Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco.

Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi CTAW11T with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but they showed values of 95.80% and 94.97% with B. cytisi CTAW11T, respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of B. cytisi CTAW11T isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies.

Molecular phylogeny of Bradyrhizobium bacteria isolated from root nodules of tribe Genisteae plants growing in southeast Poland.

The phylogeny of 16 isolates from root nodules of Genista germanica, Genista tinctoria, Cytisus ratisbonensis, and Cytisus scoparius growing in southeast Poland was estimated by comparative sequence analysis of core (16S rDNA, atpD, glnII, recA) and symbiosis-related (nodC, nodZ, nifH) genes. All the sequences analyzed placed the studied rhizobia in the genus Bradyrhizobium. Phylogenetic analysis of individual and concatenated housekeeping genes showed that the Genisteae microsymbionts form a homogeneous group with Bradyrhizobium japonicum strains. The phylogeny of nodulation and nitrogen fixation genes indicated a close relationship of the examined rhizobia with B. japonicum, Bradyrhizobium canariense, Bradyrhizobium cytisi, Bradyrhizobium rifense and Bradyrhizobium lupini strains infecting other plants of the tribe Genisteae. For the first time, the taxonomic position of G. germanica and C. ratisbonensis rhizobia, inferred from multigenic analysis, is described. The results of the phylogenetic analysis based on the protein-coding gene sequences presented in this study also indicate potential pitfalls concerning the choice of marker and reference strains, which may lead to conflicting conclusions in species delineation.

Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao.

BACKGROUND: Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches' broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. RESULTS: A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. CONCLUSIONS: In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease.

Genome-Wide Identification and Analysis of MicroRNAs Involved in Witches'-Broom Phytoplasma Response in Ziziphus jujuba.

MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches'-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches'-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches'-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches'-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5'-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches'-broom disease in Z. jujuba.

Discovery of microRNAs and transcript targets related to witches' broom disease in Paulownia fortunei by high-throughput sequencing and degradome approach.

Paulownia witches' broom (PaWB) caused by the phytoplasma is a devastating disease of Paulownia trees. It has caused heavy yield losses to Paulownia production worldwide. However, knowledge of the transcriptional and post-transcriptional regulation of gene expression by microRNAs (miRNAs), especially miRNAs responsive to PaWB disease stress, is still rudimentary. In this study, to identify miRNAs and their transcript targets that are responsive to PaWB disease stress, six sequencing libraries were constructed from healthy (PF), PaWB-infected (PFI), and PaWB-infected, 20 mg L(-1) methyl methane sulfonate-treated (PFI20) P. fortunei seedlings. As a result, 95 conserved miRNAs belonging to 18 miRNA families, as well as 122 potential novel miRNAs, were identified. Most of them were found to be a response to PaWB disease-induced stress, and the expression levels of these miRNAs were validated by quantitative real-time PCR analysis. The study simultaneously identified 109 target genes from the P. fortunei for 14 conserved miRNA families and 24 novel miRNAs by degradome sequencing. Furthermore, the functions of the miRNA targets were annotated based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The results presented here provide the groundwork for further analysis of miRNAs and target genes responsive to the PaWB disease stress, and could be also useful for addressing new questions to better understand the mechanisms of plant infection by phytoplasma in the future.

Cytisus villosus from Northeastern Algeria is nodulated by genetically diverse Bradyrhizobium strains.

Fifty-one rhizobial strains isolated from root nodules of Cytisus villosus growing in Northeastern Algeria were characterized by genomic and phenotypic analyses. Isolates were grouped into sixteen different patterns by PCR-RAPD. The phylogenetic status of one representative isolate from each pattern was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and one symbiotic gene (nodC). Analysis of 16S rRNA gene sequences showed that all the isolates belonged to the genus Bradyrhizobium. Phylogenetic analyses based on individual or concatenated genes glnII, recA, and atpD indicated that strains cluster in three distinct groups. Ten out of the sixteen strains grouped together with Bradyrhizobium japonicum, while a second group of four clustered with Bradyrhizobium canariense. The third group, represented by isolates CTS8 and CTS57, differed significantly from all other bradyrhizobia known to nodulate members of the Genisteae tribe. In contrast with core genes, sequences of the nodC symbiotic gene from all the examined strains form a homogeneous group within the genistearum symbiovar of Bradyrhizobium. All strains tested nodulated Lupinus angustifolius, Lupinus luteus, and Spartium junceum but not Glycine max. From these results, it is concluded that C. villosus CTS8 and CTS57 strains represent a new lineage within the Bradyrhizobium genus.

Disparate origins of Bradyrhizobium symbionts for invasive populations of Cytisus scoparius (Leguminosae) in North America.

To identify the geographic origin of nodule bacteria associated with invasion of the European legume Cytisus scoparius in the United States, isolates from 15 sites in six states were compared to > 200 Bradyrhizobium strains from indigenous legumes in the U.S., Mexico, Europe (six countries), Morocco, and Australia. Portions of five housekeeping loci (2849 bp) were sequenced, along with the nifD locus in the symbiosis island (SI) portion of the Bradyrhizobium chromosome. Bayesian phylogenetic analysis showed that North American C. scoparius symbionts had highly heterogeneous ancestry. Some were grouped into three distinct clades of European C. scoparius symbionts. One isolate had both housekeeping and SI genes belonging to a Bradyrhizobium clade from native legumes in western North America. Two other clades had mosaic ancestry: sequences for nifD as well as two other SI genes (nifH, nodC) were highly similar or identical to a C. scoparius strain from Spain, while their housekeeping loci belonged to American Bradyrhizobium clades. Thus, it appears that bacteria ancestrally associated with other North American legumes have evolved to utilize C. scoparius, by acquiring SI-region genes from European C. scoparius symbionts. Inoculation assays indicated that North American isolates were as competent as European strains in promoting plant growth, consistent with the findings on symbiont ancestry.

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif.

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).

Characterization of Bradyrhizobium species isolated from root nodules of Cytisus villosus grown in Morocco.

From a total of 73 bacterial strains isolated from root nodules of Cytisus villosus grown in soils of the central-western region of the Moroccan Rif, 68 strains clustered into 19 repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) groups. The nearly complete 16S rRNA gene sequence from each strain showed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis showed that 11 out of the 19 strains belong to Bradyrhizobium canariense and that another three strains were Bradyrhizobium japonicum. The remaining five strains represented new lineages within the genus Bradyrhizobium since they were not identified with any previously described species. Sequencing of the symbiotic nodC and nifH genes from each bradyrhizobial strain revealed they were all similar to those of the strains included in biovar genistearum.

Bradyrhizobium cytisi sp. nov., isolated from effective nodules of Cytisus villosus.

Several strains isolated from Cytisus villosus nodules have been characterized based on their diverse genetic, phenotypic and symbiotic characteristics. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to Bradyrhizobium canariense BTA-1(T) with 99.4% similarity. Analysis of three housekeeping genes, recA, atpD and glnII, suggested that the C. villosus strains represent a novel Bradyrhizobium species most closely related to B. canariense BTA-1(T) with similarities of 94.2, 96.7 and 94.5%, respectively. All these differences were congruent with DNA-DNA hybridization analysis, which revealed 31% relatedness between a representative strain (CTAW11(T)) isolated from C. villosus nodules and B. canariense BTA-1(T). Phenotypic differences among the strains isolated from C. villosus and B. canariense were based on assimilation of carbon and nitrogen sources. The nodC and nifH genes of strain CTAW11(T) were phylogenetically related to those of strains belonging to bv. genistearum and divergent from those of bv. glycinearum and, accordingly, they do not nodulate soybean. Based on the genotypic and phenotypic data obtained in this study, our strains should be classified as representatives of a novel species for which the name Bradyrhizobium cytisi sp. nov. is proposed; the type strain is CTAW11(T) (=LMG 25866(T)=CECT 7749(T)).

[Cloning and analysis of tuf and rp gene of the phytoplasma associated with jujube witches' -broom].

OBJECTIVE: Jujube witches' -broom (JWB) is an important plant disease caused by phytoplasma. The major objective of our research was to classify JWB in Beijing and Hebei districts and to provide reference for classification in subgroup level. METHODS: By use of PCR, the elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs fTufu/rTufu and rp(v)F1A/rp(v) R1A. Partial tuf gene and rp gene were sequenced and similarity analysed with other phytoplasmas. RESULTS: We obtained partial tuf gene sequence (824bp) and complete rp gene (1196bp) from the diseased sample. In tuf gene, JWB in Beijing shared most similarity (92.84%) with Flavescence dorée (FD) phytoplasma (Candidatus Phytoplasma vitis), however, shared a low similarity (57.29%) with JWB in Shaanxi district which had been already reported. The similarity analysis for sequences of rp gene showed a high identity (> 96%) with members of the 16SrV group phytoplasmas. It shared most identity (99.83%) with JWB strain Taishan and Hemp fiber witches' -broom phytoplasma (HFWB) of the 16SrV group. CONCLUSION: The JWB strains in Beijing and Hebei are members of 16Sr V; JWB in Beijing and Hebei share high similarity, and show a diversity with JWB in Shaanxi.

Ochrobactrum cytisi sp. nov., isolated from nodules of Cytisus scoparius in Spain.

Two strains named ESC1(T) and ESC5 were isolated from nodules of Cytisus scoparius growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene showed that these strains belong to the genus Ochrobactrum, their closest relatives being Ochrobactrum anthropi and Ochrobactrum lupini, with 100 and 99.9 % similarity to the respective type strains. Despite this high similarity, the results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses showed that these strains represent a novel species of genus Ochrobactrum. The DNA-DNA hybridization values were respectively 70, 66 and 55 % with respect to O. lupini LUP21(T), O. anthropi DSM 6882(T) and Ochrobactrum tritici DSM 13340(T). The predominant fatty acids were C(18 : 1)omega7c and C(18 : 1) 2-OH. Strains ESC1(T) and ESC5 were strictly aerobic and were able to reduce nitrate and to hydrolyse aesculin. They produced beta-galactosidase and beta-glucosidase and did not produce urease after 48 h incubation. The G+C content of strain ESC1(T) was 56.4 mol%. Both strains ESC1(T) and ESC5 contained nodD and nifH genes on megaplasmids that were related phylogenetically to those of rhizobial strains nodulating Phaseolus, Leucaena, Trifolium and Lupinus. From the results of this work, we propose that the strains isolated in this study be included in a novel species named Ochrobactrum cytisi sp. nov. The type strain is ESC1(T) (=LMG 22713(T)=CECT 7172(T)).

Molecular diversity of rhizobia nodulating the invasive legume Cytisus scoparius in Australia.

AIMS: To contribute to the understanding of Cytisus scoparius success at invading and establishing itself in Australia. METHODS AND RESULTS: Root-nodule bacteria isolated from C. scoparius, growing on five different sites and originally introduced to Australia, were compared with isolates from indigenous plants growing in France and isolates from native legumes growing on the same Australian sites as C. scoparius. Small-subunit rDNA from 251 isolates were analysed by PCR-RFLP and representatives from different genospecies were selected for sequencing. Phylogenetic analyses revealed a great diversity of lineages belonging to Bradyrhizobium, with one genospecies being specific for Cytisus both in Australia and in France, Rhizobium and Mesorhizobium and one falling outside the described genera of legume-nodulating bacteria. Principal component analysis showed that the Cytisus Australian rhizobial communities are more similar to each other than to their co-occurring native partners. CONCLUSIONS: Early established rhizobial symbionts may have an increased probability to contribute inoculum for the development of further nodules. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report comparing rhizobia nodulating C. scoparius in its native and exotic environments. Cytisus scoparius symbionts were identified outside the Bradyrhizobium genus and a new lineage of legume-nodulating bacteria was identified.

Characterization of phages virulent for Sarothamnus scoparius bradyrhizobia.

Four virulent phages--PhiDl, PhiTl, PhiCYT21, and PhiOS6, infective on Sarothamnus scoparius rhizobia--were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (PhiDl, PhiTl) and Myoviridae with long, contractile tails (PhiCYT21, PhiOS6). They were also classified into two groups by a host specificity. One of them included viruses (PhiDl and PhiTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (PhiCYT21 and PhiOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p.