 200 °C and is able to stimulate powerful beneficial biological functions that could potentially be more valuable for agricultural applications than those stimulated by Hpa1Xoo. We hypothesize that the extreme heat resistance of HpaXpm is because the structure of harpin is very stable and, therefore, the HpaXpm structure is less affected by temperature.

Evaluation of antibacterial activity of mangosteen (Garcinia mangostana L.) pericarp extract against rice leaf blight bacteria (Xanthomonas oryzae pv. oryzae) at various temperatures and durations of fruit storage

Bioactive contents of mangosteen pericarp can be used as antibacterial agent. This study aimed to examine the antibacterial activity of mangosteen pericarp extract (Garcinia mangostana L.) against rice leaf blight bacteria (Xanthomonas oryzae pv. oryzae) at different temperature and duration of fruit storage, and to identify the antibacterial bioactive metabolites present in the pericarp extract. The antibacterial experiment was arranged with Randomized Completely Block Design (RCBD) with two factors, namely: storage temperature (0, 13.5, and 27 °C) and storage duration (1, 7, and 14 days), with 3 replicates. There was significant interaction between storage temperature and storage duration towards the antibacterial activity of mangosteen pericarp extract. The best was showed by the fruits that was kept in temperature of 13,5 °C for 7 days of storage with inhibition zone of 24.46 mm and the minimum inhibitory concentration (MIC) of 25%. Gas chromatography-mass spectrometry (GC-MS) result showed antibacterial bioactive compounds including (-)-Alpha-Copaene and 2H,6H-Pyran [3,2-B] Xanthen-6-One,5,8-Dihydroxy-9-Methoxy-2,2-Dimethyl-10-(3-Methyl-2-Butenyl)-on the fruits stored in temperature of 0 °C and duration of 1 day, and Copaene on the fruits stored during 7 days on temperature of 13,5 °C. In conclusion, mangosteen pericarp is a promising source of antibacterial bioactive against Xoo pathogen.

Potential application on preparation for bio-fertilizer using Bacillus velezensis strains isolated from various regions in Vietnam

Bacillus velezensis is a species belonging to the bacterial group of B. subtilis species complex. Due to various beneficial traits to plants, the bacterium has been received considerable attention for application in disease control and crop productivity. In this study, we isolated 15 B. velezensis strains from various soils collected in regions of Hoang Lien, Cuc Phuong, Bach Ma, Chu Yang Sin and Con Dao. All of the strains demonstrated antagonistic activity against phythopathogenic fungi of Fusarium oxysporum, Sclerotium hydrophilum, Rhizoctonia solani and Phytophthora capsici and rice blight bacterium Xanthomonas oryzae. Those strains were capable of solubilizing Ca3(PO4)2 and producing plant growth hormone of indole acetic acid (IAA). Moreover, those strains produced a series of extracellular hydrolytic enzymes such as lipase, protease, amylase, CMCase, xylanase and chitinase. Analysis of genetic diversity based on rep - PCR fingerprinting technique showed that primer ERIC had greater discrimination than primer GTG5. Number of DNA banding patterns generated by primer ERIC and GTG5 were 8 and 13, respectively. Combination of DNA banding patterns of both primers showed 14 different genotypes among 15 B. velezensis strains. Using multilocus sequencing technique, genetic diversity was confirmed by a clear discrimination of strain CP 1604 and SP 1901 in two different clusters on phylogenetic tree. Due to the possession of many beneficial traits to plant growth, pesticide protection and production of many hydrolytic enzymes, the B. velezensis strains in this study have potential application on preparation of bio-fertilizer. Further investigation is needed in order to select an appropriate strain for this application.

Functional regions of HpaXm as elicitors with specific heat tolerance induce the hypersensitive response or plant growth promotion in nonhost plants.

HpaXm produced by the cotton leaf blight bacterium Xanthomonas citri subsp. malvacearum is a novel harpin elicitor of the induced hypersensitive response (HR) in tobacco. We investigated whether fragments of HpaXm, compared with fragments of Hpa1Xoo, are sufficient for HR or plant growth promotion (PGP) elicitation using four synthetic peptides (HpaXm35-51, HpaXm10-39, Hpa1Xoo36-52 and Hpa1Xoo10-40). We also heated the fragments to determine the heat tolerance of the functional fragments. HpaXm35-51 and Hpa1Xoo36-52 induced hypersensitive response (HR). Bursts of reactive oxygen intermediates (ROI) induced by HpaXm35-51 and Hpa1Xoo36-52 were earlier and stronger than those induced by HpaXm and Hpa1Xoo. In plants treated with HpaXm35-51 or Hpa1Xoo36-52, the expression of the HR marker genes Hin1 and Hsr203J and the active oxygen metabolism related gene AOX were significantly upregulated. These findings suggest that the predicted α-helical structures of the HpaXm35-51 and Hpa1Xoo36-52 fragments are crucial for HR. PGP result by soaking seeds in unheated/heated HpaXm10-39 or Hpa1Xoo10-40 solution prior to transfer, which obviously enhances root growth and the aerial parts of plants. The PGP related gene NtEXP6 was greatly enhanced when plants were sprayed with a solution of HpaXm10-39 or Hpa1Xoo10-40; heated fragment treatments induced higher levels of NtEXP6 expression than unheated HpaXm fragments. In addition, HR marker genes induced by the heated fragments had lower expression levels than when induced with unheated HpaXm fragments. Moreover, the expression levels of HR marker genes and PGP related genes induced by treatment with Hpa1Xoo fragments before or after heating were the opposite of those induced by HpaXm fragments. Different functional fragments of harpin and different harpins with the same functional region have different degrees of heat tolerance. Therefore, the heat resistance of harpin is conservative, but the degree of heat tolerance of the functional fragments is specific.

Whole-Genome Sequence of Pseudomonas fluorescens EK007-RG4, a Promising Biocontrol Agent against a Broad Range of Bacteria, Including the Fire Blight Bacterium Erwinia amylovora.

ABSTRACTHere, we report the first draft whole-genome sequence of Pseudomonas fluorescens strain EK007-RG4, which was isolated from the phylloplane of a pear tree. P. fluorescens EK007-RG4 displays strong antagonism against Erwinia amylovora, the causal agent for fire blight disease, in addition to several other pathogenic and non-pathogenic bacteria.

The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.

Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1–45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

Alternative Splicing of Rice WRKY62 and WRKY76 Transcription Factor Genes in Pathogen Defense.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins.

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Biosynthesis of Silver Nanoparticles by Phytopathogen Xanthomonas oryzae pv. oryzae Strain BXO8

Extracellular biogenic synthesis of silver nanoparticles with various shapes using the rice bacterial blight bacterium Xanthomonas oryzae pv. oryzae BXO8 is reported. The synthesized silver nanoparticles were characterized by UV-Vis spectroscopy, powder X-ray diffractometry (XRD), scanning electron microscopy, energy dispersive X-ray spectrometry, and highresolution transmission electron microscopy (HR-TEM). Based on the evidence of HR-TEM, the synthesized particles were found to be spherical, with anisotropic structures such as triangles and rods, with an average size of 14.86 nm. The crystalline nature of silver nanoparticles was evident from the bright circular spots in the SAED pattern, clear lattice fringes in the high-resolution TEM images, and peaks in the XRD pattern. The FTIR spectrum showed that biomolecules containing amide and carboxylate groups are involved in the reduction and stabilization of the silver nanoparticles. Using such a biological method for the synthesis of silver nanoparticles is a simple, viable, cost-effective, and environmentally friendly process, which can be used in antimicrobial therapy.

Phytoalexin formation in fire blight-infected apple

Biosynthesis of phytoalexins is a plant defence strategy against pathogens. Shoots of the apple (Malus × domestica) cultivar ‘Holsteiner Cox’ formed biphenyls and dibenzofurans when inoculated with the fire blight bacterium, Erwinia amylovora. The phytoalexins were only present in the transition zone of stems, whereas the leaves were devoid of the defence compounds. The scaffold of the phytoalexins is formed by biphenyl synthase (BIS), a type III polyketide synthase. In apple, BIS is encoded by a gene family, members of which fall into four subfamilies. Representative BIS cDNAs were cloned from fire blight-infected shoots of ‘Holsteiner Cox’ and functionally expressed. The preferred starter substrates were benzoyl-CoA and salicoyl-CoA, leading to the formation of 3,5-dihydroxybiphenyl and 4-hydroxycoumarin, respectively, in the presence of malonyl-CoA as extender molecule. The four subfamilies were differentially regulated after inoculation of shoots with E. amylovora. The BIS3 gene was expressed in stems, with maximum transcript levels in the transition zone. The BIS3 protein was immunochemically localized to the parenchyma of the bark. Dot-shaped immunofluorescence was restricted to the junctions between neighbouring cortical parenchyma cells. Leaves contained transcripts for BIS2 which, however, were not translated into immunodetectable BIS protein. The understanding of phytoalexin metabolism may aid in improving apple resistance to fire blight.

Variation in disease development among persian walnut cultivars, selections and crosses when inoculated with Xanthomonas arboricola pv. juglandis in Greece.

A total of 27 walnut (Juglans regia) cultivars (14 lateral and 13 terminal fruit fullness cultivars), 12 selections and 12 crosses were evaluated for their resistance to the walnut blight bacterium Xanthomonas arboricola pv. juglandis. Healthy nuts were inoculated with pure bacterial cultures isolated from diseased nuts. Two weeks after inoculation, the frequency of successful infections, as well as the disease severity was recorded and the index of varietal susceptibility (IVS) was calculated for each treatment. Based on these results walnut cultivars, selections and crosses were ranked in five classes of susceptibility (A to E). The experiment was repeated for two consecutive years, 2007 and 2008. Results showed that all the artificially infected nuts showed typical bacterial blight symptoms as compared to the negative control. Among the cultivars tested Amigo, Asley, Eliana, Franquette and Spurgcan were the less susceptible with average IVS ranging from 13.1 to 24.4, therefore ranked as class A. Walnut selections EK1, EH29 and FM16, as well as crosses PZ-14 and FxH5 also showed low susceptibility. In contrast, the terminal fruit fullness cv. Milotai and the cross S3/1 were highly susceptible and ranked as class E.

Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’

In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’ after inoculation with the fire blight bacterium, Erwinia amylovora. No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2′-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2′-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains. The most efficient compound was 3,5-dihydroxybiphenyl (MIC=115μg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.

Interaction of Common Bacterial Blight Bacteria with Disease Resistance Quantitative Trait Loci in Common Bean

Common bacterial blight (CBB) of common bean (Phaseolus vulgaris L.) is caused by Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans, and is the most important bacterial disease of this crop in many regions of the world. In 2005 and 2006, dark red kidney bean fields in a major bean-growing region in central Wisconsin were surveyed for CBB incidence and representative symptomatic leaves collected. Xanthomonad-like bacteria were isolated from these leaves and characterized based upon phenotypic (colony) characteristics, pathogenicity on common bean, polymerase chain reaction (PCR) with X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers, and repetitive-element PCR (rep-PCR) and 16S-28S ribosomal RNA spacer region sequence analyses. Of 348 isolates that were characterized, 293 were identified as common blight bacteria (i.e., pathogenic on common bean and positive in PCR tests with the X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers), whereas the other isolates were nonpathogenic xanthomonads. Most (98%) of the pathogenic xanthomonads were X. campestris pv. phaseoli, consistent with the association of this bacterium with CBB in large-seeded bean cultivars of the Andean gene pool. Two types of X. campestris pv. phaseoli were involved with CBB in this region: typical X. campestris pv. phaseoli (P) isolates with yellow mucoid colonies, no brown pigment production, and a typical X. campestris pv. phaseoli rep-PCR fingerprint (60% of strains); and a new phenotype and genotype (Px) with an X. campestris pv. phaseoli-type fingerprint and less mucoid colonies that produced brown pigment (40% of strains). In addition, a small number of X. fuscans subsp. fuscans strains, representing a new genotype (FH), were isolated from two fields in 2005. Representative P and Px X. campestris pv. phaseoli strains, an FH X. fuscans subsp. fuscans strain, plus five previously characterized X. campestris pv. phaseoli and X. fuscans subsp. fuscans genotypes were inoculated onto 28 common bean genotypes having various combinations of known CBB resistance quantitative trait loci (QTL) and associated sequence-characterized amplified region markers. Different levels of virulence were observed for X. campestris pv. phaseoli strains, whereas X. fuscans subsp. fuscans strains were similar in virulence. The typical X. campestris pv. phaseoli strain from Wisconsin was most virulent, whereas X. campestris pv. phaseoli genotypes from East Africa were the least virulent. Host genotypes having the SU91 marker-associated resistance and one or more other QTL (i.e., pyramided resistance), such as the VAX lines, were highly resistant to all genotypes of common blight bacteria tested. This information will help in the development of CBB resistance-breeding strategies for different common bean market classes in different geographical regions, as well as the identification of appropriate pathogen genotypes for screening for resistance.