Assessment of the phenotypic and genotypic diversity of endophytic strains of Bacillus and closely related genera from Carpinus betulus in the Hyrcanian forests of Iran.

BACKGROUND: Identification and characterization of the endophytic microorganism, is gaining their underestimated significance in influencing health, performance, and other biological attributions of plants in general and forest tree species in particular. Because of the scarcity of information on the endophytic microbiome of the Hyrcanian forests species, including hornbeam (Carpinus betulus L.) trees, as a major constituent thereof, the present study aimed at the identification and partial characterization of the endophytic Bacillus species of Carpinus betulus as the first step in this context. METHODS AND RESULTS: Shoot samples were collected from the Hyrcanian forest locations of Mazandaran and Golestan provinces in Iran. Bacterial strains were isolated from the surface-disinfected shoot segments and subjected to phenotypic characterization. Following assessment of the genetic diversity of the isolates by BOX-PCR fingerprinting, the representative isolates of each of the 15 groups were used for further characterization. Analysis of the nucleotide sequences of the 16S rDNA and HSP60 gene of the isolates led to the identification of 10 species. The predominant species was B. cereus followed by B. subtilis. The other species encountered were B. thuringiensis, Priestia filamentosa, B. velezensis, B. mojavensis, B. amyloliquefaciens, B. safensis, P. aryabhattai, and Gottfriedia acidiceleris. Most isolates possessed characteristics which could contribute to the biocontrol potential of the isolates, including formation of biofilm, production of hydrogen cyanide, tolerant to relatively high concentration of sodium chloride, and antibacterial activity. CONCLUSIONS: Ten Bacillus species were identified as the prevailing endophytic species of C. betulus in the Hyrcanian forest of northern Iran, most turned up to possess biological activities involved in biocontrol capability of the isolates against some plant pathogens. These potentially capable bacteria could be implemented in the promotion of plant growth as well as in the biological control of pathogens. This is the first report on the characterization and elucidation of the diversity of the potentially beneficial endophytic species of Bacillus and the closely related genera living in the internal tissues of hornbeam trees.

Complete Genome Sequencing and Targeted Mutagenesis Reveal Virulence Contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in Bacterial Leaf Streak of Wheat.

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas, but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas, including KG, NF, Y∗, YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b, only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae phylogenetic group.

In a screening by multilocus sequence analysis of Pseudomonas strains isolated from diverse origins, 4 phylogenetically closely related strains (FBF58, FBF102T, FBF103, and FBF122) formed a well-defined cluster in the Pseudomonas syringae phylogenetic group. The strains were isolated from citrus orchards in northern Iran with disease symptoms in the leaves and stems and its pathogenicity against citrus plants was demonstrated. The whole genome of the type strain of the proposed new species (FBF102T=CECT 9164T=CCUG 69273T) was sequenced and characterized. Comparative genomics with the 14 known Pseudomonas species type strains of the P. syringae phylogenetic group demonstrated that this strain belonged to a new genomic species, different from the species described thus far. Genome analysis detected genes predicted to be involved in pathogenesis, such as an atypical type 3 secretion system and two type 6 secretion systems, together with effectors and virulence factors. A polyphasic taxonomic characterization demonstrated that the 4 plant pathogenic strains represented a new species, for which the name Pseudomonas caspiana sp. nov. is proposed.

New Pseudomonas spp. Are Pathogenic to Citrus.

Five putative novel Pseudomonas species shown to be pathogenic to citrus have been characterized in a screening of 126 Pseudomonas strains isolated from diseased citrus leaves and stems in northern Iran. The 126 strains were studied using a polyphasic approach that included phenotypic characterizations and phylogenetic multilocus sequence analysis. The pathogenicity of these strains against 3 cultivars of citrus is demonstrated in greenhouse and field studies. The strains were initially grouped phenotypically and by their partial rpoD gene sequences into 11 coherent groups in the Pseudomonas fluorescens phylogenetic lineage. Fifty-three strains that are representatives of the 11 groups were selected and analyzed by partial sequencing of their 16S rRNA and gyrB genes. The individual and concatenated partial sequences of the three genes were used to construct the corresponding phylogenetic trees. The majority of the strains were identified at the species level: P. lurida (5 strains), P. monteilii (2 strains), P. moraviensis (1 strain), P. orientalis (16 strains), P. simiae (7 strains), P. syringae (46 strains, distributed phylogenetically in at least 5 pathovars), and P. viridiflava (2 strains). This is the first report of pathogenicity on citrus of P. orientalis, P. simiae, P. lurida, P. moraviensis and P. monteilii strains. The remaining 47 strains that could not be identified at the species level are considered representatives of at least 5 putative novel Pseudomonas species that are not yet described.

Chitinolytic and antifungal activity of a Bacillus pumilus chitinase expressed in Arabidopsis.

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin-glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.

RP-HPLC prefractionation and its application in expressional proteomics analysis of an in vitro viral infection model.

Prefractionation of complex protein mixtures is an efficient method for increasing the separation power of 2-DE. RP-HPLC has been successfully utilized as a prefractionation method prior to 2-DE. Here we describe the optimization of an efficient RP-HPLC method for prefractionation of baby hamster kidney cell solubilized proteins. A step gradient elution of acetonitrile was optimized and collected fractions were further examined by SDS-PAGE and 2-DE. By utilizing this method an effective increase in separation power of 2-DE is accomplished. Moreover, we describe the application of this method to expressional proteome analysis of a virally infected cell model.