Bean Pathogen Research Articles

Seed-Borne Pathogens of Faba Bean in Egypt: Detection and Pathogencity

This study was undertaken to study the seed-borne fungi of faba bean that attack the plants and reduce their yield in Egypt. The results provide a database for further study to control the pathogens. Twenty-six seed samples representing six faba bean cultivars collected from different parts of Egypt were used in this investigation. The blotter and deep-freezing methods were used. Surface- and non-surface-sterilized faba bean seeds were tested to detect and isolate the associated seed-borne fungi. The following 20 fungal species belonging to 13 genera were observed and identified: Aspergillus flavus (Link ex. Gray), Aspergillus niger (Van Tieghem), Aspergillus ochraceus (Wilhelm), Penicillium digitatum (Pers.:Fr.) Sacc., Penicillium italicum (Wehmer), Alternaria alternata (Fr.) Keissler, Botrytis faba (Sardina), Cephalosporium sp., Cladosporium cladosporioides (Frensen. de Vries), Epicoccum nigrum (Link), Fusarium oxysporum (Schlechlendahl), Fusarium semitectum (Berkeley and Ravenel), Fusarium solani (Mart.) Sacc., Fusarium verticillioides (moniliforme) (Sheld), Rhizoctonia solani (Kuhn), Rhizopus stolonifer (Ehr. ex Fr.), Stemphylium globuliferum (Vestergr.) E.G. Simmons), Trichothecium roseum (Pers.) Link, Verticillium dahliae (Ehrenp) Vuill. The blotter method yielded a greater number of fungi than the deep-freezing method on both surface and non-surface sterilized seeds, but the deep-freezing method was better for slow-growing fungi. The pathogenicity test revealed that the most commonly isolated fungi from pre- and post-emergence damping-off and stunted seedlings were F. verticillioides, R. solani, Cephalosporium sp. and V. dahliae. These fungi significantly reduced the photosynthetic pigments in faba bean leaves. Fusarium verticillioides caused the greatest reduction in chlorophyll content (A, B and total chlorophyll). Fusarium oxysporum and V. dahliae significantly reduced carotenoid content. R. solani significantly reduced total phenols content when compared with the other tested fungi.

Pythium solare sp. nov., a new pathogen of green beans in Spain

A new disease causing wilt and death of adult plants of Phaseolus vulgaris was discovered in plastic-house crops of southeast Spain in 2004. The causal agent was shown to be a Pythium species with a unique type of oogonium ornamentation different from any of the described species. Zoospores were not observed, but globose or subglobose hyphal swellings, intercalary or terminal, were frequently found. Moreover, the ribosomal ITS region showed a unique sequence, significantly different (>14 %) from any other known species of Pythium. This paper describes and illustrates the morphology of the new Pythium species and its pathogenicity to green beans. Its taxonomic position and phylogenetic relationships with other Pythium species are discussed.

Molecular Genotyping of Macrophomina phaseolina Isolates: Comparison of Microsatellite Primed PCR and Repetitive Element Sequence‐based PCR

AbstractSeventy isolates of Macrophomina phaseolina recovered from different host plants were assessed for DNA polymorphism using two molecular techniques: microsatellite primed polymerase chain reaction (MSP‐PCR) under both touchdown (T) and non‐touchdown (NT) PCR conditions and primers corresponding to disperse repetitive sequence‐based polymerase chain reaction (rep‐PCR). Fingerprints obtained by rep‐PCR were compared with those of MSP‐PCR. Even though these methods yielded intraspecific polymorphisms, yet different levels of discrimination could be obtained. A partial correlation was apparent between the molecular techniques used. Some of the genetic groups/genotypes were supported by both the molecular markers employed in the study, thus confirming their relationship. Thirty nine MSP (T), 55 MSP (NT) and 53 rep‐PCR genotypes were identified with discrimination indices of 0.962, 0.993 and 0.99, respectively. Our results have shown that rep‐PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as MSP‐PCR for genotyping M. phaseolina isolates and is highly suitable for understanding disease epidemiology at molecular level. Suggesting, thereby, that it is a robust technique employed for genotypical and phylogenetic studies for determining taxonomical diversity and phylogenetic structure of the economically important fungal pathogen of cluster bean. The data presented here will help researchers to design effective strategies for deployment of resistant germplasm in cluster bean (Cymopsis tetragonoloba) growing regions in the country and worldwide.

First Report of Mefenoxam-Resistant Isolates of Phytophthora capsici from Lima Bean Pods in the Mid-Atlantic Region.

Phytophthora capsici Leonian, the causal agent of lima bean pod rot, was first identified as a pathogen of lima bean in 2002 (1) and poses a new threat to lima bean (Phaseolus lunatus L.) production in the Mid-Atlantic Region. The phenylamide fungicide mefenoxam (Ridomil Gold; Syngenta Crop Protection) is widely used in the region for controlling foliar and soilborne diseases caused by Oomycetes. Isolates of P. capsici were collected from lima bean pods from production fields in Delaware, Maryland, and New Jersey from 1998 to 2004. These isolates originated from survey samples of lima bean fields for another pathogen, P. phaseoli, in 1999 and 2000 and diagnostic samples were submitted to the Plant Disease Clinic. Isolates were from lima bean, except for one from pepper (basal stem). Identification was made on the basis of morphometric characteristics. No known sensitive or insensitive isolates were included in the evaluation. Single zoospore cultures were evaluated for mefenoxam sensitivity on V8 agar plates amended with 100 ppm of mefenoxam, a previously tested concentration (2). Seven-millimeter-diameter agar plugs of each isolate were cut from the edge of actively expanding cultures of P. capsici with a cork borer and transferred to three V8 agar plates amended with mefenoxam and three unamended V8 plates. The plates were arranged in a completely randomized design and incubated at 25°C in the dark for 3 days. After incubation, colony growth was measured in millimeters and averaged for the three replicate plates of each isolate and percent growth relative to the unamended control was calculated. Mefenoxam sensitivity was assigned according to methods of Lamour et al. (2). The experiment was repeated once, and also run with a treatment of 200 ppm of mefenoxam. Of sixteen isolates screened, nine were rated as sensitive, four were intermediately resistant, and three were resistant. There was no difference between the 100 and 200 ppm results, except for a slight increase in sensitivity for one isolate. A subsequent experiment tested five isolates at concentrations of 1, 10, 100, and 1,000 ppm. Results were consistent with previous tests, with resistant isolates exhibiting some growth at the highest concentration of mefenoxam. One resistant isolate was from a field in Delaware previously cropped to slicing cucumbers with a history of mefenoxam applications. The second was from Caroline County, MD, which is heavily cropped to pickling cucumbers and likely to have been exposed to mefanoxam applications for the control of fruit rot; the origin of the third insensitive isolate from lima bean is unknown. Mefanoxam usage on lima bean is usually limited to one foliar application of mefenoxam+copper hydroxide to control downy mildew in the fall crop in wet seasons. This study indicates that mefenoxam resistance is present in populations of P. capsici in lima bean fields in the Mid-Atlantic Region, presumably as a result of mefenoxam applications to other vegetable crops, principally cucurbits, which are planted in rotation with lima beans or from nearby cucurbit fields. Implementing strategies to minimize fungicide resistance in other vegetables is important to slow resistance development associated with this emerging pathogen on lima beans. Lima bean pod rot continues to be seen sporadically each year in fields with a history of P. capsici and abundant rainfall or excessive irrigation.

Assessment of the Genetic Diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a Basis To Identify Putative Pathogenicity Genes and a Type III Secretion System of the SPI-1 Family by Multiple Suppression Subtractive Hybridizations

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.

Aphanomyces root rot on faba bean in northern NSW

Aphanomyces euteiches was found to be the causal agent of faba bean root rot in northern NSW, both in experimental and commercial fields. Resistance to Aphanomyces root rot appears to be common in the faba bean germplasm pool, but the susceptibility of some varieties justifies further research on the pathogen. This is the first report of A. euteiches as a faba bean pathogen in Australia.

A Specific and Sensitive Method for the Detection of Colletotrichum lindemuthianum in Dry Bean Tissue.

To facilitate early diagnosis and improve control of bean anthracnose, a rapid, specific, and sensitive polymerase chain reaction (PCR)-based method was developed to detect the causal agent, Colletotrichum lindemuthianum, in bean (Phaseolus vulgaris) seed. Based on sequence data of the rDNA region consisting of the 5.8S gene and internal transcribed spacers (ITS) 1 and 2 of four C. lindemuthianum races and 17 Colletotrichum species downloaded from GenBank, five forward primers were designed and evaluated for their specificity. Among them, one forward primer was selected for use in combination with ITS4 to specifically detect C. lindemuthianum. A 461-bp specific band was amplified from the genomic DNA template of 16 representative isolates of C. lindemuthianum, but not from 58 representative isolates of 17 other Colletotrichum species or 10 bean pathogens. Moreover, to enhance the sensitivity of detection, nested PCR was applied, which allowed the detection of as little as 10 fg of C. lindemuthianum genomic DNA and 1% infected seed powder, which was mixed with 99% healthy seed powder. The diagnostic analysis can be completed within 24 h, compared with about 2 weeks required for culturing. Furthermore, this method can be performed and interpreted by personnel with no specialized taxonomic expertise.

Layered basal defenses underlie non‐host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola

Arabidopsis is a non-host for Pseudomonas syringae pv. phaseolicola NPS3121 (Pph), a bacterial pathogen of bean. Pph does not induce a hypersensitive response in Arabidopsis. Here we show that Arabidopsis instead resists Pph with multi-layered basal defense. Our approach was: (i) to identify defense readouts induced by Pph; (ii) to determine whether mutations in known Arabidopsis defense genes disrupt Pph-induced defense signaling; (iii) to determine whether heterologous type III effectors from pathogens of Arabidopsis suppress Pph-induced defense signaling, and (iv) to ascertain how basal defenses contribute to resistance against Pph by individually or multiply disrupting defense signaling pathways with mutations and heterologous type III effectors. We demonstrate that Pph elicits a minimum of three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 protein accumulation and morphologically distinct types of callose deposition. Further, they require distinct defense genes, including PMR4, RAR1, SID2, NPR1, and PAD4. Finally, they are suppressed differentially by heterologous type III effectors, including AvrRpm1 and HopM1. Pph growth is enhanced only when multiple defense pathways are disrupted. For example, mutation of NPR1 or SID2 combined with the action of AvrRpm1 and HopM1 renders Arabidopsis highly susceptible to Pph. Thus, non-host resistance of Arabidopsis to Pph is based on multiple, individually effective layers of basal defense.

Host Specificity of Ascochyta spp. Infecting Legumes of the Viciae and Cicerae Tribes and Pathogenicity of an Interspecific Hybrid

ABSTRACT Ascochyta spp. (teleomorphs: Didymella spp.) infect a number of legumes, including many economically important species, and the diseases they cause represent serious limitations of legume production worldwide. Ascochyta rabiei, A. fabae, A. pisi, A. lentis, and A. viciae-villosae are pathogens of chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), lentil (Lens culinaris), and hairy vetch (V. villosa), respectively. Inoculations in the greenhouse and in growth chambers demonstrated that A. fabae, A. lentis, A. pisi, A. rabiei, and A. viciae-villosae were host specific. Isolates caused no visible disease symptoms on "nonhost" plants (plants other than the hosts they were originally isolated from) but were recovered consistently from inoculated, surface-disinfested, nonhost tissues. Interspecific crosses of A. pisi x A. fabae and A. viciae-villosae x A. lentis produced pseudothecia with viable ascospores, and the hybrid status of the ascospore progeny was verified by the segregation of mating type and amplified fragment length polymorphism (AFLP) markers. Interspecific progeny were morphologically normal in culture but exhibited more phenotypic variation compared with progeny from intraspecific crosses. Mating type and the majority of AFLP markers segregated in Mendelian 1:1 ratios in both intraspecific and interspecific crosses. A total of 11 and 7% of AFLP markers showed segregation distortion among progeny from interspecific crosses and intraspecific crosses, respectively; however, this difference was not significant (P = 0.90). Only 30 of 114 progeny isolates from the A. fabae x A. pisi cross inoculated in the greenhouse caused lesions on pea and only 4 caused disease on faba bean. In all, 15 of 110 progeny isolates were pathogenic to pea and none were pathogenic to faba bean under growth chamber conditions. Although no obvious postzygotic, intrinsic isolating barriers were identified in any of the interspecific crosses, it appears that host specialization may act as both a prezygotic, ecological isolating barrier and a postzygotic, extrinsic, ecological isolating barrier in these fungi. Host specificity, coupled with low pathogenic fitness of hybrids, may be an important speciation mechanism contributing to the maintenance of hostspecific, phylogenetic lineages of these fungi.

The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non‐host plants

The bacterial plant pathogen Pseudomonas syringae injects a large repertoire of effector proteins into plant cells using a type III secretion apparatus. Effectors can trigger or suppress defences in a host-dependent fashion. Host defences are often accompanied by programmed cell death, while interference with defences is sometimes associated with cell death suppression. We previously predicted the effector repertoire of the sequenced bean pathogen P. syringae pv. syringae (Psy) B728a using bioinformatics. Here we show that PsyB728a is also pathogenic on the model plant species Nicotiana benthamiana (tobacco). We confirm our effector predictions and clone the nearly complete PsyB728a effector repertoire. We find effectors to have different cell death-modulating activities and distinct roles during the infection of the susceptible bean and tobacco hosts. Unexpectedly, we do not find a strict correlation between cell death-eliciting and defence-eliciting activity and between cell death-suppressing activity and defence-interfering activity. Furthermore, we find several effectors with quantitative avirulence activities on their susceptible hosts, but with growth-promoting effects on Arabidopsis thaliana, a species on which PsyB728a does not cause disease. We conclude that P. syringae strains may have evolved large effector repertoires to extend their host ranges or increase their survival on various unrelated plant species.

The use of chemical genomics to detect functional systems affecting the non-host disease resistance of pea to Fusarium solani f. sp. phaseoli

Non-host resistance is more durable than race-specific resistance and may involve more signaling systems than race-specific resistance. An array of chemicals capable of specifically inhibiting/affecting most of the vital systems of the plant cell was employed to evaluate a range of systems vital in promoting non-host resistance in the Fusarium solani f. sp. phaseoli/pea endocarp interaction. The parameters measured included pisatin synthesis, hypersensitive discoloration, fungal growth, PR gene induction, and DNA damage. Specific inhibitors of protein phosphatases 1 and 2A (calyculin A, okadaic acid, cantharidin and endothall) and two kinase inhibitors (staurosporine and K-252a from Nocardiopsis sp.) were comparable to fungal challenge in inducing pisatin accumulation. These treatments could often break non-host resistance to a bean pathogen, F. solani f. sp. phaseoli. At low concentrations the treatments transiently enhanced resistance to the pea pathogen, F. solani f. sp. pisi. Nitric oxide and superoxide-generating compounds, salicylic acid, methyl salicylate, and jasmonic acid implicated, as effectors in other systems had no major detectable effect. Thus the broad array of inhibitors delineated cellular functions associated with non-host disease resistance in pea and tentatively excluded some signaling systems reported in other systems.

The Use of Bioluminescence for Monitoring in planta Growth Dynamics of a Pseudomonas syringae Plant Pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Accumulation of cell-wall bound phenolic compounds and phytoalexin in Arabidopsis thaliana leaves following inoculation with pathovars of Pseudomonas syringae

Here we have examined genetically well-defined interactions in leaves of Arabidopsis ecotype Col-5 during compatible and incompatible interactions with isogenic pairs of Pseudomonas syringae pathovars tomato ( Pst) DC3000 and phaseolicola ( Pph) 1448A strains differing only in the presence or absences of cloned avr genes that determine the compatibility and incompatibility of the reaction. Strain Pst DC3000 was virulent causing formation of a spreading lesion surrounded by a chlorotic halo. The bean pathogen Pph 1448A (as a non-host pathogen) caused no visible symptoms apart from very patchy discolouration after 3 days. The presence of the avirulence genes avrPpiA and avrPphB in Pph 1448A led to a rapid or slow HR owing to interaction with the resistance genes RPM1 and RPS5 respectively. The hrpL mutant of Pph failed to produce symptoms. Microscopical and biochemical studies were used for in situ localization of plant molecules involved in plant resistance such as autofluorescent phenolics, lignin and camalexin. A strong correlation was observed between the timing and extent of cell death and high levels of phenolic and lignin accumulation in cell-walls and cytoplasm of cells undergoing the HR, whereas no staining of lignin-like material was observed during the compatible interaction. Accumulation of phytoalexin, camalexin, was associated with tissue necrosis whether during the HR or a susceptible response. Although high levels of camalexin were found within tissues inoculated with the virulent strain DC3000, the earliest detection of camalexin occurred during HR following inoculation with avirulent strain. The results indicate that accumulation of phenolics but not camalexin may be involved in resistance against bacterial pathogens used in this study.

Characterization of Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using conventional techniques and PCR‐based molecular markers

Phenotypic and genetic diversity of 59 Macrophomina phaseolina isolates collected from various host species growing in or near cluster bean (Cyamopsis tetragonoloba) fields in four states of north and north‐west India were characterized using RAPD and PCR–RFLPs of the ITS region. These isolates, and 11 from various hosts from culture collections, were classified into three mycelial phenotypes: dense, feathery and restricted, based on variable growth patterns on nutrient agar containing 120 mm chlorate. Pathogenicity of isolates was evaluated by measuring the length of stem lesions 21 days post‐inoculation on the susceptible cluster bean genotype FS 277. Isolates showed considerable variation in aggressiveness, with the isolates from cluster bean with dense chlorate phenotype producing relatively higher lesion lengths on cluster bean plants. The results of the RAPD assay clearly distinguished the isolates on the basis of chlorate phenotype and host origin. Isolates from a single host were generally similar to each other, but differed distinctly from those from other hosts. Chlorate‐sensitive isolates were distinct from chlorate‐resistant isolates within a given host. A high degree of polymorphism in restriction patterns of the ITS region, including part of 25S rDNA, has been reported for the first time in the charcoal rot fungus.

Bacterial Wilt of Beans (Phaseolus vulgaris) Caused by Curtobacterium flaccumfaciens in Southeastern Spain

Symptoms of bacterial wilt were observed on common beans (cv. Donna) in southeastern Spain. From samples collected in four different fields (coast of Granada), a bacterium was isolated with the following characteristics: gram positive, aerobic rods with yellow colonies, strictly oxidative, oxidase negative, galactose, sucrose, erythritol, mannitol, sorbitol and m-inositol were not used as a sole carbon source, and hydrolysis of casein was positive. These results coincide with what is expected for Curtobacterium flaccumfaciens pv flaccumfaciens (3). One isolate from each field was selected for pathogenicity tests using two different methods. Bacterial suspensions (approximately 108 CFU/ml) were spray inoculated on bean seedlings of cv. Andecha (10 plants with three true leaves for each isolate). Beans were covered with transparent plastic bags for 2 days and held at 25°C and 80% relative humidity with a 12-h photoperiod. In addition, 10 healthy seeds of cv. Andecha were soaked in bacterial suspensions (approximately 108 CFU/ml) for 1 h and incubated at 25°C (2). Seedlings sprayed with distilled sterile water and seeds soaked in water served as controls. With both methods of inoculation, assays were conducted twice. Results were recorded after 3 weeks. Symptoms that developed on plants after infection with the four isolates were similar to those observed in the field. They included golden yellow necrotic leaf lesions and wilting. Wilting was more pronounced in the field and when inoculation was performed by spraying seedlings rather than by soaking seeds. Control plants did not develop symptoms and grew bigger than the inoculated plants. Two pathogenic isolates were identified through sequencing of the 16S rRNA gene. The genes were amplified by polymerase chain reaction (1) and their nucleotide sequences (1,418 bp) proved to be identical (Accession No. AJ879110). Comparison of these sequences with databases showed that they were also identical to those of C. flaccumfaciens strains LMG 3645 and P 259/26 (Accession Nos. AJ312209 and AJ310414) and Curtobacterium sp. strains 2384 and 3426 (Accession Nos. AY688359 and AY688360). In Spain, the bean pathogen C. flaccumfaciens was first isolated from seeds during 2001 (4). However, to our knowledge, this is the first report of damage caused by this bacterium in the field. Bacterial wilt has been recorded, but often not substantiated, in several countries from North and South America, Africa, Asia, Oceania, and Europe.

Nonpathogenic strains of Colletotrichum lindemuthianum trigger progressive bean defense responses during appressorium-mediated penetration.

The fungal bean pathogen Colletotrichum lindemuthianum differentiates appressoria in order to penetrate bean tissues. We showed that appressorium development in C. lindemuthianum can be divided into three stages, and we obtained three nonpathogenic strains, including one strain blocked at each developmental stage. H18 was blocked at the appressorium differentiation stage; i.e., no genuine appressoria were formed. H191 was blocked at the appressorium maturation stage; i.e., appressoria exhibited a pigmentation defect and developed only partial internal turgor pressure. H290 was impaired in appressorium function; i.e., appressoria failed to penetrate into bean tissues. Furthermore, these strains could be further discriminated according to the bean defense responses that they induced. Surprisingly, appressorium maturation, but not appressorium function, was sufficient to induce most plant defense responses tested (superoxide ion production and strong induction of pathogenesis-related proteins). However, appressorium function (i.e., entry into the first host cell) was necessary for avirulence-mediated recognition of the fungus.

Phenotypic and molecular characterization of cultivar BRSMGTalismã regarding the principal common bean pathogens

Information about common bean cultivars recommended for commercial use in Brazil regarding the reaction to pathogens is essential for farmers besides furthering future breeding efforts. Our research evaluated the reactions of the common bean cultivar BRSMG-Talisma to several pathotypes evaluated of Uromyces appendiculatus, Colletotrichum lindemuthianum and Phaeoisariopsis griseola. The cultivar was further characterized through RAPD and SCAR molecular markers used in the marker-assisted selection (MAS) in the bean breeding program of BIOAGRO/UFV which deals with gene pyramiding of rust, anthracnose and angular leaf spot resistance genes. Results showed that BRSMG-Talisma is susceptible to different genotypes of the tested pathogens. All analyzed molecular markers were polymorphic, between BRSMG-Talisma and the resistance sources. Consequently, these markers can be used to monitor a simultaneous introgression of genes with a broad resistance spectrum into the genetic background of BRSMG-Talisma.

Harpin of Pseudomonas syringae pv. phaseolicola Harbors a Protein Binding Site

Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis.

Functional analysis ofCLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogenColletotrichum lindemuthianum

In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation. To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains. This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae. A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C. lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose. The same strategy was used to overexpress the wild-type CLPT1 allele. During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains. Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins. Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells. These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures.

Polysaccharides of pseudomonas pathovar strains that infect pea, tomato, and soya bean.

In order to understand the mode of action of taxonomically related Pseudomonas syringae pathovar strains that infect pea, tomato, and soya bean, we examined their extracellular polysaccharides (EPS). Maximum production of polysaccharide in shake culture of these pathogens was observed between 24 and 60 h. P. syringae pv. pisi 519, the bacterial blight pathogen of pea, produced a higher amount of polysaccharide (34.87 microg/mL) at 60 h compared with 32.67 microg/mL produced by P. syringae pv. glycinea NCPPB 1783, the bacterial blight pathogen of soya bean, and 30.03 microg/mL produced by P. syringae pv. tomato NCPPB 269, the bacterial speck pathogen of tomato. EPS produced by P. syringae pv. pisi 519, P. syringae pv. tomato NCPPB 269, and P. syringae pv. glycinea NCPPB 1783 was characterized with infrared (FTIR), nuclear magnetic resonance (NMR), high performance thin layer chromatography, (HPTLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. HPTLC profiles revealed the presence of glucose and glucuronic acid in all bacteria and mannose only in P. syringae pv. tomato. Molecular mass of EPS of P. syringae pv. pisi (m/z 933.8), P. syringae pv. tomato (m/z 950.4), and P. syringae pv. glycinea (m/z 933.5) was confirmed by MALDI-TOF mass spectrometry.