Molecular characterization and identification ofSaprolegnia by restriction analysis of genes coding for ribosomal RNA

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species ofSaprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except forS. hypogyna, S. delica, S. lapponica, andS. mixta. The rDNA data indicate thatS. lapponica andS. mixta are conspecific withS. ferax, whereas there is no support for the proposed synonymies ofS. diclina withS. delica and ofS. mixta withS. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in theS. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains ofS. parasitica originating from the U.K. and Japan.HhaI andBsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonucleaseBstUI was diagnostic forS. parasitica, generating identical fingerprints for all strains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved inS. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed thatS. diclina andS. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification ofSaprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.     

Population biology ofHortaea werneckii based on restriction patterns of mitochondrial DNA

Fifteen isolates ofHortaea werneckii, causative agent of tinea nigra in man, were examined with respect to restriction fragment length polymorphisms of mitochondrial DNA. Seven types of mtDNA, interpreted as populations, could be distinguished, with similarities between the restriction patterns ranging from 32 to 79%. Much of the variance originated from length mutations. Of the seven populations four represented isolates from man, two of which also comprised isolates from other sources. This makes adaptation ofH. werneckii towards association with man in its evolution unlikely; similarity in the chemical and/or physical characteristics of the different isolation sources, viz. salinity, seems more probable. mtDNA types were not correlated with geographic origin. Isolates with the same mtDNA type are widely geographically distributed.     

Species-specific banding patterns of restriction endonuclease-digested mitochondrial DNA from the genusPythium

Restriction endonuclease-digested mitochondrial DNA from 29Pythium spp. showed distinctly different species-specific electrophoretic banding patterns. Numerical comparisons among species were conducted by calculating the percentage of restriction fragments having the same apparent molecular size. The greatest interspecific similarity in banding patterns (67%) was observed betweenHindIII digests ofPythium heterothallicum andP. sylvaticum. However, comparisons among other species generally revealed similarities of less than 50%, and often less than 30%. The lack of similarity of restriction banding patterns was observed even with several species that share many common morphological features:P. arrhenomanes vsP. graminicola (20%),P. myriotylum vsP. aristosporum (28%), andP. torulosum vsP. vanterpoolii (32%). In contrast to the fragment size heterogeneity among different species, isolates of the same species have highly conserved restriction patterns. Ten isolates ofP. oligandrum, collected from the United States, South Africa, and Czechoslovakia, had a minimum of 86% similarity inHindIII banding patterns. Similar results were observed with eight isolates ofP. ultimum, five ofP. acanthicum, six ofP. spinosum, five ofP. sylvaticum, and eight ofP. irregulare. However, two isolates ofP. irregulare exhibited a higher degree of heterogeneity and shared only 64 to 76% comigrating bands with the eight other isolates of this species.     

Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

Contributions to the genus phytophthora de bary from india

The paper deals with some of the contributions made from India by various research workers towards our knowledge of the fungus genusPhytophthora de Bary, in the fields of morphology, disease production, occurrence and distribution of species, host range, physiology, taxonomy and control measures. Out of the total of fifteen species and five varieties ofPhytophthora reported from India, five species (viz.,P. arecae, P. palmivora, P. colocasiae, P. parasitica andP. infestans, and four varieties ofP. parasitica, viz.,P. parasitica var.macrospora, var.sesami, var.piperina and var.nicotianae) assume considerable parasitic roles inciting serious diseases like seedling-blights, wilts, cottoney-leaks, foot-rots, blights, budrot and rots of fruits, nuts, tubers and corms etc. They are widely distributed in various parts of this country, and are mostly prevalent in rainy seasons.Phytophthora palmivora, andP. parasitica were found to have a wide host range.     

Starch utilization by Phytophthora spp

Summary Phytophthora spp. were grown on artificial starch agar medium. In some cases, the capacity of starch utilization could be a useful tool in species separation ofPhytophthora. Based on the ability to hydrolyse starch,P. palmivora andP. parasitica could be readily distinguished, whereasP. parasitica andP. parasitica var.nicotianae, P. megasperma andP. megasperma var.sojae (P. sojae) behaved similarly. Starch hydrolysis was indicated by a clear unstained zone within the fungal colony when treated with iodine solution. Simple quantitative analysis of starch hydrolysis was made feasible by the following formula:Starch Hydrolysis Index (S.H.I.) = Mean diameter of clear starch hydrolysis zone (d) / Mean diameter of fungal colony (D)     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Patterns of genetic differentiation in two annual bromegrasses,Bromus lanceolatus andB. hordeaceus (Poaceae)

Infraspecific genetic differentiation was analysed in two tetraploid annual bromegrasses,Bromus lanceolatus (from N Africa) andB. hordeaceus (from N Africa and France). Genetic analysis of populations was based on allozyme polymorphisms at 17 loci. Different fixed heterozygous phenotypes were scored in both species, according to their allopolyploid origin. In N Africa, more variation occurred among populations ofB. lanceolatus than ofB. hordeaceus. The variation was not randomly distributed among populations of both species. InB. lanceolatus, differentiation was correlated with climatic variables rather than with geographic distance between populations. Higher correlation of genetic differentiation with geographic distance occurred inB. hordeaceus, particularly at large geographic scale, between French and N African populations. Within each region, the populations appeared weakly genetically differentiated, even when belonging to different subspecies.     

In vitro expression of partial resistance toPhytophthora palmivora by shoot cultures of papaya

Shoot apices ofCarica papaya were multiplied in vitro on solidified nutrient media supplemented with -naphthyl-acetic acid and 6-benzylaminopurine. The micropropagated shoots were inoculated in vitro, through a stem wound, with a sporangial suspension (1.2×10⁴ sporangia ml^-1) ofPhytophthora palmivora. The symptoms exhibited by the shoots in vitro were similar to those described previously for infection of the whole plant in the field. The time taken for the host tissue to become brown and to wilt and the time to sporulation of the pathogen were all recorded for each shoot of four varieties of papaya challenged with each of ten isolates ofP. palmivora. Significant differences were observed between host-pathogen combinations for these variables and host-specificity was detected amongst the isolates ofP. palmivora. The time taken for the shoot to wilt was positively correlated with the time to sporulation of the isolated but both these variables were negatively correlated with the time to browning of the shoot. In vitro selection for disease resistance will be useful during breeding programmes involving papaya genotypes which are maintained through clonal propagation.     

Speciation and paraphyly in western mediterranean hares (Lepus castroviejoi,L. europaeus,L. granatensis,andL. capensis) revealed by mitochondrial DNA phylogeny

Mitochondrial DNA (mtDNA) variation among specimens of the northwestern African hare (Lepus capensis schlumbergeri) and three European hares sampled in Spain (L. castroviejoi andL. granatensis, which are endemic to the Iberian Peninsula, andL. europaeus) was analyzed using seven restriction endonucleases. Fourteen haplotypes were found among the 34 animals examined. Restriction site maps were constructed and the phylogeny of the haplotypes was inferred. mtDNA ofL. capensis was the most divergent, which is consistent with its allopatric African distribution and with an African origin of European hares. We estimated that mtDNA in hares diverges at a rate of 1.5–1.8% per MY assuming that the European and African populations separated 5–6 MYBP. Maximum intraspecies nucleotide divergences were 1.3% inL. capensis, 2.7% inL. castroviejoi, and 2.3% inL. granatensis but 13.0% inL. europaeus. The latter species contained two main mtDNA lineages, one on the branch leading toL. castroviejoi and the other on that leading toL. granatensis. The separation of these two lineages from theL. castroviejoi orL. granatensis lineages appears to be much older than the first paleontological record ofL. europaeus in the Iberian peninsula. This suggests that the apparent polyphyly ofL. europaeus is due not to secondary introgression, but to the retention of ancestral polymorphism inL. europaeus. The results suggest thatL. europaeus either has evolved as a very large population for a long time or has been fractionated. Such a pattern of persistence of very divergent lineages has also been reported in other species of highly mobile terrestrial mammals. As far as mtDNA is concerned,L. europaeus appears to be the common phylogenetic trunk which has diversified during dispersion over the European continent and from whichL. castroviejoi andL. granatensis speciated separately in southwest Europe.     

辣椒疫霉RXLR型效应子PcAvh2的序列多态性、基因转录特征及功能

【目的】分析辣椒疫霉中RXLR型效应子PcAvh2的序列多态性,研究该效应子在辣椒疫霉生长发育和侵染阶段的转录特征及其生物学功能。【方法】本研究通过高保真扩增,分析2个烟草疫霉、1个恶疫霉和31个辣椒疫霉菌株的PcAvh2序列;提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点(1.5、3、6、12、24、36、72 h)的本氏烟根部总RNA,利用RT-qPCR分析PcAvh2的转录表达水平;利用PVX瞬时表达系统,分析PcAvh2是否抑制6种效应子(BAX、INF1、PsojNIP、PsCRN63、PsAvh241、R3a/Avr3a)激发的植物免疫反应;利用CaCl_2-PEG介导的原生质体稳定转化技术,沉默PcAvh2基因,分析辣椒疫霉致病力的变化。【结果】PcAvh2为典型的RXLR效应子,在辣椒疫霉群体中该效应子具有10个等位基因,而且烟草疫霉和恶疫霉中也存在该效应子。该基因在辣椒疫霉的侵染阶段上调表达,它能够抑制6种效应子激发的植物免疫反应,进一步研究发现基因沉默导致辣椒疫霉的致病力显著下降。【结论】RXLR型效应子PcAvh2是辣椒疫霉中一个重要的侵染致病因子。     

PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.     

Aggressiveness and diversity of Phytophthora capsici on vegetable crops in Georgia

Phytophthora blight induced by Phytophthora capsici causes significant yield loss in a number of vegetable crops. It is imperative to understand the diversity and aggressiveness of the pathogen to design more efficient disease management programs. A collection of P. capsici strains isolated from different vegetable crops in Georgia, USA, were characterised in this study. Of the 49 isolates tested, 24 were A1 and 25 were A2 mating type, respectively, with both mating types found in the same fields. Variability of the isolates was assessed in terms of their aggressiveness on six pepper genotypes. The isolates differed in their aggressiveness on different pepper cultivars with 10 pathotypes identified. No correlation between aggressiveness of the isolates and their host origin or geographical location of isolation was observed. Randomly amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic variability among P. capsici populations. RAPD analysis using 15 random primers resulted in 133 reproducible bands and cluster analysis separated the isolates into 5 groups. Analysis of molecular variance showed that there was moderate genetic differentiation associated with host origin and geographical location of the isolates. No correlation was found between RAPD groups and pathotypes or mating types. These results indicate that P. capsici populations infecting vegetable crops in Georgia were genetically diverse, which should be taken into account in developing resistant cultivars or other disease management programmes.     

The phytogeography of leaf morphology in two species of North American Asters: Lasallea Novae-Angliae and L. Oblongifolia (Compositae)

Width-to-length ratios of upper stem leaves ofLasallea novaeangliae andL. oblongifolia were determined from herbarium specimens and field collections. No distinct subgroups based on suchratios were found in either species. InL. novaeangliae the curve of the ratio versus number of individuals was skewed toward the narrow-leaf range. This phenomenon was present but less pronounced inL. oblongifolia. The histogram of leaf ratios forL. novaeangliae was somewhat weakly bimodal, suggesting a possible broad-leaf race, which has not been recognized nomenclaturally. The absence of bimodality in the histogram of leaf ratios forL. oblongifolia indicates the need to place Shinners’ narrow-leaved varietyangustatus in synonymy. Other traits such as height, degree of branching and the nature of the pubescence were also found to be of no significance. Slight geographic trends in leaf width occur in both species. No real evidence was found for nomenclaturally recognizing any eastern variant in either species. Thus, Shinners’ var.orientis is not recognized inL. oblongifolia either.     

Species-specific polymorphisms in transcribed ribosomal DNA of fivePythium species

Twenty-five isolates representing fivePythium species collected from diverse hosts and geographic origins were evaluated using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. DNA regions coding for the small-subunit ribosomal RNA (SrDNA) and the internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The amplified SrDNA was about 1800 bp long and uniform in length among the five species. However, restriction digestion revealed three polymorphic groups. They areP. arrhenomanes andP. graminicola,P. irregulare andP. spinosum, andP. ultimum. The amplified-ITS region showed three different lengths which corresponded to the three polymorphic groups of SrDNA. Each length variant of the ITS showed distinct banding patterns after restriction enzyme digestion. In addition,P. irregulare andP. spinosum each showed distinct banding patterns after digestion with enzymesHinfI andMboI. Physical maps of the restriction sites in the SrDNA and the ITS were determined. Length variation occurred primarily in the spacer between the SrDNA and 5.8 S rDNA; although, it also was detected in the ITS-2 region. Little intraspecific variation was observed in the SrDNA and ITS, and species could be reliably distinguished by RFLP analysis of the amplified rDNA regions. Data presented do not support the maintenance ofP. arrhenomanes andP. graminicola as distinct species. Results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for ecological studies ofPythium species.     

Some biochemical characteristics of fungi isolated from salmonid eggs

Fungal isolates from salmonid eggs displayed apparently unique patterns of biochemical characteristics at both the generic and specific levels. of the five genera examinedAchlya andPythium were able to assimilate 13–16 out of 19 carbohydrates.Aphanomyces was able to assimilate only glucose and starch, which was assimilated by all isolates. Members ofSaprolegnia displayed identical patterns of carbohydrate assimilation, except forS. hypogyna, which was also able to assimilate melibiose, in common withAchlya, Pythium, andLeptolegnia. Pythium was the only genus capable of assimilating salicin. OnlyAchlya andP. monospermum were able to assimilate rhamnose. In terms of amino acid assimilation isolates ofSaprolegnia ferax andS. diclina displayed an identical patterns, as did isolates ofS. parasitica andS. hypogyna. OnlyAphanomyces frigidophilus isolate was capable of assimilating cysteine. All genera exceptPythium assimilated glutamine, a fundamental amino acid. All isolates exhibited lipase and fatty acid esterase activities but no cellulase acitivity. The biochemical characteristics discovered in this study offer possibilities for identification and classification of these fungi, which are discussed herein.     

Phytophthora palmivora from Sulawesi and Java Islands,Indonesia, reveals high genotypic diversity and lack of population structure

Phytophthora palmivora is the causal agent of cocoa black pod disease, one of the primary diseases of cocoa in Indonesia. A better understanding of P. palmivora population genetics is needed to aid the development of relevant disease management strategies. This study is the first population genetic study of P. palmivora in Indonesia using microsatellite markers based on the alleles genotyping method. The microsatellite markers were used to determine the genotype of 44 P. palmivora isolates from Sulawesi (24) and Java (20) islands. The total number of observed multilocus genotypes (MLG) from both populations was 34. The genotypic diversity of P. palmivora from Sulawesi (2.90; 16.0; 0.938) and Java (2.76; 14.3; 0.930) islands was high as seen from Shannon's diversity index (H), Stoddart and Taylor's Index (G), and Simpson's Index (λ) respectively. Evenness and Nei's unbiased gene diversity exhibited similarly high levels from both populations. The linkage disequilibrium test indicated that sexual recombination occurred in the Java population (P = 0.312). Analysis of molecular variance (AMOVA) and Bayesian clustering revealed five genetic clusters, and isolates from both islands were evenly distributed across the five gene clusters. All genetic diversity was from within individuals. P. palmivora from Sulawesi and Java showed a high genotypic diversity but a lack of genetic differentiation among populations (Fst = 0.006). Both populations formed one highly diverse group. Minimum spanning network analysis showed no particular grouping of MLGs, and shared MLGs from both populations indicated long-distance migration of P. palmivora facilitated by human activities.     

Intraspecific Variation within Populations of Fusarium oxysporum Based on RFLP Analysis of the Intergenic Spacer Region of the rDNA

Appel, D. J., and Gordon, T. R. 1995. Intraspecific variation within populations of Fusarium oxysporum based on RFLP analysis of the intergenic spacer region of the rDNA. Experimental Mycology 19, 120-128. Fifty-six isolates of Fusarium oxysporum, including F. oxysporum f. sp. melonis and nonpathogenic strains, were chosen from a larger collection to represent diversity in vegetative compatibility groups (VCGs), mitochondrial DNA (mtDNA) haplotype, geographic distribution, and virulence. Using PCR, a 2.6-kb fragment including the intergenic spacer (IGS) region of the ribosomal DNA was amplified from each isolate. The enzymes EcoRI, Sau 3A, Cfo1, and Ava1I, cut this fragment differentially, revealing 5, 6, 6, and 7 patterns, respectively. Among the 56 isolates, a total of 13 unique IGS haplotypes was identified. Among most F. o. melonis isolates. IGS haplotype correlated with VCG and mtDNA haplotype, but did not differentiate among races. However, a race 1 isolate found in VCG 0131 shared virulence, mtDNA, and IGS haplotypes characteristic of VCG 0134; this isolate may represent a conversion in VCG from 0134 to 0131. Four nonpathogens shared the pathogen vegetative compatibility phenotypes. One race 1,2 isolate associated with VCG 0134 shared both IGS haplotype and VCG with a nonpathogen, but these isolates did not share the same mtDNA haplotype. Another nonpathogenic isolate shared mtDNA and IGS haplotypes with pathogen group 0131 and may simply be an avirulent mutant of a pathogenic strain. For the other two nonpathogenic isolates, vegetative compatibility indicated a close relationship to the pathogen, but differences in both mtDNA and IGS haplotype suggest otherwise. Overall, the IGS haplotype was more variable among the nonpathogenic F. oxysporum VCGs among which 12 of the 13 IGS haplotypes were found. Nonpathogenic isolates that shared a common mtDNA haplotype, but were associated with different VCGs, often had different IGS haplotypes.     

Evidence of genetically diverse virulent mating types of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> from <Emphasis Type="Italic">Capsicum annum</Emphasis> L.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), β-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida,strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.