Bean Pathogen Research Articles

Changes in spatial and temporal gene expression during incompatible interaction between common bean and anthracnose pathogen

Common bean, one of the most important legumes for human consumption, may have drastic reduction in yield due to anthracnose, a disease caused by the fungus Colletotrichum lindemuthianum. Rapid induction of the plant defense mechanisms is essential to establish an incompatible interaction with this pathogenic fungus. In this study, we evaluated spatial (leaves, epicotyls and hypocotyls) and temporal (24, 48, 72 and 96 hours after inoculation [HAI]) relative expression (RE) of 12 defense-related transcripts selected from previously developed ESTs libraries, during incompatible interaction between the resistant common bean genotype SEL 1308 and the avirulent anthracnose pathogen race 73, using real time quantitative RT-PCR (RT-qPCR) analysis. All selected transcripts, including the ones coding for pathogenesis-related (PR) proteins (PR1a, PR1b, PR2, and PR16a and PR16b) were differentially regulated upon pathogen inoculation. The expression levels of these transcripts were dependent on the tissue and time post inoculation. This study contributes to a better understanding of the kinetics of induced defenses against a fungal pathogen of common bean and may be used as a base line to study defenses against a broad range of pathogens including bacteria as well as non-host resistance.

Domestication affected the basal and induced disease resistance in common bean (Phaseolus vulgaris)

Crop plants exhibit reduced levels of disease resistance, but little is known about the specific resistance mechanisms that are affected by breeding for increased yields. We investigated basal and chemically induced resistance of two wild accessions and four cultivars (including one landrace and three ‘modern’, yield-improved cultivars that have been produced by hybridisation and pedigree breeding) of common bean (Phaseolus vulgaris) under greenhouse and field conditions. After treatment with benzothiadiazole, a widely used inducer of systemic acquired resistance, plants were challenged with one of two bacterial pathogens (Pseudomonas syringae pv. syringae and Enterobacter sp. strain FCB1). Basal resistance to Pseudomonas in the wild accessions was significantly higher than in the cultivars. Moreover, benzothiadiazole-treatment elevated resistance to the same pathogen in a wild accession and the landrace, but not in the yield-improved cultivars. Similarly, benzothiadiazole-induced resistance to Enterobacter FCB1 was detected in both wild accessions and the landrace, whereas the same treatment enhanced susceptibility to Enterobacter FCB1 in two of the yield-improved cultivars. Basal resistance to Pseudomonas was highly (but negatively) correlated to induced resistance over all accessions, and basal and inducible resistance to Enterobacter FCB1 were negatively correlated for the cultivars, but not when considering all six accessions. Benzothiadiazole-treatment increased growth rates under pathogen pressure of the wild accessions but not the cultivars. Apparently, the yield-improved cultivars investigated here have lost a considerable part of the basal and induced broad-spectrum disease resistance that characterises their wild relatives and to some degree also the landrace. Two of the yield-improved cultivars even became highly susceptible to infection by an Enterobacter strain that has not yet been described as a pathogen of bean and that is likely to represent a common environmental or phyllosphere bacterium. Future studies should disentangle the effects of domestication on the various layers of plant resistance to pathogens and consider the potential of wild accessions and landraces for future breeding programmes.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86 % as Trichoderma asperellum, 33.33 % as Trichoderma harzianum, 14.29 % as Trichoderma tomentosum, 4.76 % as Trichoderma koningiopsis, and 4.76 % as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.

First Report of Amphobotrys ricini Causing Gray Mold Disease on Acalypha australis in Central China.

In 2008, we isolated a strain of an Amphobotrys sp., CotAr-12, from a cotton plant showing Verticillium wilt symptoms in Hubei Province, China. In October 2010 and 2011, surveys for Amphobotrys sp. on cotton were conducted in 14 counties in Hubei. No signs of an Amphobotrys sp. was observed on cotton. However, a gray mold disease was found on the weed, Acalypha australis in 40 of 51 cotton fields with an average disease incidence of 18.6%. The disease started on the inflorescences at the top of stems or branches and then spread downward to the main stems, branches, and leaves. Abundant sporulation on the necrotic tissues and formation of sclerotia underneath the epidermis or in the stem pith were evident. A total of 128 isolates, including CopAr-5, were obtained from A. australis (1 to 10 isolates per field). All isolates from A. australis and CotAr-12 appeared similar in colony morphology; ragged colony margins, erect conidiophores with long stipes and dichotomous branches at the top, globose and subhyaline conidia with an average diameter of 5.3 to 8.5 μm, and black, oval sclerotia of 0.6 to 26.2 × 0.5 to 19.0 mm. These characteristics matched the description for Amphobotrys ricini (Buchw.) Hennebert (teleomorph Botryotinia ricini [Godfrey] Whetzel) (2). Strains CopAr-5 and CotAr-12 were selected for molecular identification. DNA was extracted from mycelia and used for cloning of three nuclear genes (e.g., G3PDH, HSP60, and RPB2) using the procedures described by Staats et al. (4). The resulting DNA sequences were used for phylogenetic analysis with the corresponding sequences for Botrytis spp. (4). Results showed that strains CopAr-5 (GenBank Accession Nos. JN681883, JN681881, and JN6981879), CotAr-12 (GenBank Accession Nos. JN681882, JN681880, and JN6981878), and Botrytis (teleomorph Botrytinia) ricini (GenBank Accession Nos. GQ860998, GQ860996, and GQ860997) formed a separate clade that was distinct from Botrytis spp., supporting the distinction of Amphobotrys from Botrytis and suggesting that the two strains were Amphobotrys ricini. Pathogenicity was determined by placing mycelia of strains CopAr-5 and CotAr-12 on 10 detached leaves of A. australis, castor bean, and cotton. Control leaves of these plants were inoculated with potato dextrose agar alone. After incubation at 20°C under moist conditions (>90% relative humidity) for 60 h, the control leaves remained healthy, while the leaves of A. australis and castor bean inoculated with both strains formed extensively expanded lesions of 20 and 27 mm in diameter on average, respectively. Both strains also caused disease on cotton leaves with discontinuous and localized lesions of 18 mm in average diameter. A fungus was reisolated from the leaf lesions on these plant species and were identical to Amphobotrys ricini in colony morphology and conidial characteristics. We conclude that Amphobotrys ricini is a major pathogen on A. australis in central China. It is an important pathogen of castor bean (1) and has been reported to infect several euphorbiaceous plants, including A. hispida (3). To our knowledge, this is the first report of Amphobotrys ricini on A. australis.

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

Genetic diversity analysis of isolates of the fungal bean pathogen Pseudocercospora griseola from central and southern Brazil

Planting resistant varieties is the most effective control measure against the angular leaf spot of dry beans, a fungal disease caused by Pseudocercospora griseola. However, dry bean varieties with durable resistance are not easily obtained. Knowledge about the genetic variability of the pathogen population is key for the success of dry bean breeding programs aimed at developing resistant materials, but finding suitable operationally simple and genetically accurate markers is not an easy task. The aim of this study was to assess the suitability of the ISSR-PCR technique to quantify the genetic variability of P. griseola isolates. Total DNA of 27 P. griseola isolates from Goiás, Minas Gerais, Espírito Santo, and Paraná States was extracted and amplified using specific primers for ISSR. Using cluster analysis, 27 genotypes were identified. The ISSR-PCR technique was suitable for assessing intraspecific variability of P. griseola. The ISSR-PCR marker was found to be highly sensitive to genetic variation and can aid in elucidating the genetic structure of the population of this plant pathogen as a support tool for the dry bean breeding programs.

Miniature Transposable Sequences Are Frequently Mobilized in the Bacterial Plant Pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10−5 and 1.1×10−6, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Development, characterization and linkage analysis of microsatellite loci for the Ascochyta blight pathogen of faba bean, Didymella fabae

Eighteen microsatellite markers were developed for Didymella fabae, seventeen of which were highly polymorphic among a sample of D. fabae isolates from Syria. Genetic linkage analysis assigned the markers to eight linkage groups. These markers will facilitate population and evolutionary studies of D. fabae and related species.

Identification of QTL for agronomic traits and resistance to white mold in wild and landrace germplasm of common bean

With 1 figure and 5 tablesAbstractWhite mold, caused by Sclerotinia sclerotiorum (Lib.) De Bary, is a serious yield reducing fungal pathogen of common bean (Phaseolus vulgaris L.). Our objective was to identify QTL for resistance to white mold from wild and landrace accessions of common bean using two inbred backcross line (IBL) populations derived from the recurrent black bean parent ‘Tacana’. Selective phenotyping failed to detect QTL for field disease resistance but other agronomic traits less sensitive to environmental conditions or population size were detected. Four novel QTLs for white mold resistance WM3.3TW, WM7.5TL, WM9.2TW, and WM11.1TL were identified in the greenhouse straw test on linkage groups B3, B7, B9, and B11, respectively, and two previously mapped QTL were also validated on B2 and B4. QTL, SY2.1TL that accounted for 19–37% of the variation for yield under white mold pressure over 3 years, was detected on B2 in the TL population. Enhanced resistance to white mold in common bean could be achieved by combining different QTL associated with physiological resistance with yield under disease pressure.

Genetic diversity of the common bacterial blight pathogen of bean, Xanthomonas axonopodis pv. phaseoli, in Iran revealed by rep-PCR and PCR–RFLP analyses

Xanthomonad-like bacteria that are associated with common bacterial blight of bean in Iran were identified on the basis of their colonial morphology, biochemical and serological properties, presence of a specific DNA fragment using PCR primers and pathogenicity on bean. Xanthomonas axonopodis pv. phaseoli (Xap) strains were further characterized using rep-PCR and restriction fragment length polymorphism (RFLP). RFLP profiles generated by the restriction endonucleases RsaI, TaqI, HaeIII and Sau96I and rep-PCR analysis revealed that Iranian strains were relatively genetically homogenous. The similarity coefficients among the strains ranged from 0.87 to 1. The genetic diversity coefficients among strains from three infected provinces, Isfahan, Markazi and Lorestan, were 0.019, 0.072 and 0.033, respectively. The low overall level of polymorphism within Xap isolates collected from the three Iranian infected regions could suggest that few initial inoculum introductions might have distributed among these different bean-growing areas in Iran.

Screening of Rhizobacteria for Their Plant Growth Promotion Ability and Antagonism Against Damping off and Root Rot Diseases of Broad Bean (Vicia faba L.)

Development of microbial inoculants from rhizobacterial isolates with potential for plant growth promotion and root disease suppression require rigorous screening. Fifty-four (54) fluorescent pseudomonads, out of a large collection of rhizobacteria from broad bean fields of 20 different locations within Imphal valley of Manipur, were initially screened for antifungal activity against Macrophomina phaseolina and Rhizoctonia solani, of diseased roots of broad bean and also three other reference fungal pathogens of plant roots. Fifteen fluorescent pseudomonas isolates produced inhibition zone (8-29mm) of the fungal growth in dual plate assay and IAA like substances (24.1-66.7μg/ml) and soluble P (12.7-56.80μg/ml) in broth culture. Among the isolates, RFP 36 caused a marked increase in seed germination, seedling biomass and control of the root borne pathogens of broad bean. PCR-RAPD analysis of these isolates along with five MTCC reference fluorescent pseudomonas strains indicated that the RFP-36 belonged to a distinct cluster and the PCR of its genomic DNA with antibiotic specific primers Phenazine-1-carboxylic acid and 2, 4-diacetyl phloroglucinol suggested possible occurrence of gene for the potent antibiotics. Overall, the result of the study indicated the potential of the isolate RFP 36 as a microbial inoculant with multiple functions for broad bean.

Genetic Dissection of Basal Resistance to Pseudomonas syringae pv. phaseolicola in Accessions of Arabidopsis

We have examined the genetics of nonhost resistance in Arabidopsis, using the bean pathogen Pseudomonas syringae pv. phaseolicola race 6 1448A to probe accessions for natural variation in basal defense. Symptoms rarely developed in leaves of Niedersenz (Nd), some yellowing and occasional necrosis developed in Columbia (Col), whereas tissue collapse was observed in Wassilewskija (Ws) after inoculation by infiltration. Analysis of F2 progeny and recombinant inbred lines (RIL) from a cross between Col and Nd revealed a pattern of continuous symptom increase, indicating the operation of quantitative determinants of resistance. By mapping quantitative trait loci (QTL), significant linkage was determined for resistance (low symptom score) to markers on chromosome 4. Segregation in the F2 cross from Nd × Ws indicated the operation of two dominant genes for resistance, one of which was FLS2 encoding the flagellin receptor. The requirement for FLS2 to confer resistance was confirmed by transgenic experiments, and we showed that the response to P. syringae pv. phaseolicola was affected by FLS2 gene dosage. Using RIL, the second locus was mapped as a QTL to a large interval on chromosome 1. Both FLS2 and the QTL on chromosome 1 were required for the highest level of resistance to bacterial colonization and symptom development in Nd.

Positive Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

Disease in compatible hosts and induction of the hypersensitive response in resistant plants by most plant-pathogenic bacteria require a functional type III secretion system (T3SS). Expression of T3SS genes responds to host and environmental factors and is induced within the plant. In Pseudomonas syringae, expression of the T3SS requires HrpL, which is transcriptionally upregulated by HrpR and HrpS. In some pathovars, expression of the hrpRS genes is upregulated by the GacA/S two-component system. Additionally, HrpA, the major component of the T3SS pilus, has also been linked to the regulation of the hrpRS gene expression. Previous studies concerning regulation of hypersensitive response and pathogenesis/hypersensitive response conserved (hrp/hrc) gene expression have used mostly in vitro inducing conditions, different pathovars, and methodology. Here, we analyze the roles of HrpL, GacA, and HrpA in the bean pathogen, using single, double, and triple mutants as well as strains ectopically expressing the regulators. We use real-time polymerase chain reaction analysis in vitro and in planta to quantify gene expression and competitive indices and other assays to assess bacterial fitness. Our results indicate that i) HrpL acts as a general virulence regulator that upregulates non-T3SS virulence determinants and downregulates flagellar function; ii) GacA modulates the expression of hrpL, and its contribution to virulence is entirely HrpL dependent; iii) there is a basal HrpL-independent expression of the T3SS genes in rich medium that is important for full activation of the system, maybe by keeping the system primed for rapid activation upon contact with the plant; and iv) HrpA upregulates expression of the T3SS genes and is essential to activate expression of the hrpZ operon upon contact with the plant.

Immunological detection of bean common mosaic virus in French bean (Phaseolus vulgaris L.) leaves

Bean common mosaic potyvirus (BCMV) is an important seed borne pathogen of French bean. Differential inoculation with bean common mosaic virus at cotylodonary trifoliate leaf stage and pre-flowering stage of crop growth revealed that cotyledonary leaf infection favored maximum disease expression. Under immunosorbent electron microscopy (ISEM) the virus particles of filamentous structure having a diameter of 750 nm (l) and 15 nm (w) were observed. These particles gave positive precipitin tests with polyclonal antiserum of Potato virus Y.

First Report of Anthracnose Caused by Colletotrichum acutatum on Mung Bean Sprouts in Taiwan.

Mung bean sprouts (Vigna radiata (L.) R. Wilczek) are a commonly consumed vegetable in Asian countries. Anthracnose lesions on mung bean sprouts (cv. You-lu-dou from Mayamar) were found in an indoor sprouting facility in Taichung County, Taiwan in May of 2009. The incidence of disease exceeded 90% in some lots. Infected hypocotyls had smooth, diamond-shaped to fusiform brown spots, which became further depressed and enlarged with age. A fungus was isolated on potato dextrose agar (PDA) from symptomatic hypocotyls after surface sterilization in 0.6% NaOCl. Fungal colonies were initially salmon to orange in color and became greenish gray on the surface within a week. Setae were not produced in acervuli that developed on PDA. Conidia in the acervuli were one-celled, cylindrical, and hyaline with an average length and width of 14.8 (7.0 to 23.7) × 4.9 (3.3 to 6.7) μm (n = 53). The internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (4). The sequence was deposited in GenBank (Accession No. GQ889269). The sequence was 99% identical to that of ATCC 56816 strain of Glomerella acutata (Guerber & J. C. Correll), the teleomorph of Colletotrichum acutatum (J. H. Simmonds) over a 522-bp alignment. Thus, the mung bean pathogen was identified as C. acutatum based on morphological (1) and molecular characters. Pathogenicity of the strain was determined by inoculating mung bean seeds from I-Mei Foods Co. (imported from Australia). The seeds were disinfested in 0.6% NaOCl for 10 min, rinsed with sterile distilled water twice, and immersed in a conidial suspension (1.5 × 104 conidia/ml) of C. acutatum for 10 min. Fifteen inoculated seeds were placed on moistened paper towels, distributed into three flasks, and stored in the dark at 32°C. Symptoms similar to those observed on the original sprouts appeared 3 days later on the hypocotyls of the seedlings and all seedlings were infected by day five. Conidia of C. acutatum were produced on all lesions and colonies of C. acutatum were recovered from symptomatic tissues, fulfilling Koch's postulates. Controls (seed immersed in water) remained symptomless. The pathogenicity test was repeated with similar results. The pathogen has been recorded on mung bean sprouts in Korea (2) and on other Vigna spp. in India (3). To our knowledge, this is the first report of sprout rot of mung bean caused by C. acutatum in Taiwan.

Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences

Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox(-)) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox(-) and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.

Contributions of the effector gene hopQ1‐1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

SUMMARYTo study the role of type III‐secreted effectors in the host adaptation of the tobacco (Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci, a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean (Phaseolus vulgaris) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1‐1Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1‐1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1‐1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine‐tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non‐host species.

Bacterial Evolution by Genomic Island Transfer Occurs via DNA Transformation In Planta

Our understanding of the evolution of microbial pathogens has been advanced by the discovery of "islands" of DNA that differ from core genomes and contain determinants of virulence. The acquisition of genomic islands (GIs) by horizontal gene transfer (HGT) is thought to have played a major role in microbial evolution. There are, however, few practical demonstrations of the acquisition of genes that control virulence, and, significantly, all have been achieved outside the animal or plant host. Loss of a GI from the bean pathogen Pseudomonas syringae pv. phaseolicola (Pph) is driven by exposure to the stress imposed by the plant's resistance response. Here, we show that the complete episomal island, which carries pathogenicity genes including the effector avrPphB, transfers between strains of Pph by transformation in planta and inserts at a specific att site in the genome of the recipient. Our results show that the evolution of bacterial pathogens by HGT may be achieved via transformation, the simplest mechanism of DNA exchange. This process is activated by exposure to plant defenses, when the pathogen is in greatest need of acquiring new genetic traits to alleviate the antimicrobial stress imposed by plant innate immunity.

The non-host disease resistance response in peas; alterations in phosphorylation and ubiquitination of HMG A and histones H2A/H2B

The non-host disease resistance response of cuticle-free pea endocarp tissue against Fusarium solani f. sp. phaseoli, a bean pathogen, is a synchronous cellular response that completely inhibits the challenging fungal growth within 6 h. This tissue becomes susceptible to the true pathogen, F. solani f. sp. pisi, within 18 h. Previous research indicated that there is an early effect by both pathogens on pea chromatin possibly via the architectural transcription factor, HMG A. HMG A binds with AT-rich regions of the pea DRR206 gene (a PR gene) promoter. The total PR gene-based defense response is activated by three eliciting agents, Fsph DNase, chitosan oligomers and inhibitors of phosphatase such as calyculin A. This report monitors the presence of HMG A and histone and their phosphorylation and ubiquitination, both within the total cytoplasm and in association with isolated chromatin. Inoculation results in a diminution of both HMG A and histones H2A/H2B within 5 h, being more extensive in the susceptible interaction. The detectable phosphorylated HMG A and some accompanying nuclear proteins are diminished within 4 h after pathogen challenge. Changes in the molecular weight of the nuclear components were ELISA-monitored with anti-HMG A, anti-histone H2A/H2B and anti-ubiquitin antisera and indicated that some fungal-caused global changes in chromatin occur via ubiquitin complexing. These modifications of the pea's nuclear proteins could have major transcriptional-related roles in disease resistance since they are temporally associated with the onset of PR gene activation.