Production and partial characterization of chitinase from a halotolerant Planococcus rifitoensis strain M2-26

peer reviewedThis paper is the first to investigate the production and partial characterization of the chitinase enzyme from a moderately halophilic bacterium Planococcus rifitoensis strain M2-26, earlier isolated from a shallow salt lake in Tunisia. The impact of salt, salinity concentration, pH, carbon and nitrogen sources on chitinase production and activity have been determined. This is the first report on a high salt-tolerant chitinase from P. rifitoensis, since it was active at high salinity (from 5 to 30% NaCl) as well as in the absence of salt. This enzyme showed optimal activity at 70 C and retained up to 82 and 66% of its original activity at 80 or 90 C, respectively. The activity of the enzyme was also shown over a wide pH range (from 5 to 11). For characterization of the enzyme activity, the chitinase secreted in the culture supernatant was partially purified. The preliminary study of the concentrated dialysed supernatant on native PAGE showed at least three chitinases produced by strain M2-26, with highest activity approximately at 65 kDa. Thus, the thermo-tolerant and high salt-tolerant chitinases produced by P. rifitoensis strain M2-26 could be useful for application in diverse areas such as biotechnology and agro-industry

Assessment of the genetic diversity of Frankia microsymbionts of Elaeagnus angustifolia L. plants growing in a Tunisian date-palm oasis by analysis of PCR amplified nifD-K intergenic spacer

Diversity of Frankia microsymbionts of non-native Elaeagnus angustifolia L. plants spontaneously growing in a Tunisian desertic retreat area, the date-palm oasis of Tozeur, was investigated by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and PCR-sequencing techniques targeting the nifD-K intergenic spacer. Three PCR-RFLP haplotypes (I, II, and III) were detected among collected nodules. Haplotype I was detected at all five sampling sites and dominated the other haplotypes present at these sites. This haplotype was also exhibited by strain BMG5.10, which was isolated by a plant-capturing assay in 1998 from soil collected in the same locality, qualifying it to be the most competitive haplotype in the edapho-climatic condition of the studied desertic date-palm oasis. nifD-K sequences of the three haplotypes formed a closely related phylogenetic subgroup. These results suggest that Frankia variability is constrained by severe edapho-climatic conditions of retreated desert in Tunisian area

Occurrence and diversity of Frankia in Tunisian soil

There is a lack of studies on the occurrence and diversity of Frankia in African soils, including those in northern African regions. The present study on Tunisian soils is an attempt to address this issue using Alnus glutinosa, Elaeagnus angustifolia and Casuarina glauca in a plant capturing bioassay on 30 soil samples, followed by amplified 16S ribosomal DNA restriction pattern analysis (ARDRA). A total of seven ARDRA haplotypes of Frankia have been detected in root actinorhizas that have been affiliated to theoretical ARDRA haplotypes upon in silico digestion of selected 16S ribosomal RNA (rRNA) gene sequences retrieved from GeneBank and confirmed by their partial 16S rRNA gene sequencing. Elaeagnus-compatible Frankia isolates were widespread and form four ARDRA haplotypes affiliated to Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups. Alnus-compatible strains occurring in northern subhumid area were closely related to Alnus-Morella-compatible strains and clustered in two ARDRA haplotypes. Casuarina-compatible strains lack variability in several northern arboreta. The relatively wide diversity of Tunisian Frankia strains opens the perspective that African soil could be an interesting reservoir for the isolation of new actinorhizal strains that could be used as potential biofertilizers to counteract the progressive soil desertification which indeed is a crucial environmental problem in Northern Africa

Elimination of bacteria from in vitro shoot cultures of 'Douce de Djerba', a micropropagated apple cultivar

5 Pag., 2 Fig. The definitive version is available at: http://www.aida-itea.org/revista.html[ES] Se describe la aplicación de un método sencillo y eficaz para controlar una contaminación endógena bacteriana en cultivos in vitro de brotes de manzano, que mostraron crecimiento de bacterias de origen endógeno, mediante el subcultivo sucesivo de los ápices en medio de cultivo con cefotaxima. Las bacterias endógenas son frecuentes en cultivos in vitro iniciados a partir de explantos tomados de árboles adultos crecidos en campo y afectan tanto al crecimiento de brotes como a la organogénesis de brotes y raíces adventicias. Utilizamos brotes de manzano del cv. 'Douce de Djerba' cultivados in vitro que mostraban crecimiento bacteriano en la base de los brotes. Mientras que tratamientos de 1 h con soluciones de hipoclorito de sodio al 0.1 % o al 1.0 % dañaron los brotes y fueron ineficaces, la adición de cefotaxima estéril al medio autoclavado (MS modificado) a 150 mg l-1 inhibió el crecimiento bacteriano y mejoró tanto el crecimiento de los brotes como la expansión de las hojas. Tras el tercer subcultivo en medio con cefotaxima, no se detectó ningún crecimiento bacteriano en el medio, tanto en el medio de cultivo como en placas PYGA. La longitud de los brotes tratados con cefotaxima fue el doble que la de los brotes sin tratar (32.7 ± 2.71 mm vs. 15.95 ± 1.48 mm respectivamente). El método descrito aquí permitirá el control de bacterias endógenas sensibles a la cefotaxima in vitro, eliminando una fuente de variabilidad incontrolada que puede afectar diferentes estudios.[EN] We describe here the application of a simple and efficient method to control an endogenous bacterial contamination of in vitro shoot cultures of apple through successive subcultures of shoot apices on media with cefotaxime. Endogenous bacteria are frequent in cultures derived from explants taken from adult trees grown in the field and affect both shoot growth and organogenesis as in the adventitious regeneration of shoots and roots. We used apple shoots cv. 'Douce de Djerba' cultured in vitro that showed bacterial growth at the base of the shoots. While 1 h treatments with 0.1 % or 1.0 % sodium hypochlorite were harmful and ineffective, the addition of sterile cefotaxime to autoclaved culture medium (a modified MS medium) at 150 mg l-1 improved both shoot length and leaf expansion inhibiting bacterial growth. After the third subculture on medium with cefotaxime no visible bacterial growth was detected and shoots grown on MS without cefotaxime did not show any bacterial growth both in the culture medium and in PYGA plates. Length of cefotaxime-treated shoots was twofold that of untreated shoots (32.7 ± 2.71 mm vs. 15.95 ± 1.48 mm respectively). The method described here would allow the control of endogenous bacteria that are sensitive to cefotaxime in in vitro cultures, removing a source of uncontrolled variability that could affect different studies.Este trabajo ha sido financiado en parte por la ayuda recibida del Gobierno de Aragón como Grupo de Excelencia A-43. M.B. disfrutó de una ayuda del Gobierno de Túnez.Peer reviewe

Frankia nodulating Alnus glutinosa and Casuarinaceae in Tunisia

The capacity of some Tunisian soils to induce nodulation on Casuarina spp. and Alnus glutinosa was investigated through survey at fields and by plant-trapping bioassay. Frankia nodules were detected only in the north of Tunisia in some experimental forest stations for Casuarinaceae, and in natural endemic A. glutinosa stands. Frankia genetic diversity was assessed by PCR-RFLP of nifD-K region and, for Casuarinaceae, also of 16S-23S rDNA internal transcribed spacers, amplified from DNA directly extracted from root nodules. Restriction patterns showed that one and two haplotypes of Frankia colonise Casuarinaceae and A. glutinosa, respectively. Frankia in nodules of Casuarinaceae were found to be closely related to the group 1 of Casuarinaceae nodulating strains previously identified in Australia, corroborating the hypothesis of a recent introduction of these strains into Tunisia, probably with their hosts

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within single Frankia strains

Aims: Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. Methods and Results: Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5′-TGG-3′. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5′-CTTGGG-3′. Conclusions: We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. Significance and Impact of the Study: We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria

Soil parameters drive the diversity of Citrus sinensis rhizosphere microbiota which exhibits a potential in plant drought stress alleviation

Plant associated microorganisms, particularly those exhibiting a plant growth promoting (PGP) effect, play an important role in plant nutrition and health and the adaptation to unfavorable climatic conditions, such as drought which threatens the productivity of agricultural crops. The selection of specific microbial populations in the soil habitats associated to plants depends upon the soil physico-chemical parameters besides the \u2018rhizosphere effect\u2019 played by each plant species through rhizodepositions. In this study, we investigated the community structure and PGP potential of the microbiota associated to Citrus sinensis plants located in different geographical regions of Tunisia. The bacteria community structure was correlated to soil physiochemical parameters and we identified potassium, carbon and organic matter content as drivers of the C. sinensis microbiota composition. The evaluation of the potential of selected bacteria as biofertilizer and bio-stimulator under drought stress was achieved through the phylogenetic and functional characterization of a large collection of bacterial strains isolated from the rhizosphere of C. sinensis. The strains were screened in vitro for putative plant growth promoting traits, and the six most promising isolates were tested in vivo on Solanum lycopersicum and Capsicum annuum model plants. The bacterized plants were cultivated under drought stress and compared with not bacterized and fully irrigated control plants. All the tested bacteria induced a significant increase in the number of leaves and in root biomass of both plant species compared to not inoculated plants. Our results highlighted that the strains Ensifer adhaerens S1B1.5 and Pseudomonas resinovorans S4R2.6 were, in particular, effective in promoting plant growth under water shortage, indicating them as promising strains for the development of sustainable biofertilizers suited for agriculture in arid and semi-arid regions characterized by water scarcity

Effect of Rhizobium Isolates on Isoflavonoid Levels in Chickpea Plants Infected with Fusarium oxysporum f.sp. ciceris

The aim of the present studies was to determine the effect of two biocontrol agents, belonging to the genus Rhizobium, PchDMS and Pch43, on the accumulation of soluble phenolic compounds, and particularly constitutive isoflavonoids, in chickpea roots infected with Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea. Pretreatment of roots with the bacterial isolates before challenge with Foc significantly increased levels of soluble phenolic compounds in both the susceptible ILC482 and the moderately resistant INRAT87/ 1 chickpea cultivars. High performance liquid chromatography analysis revealed the isoflavones biochanin A and formononetin in the chickpea roots, in both the free and the glycosidically bound forms. Bacterization of the roots with Rhizobium isolates before challenge with Foc increased levels of these isoflavones in plant roots. The antifungal activity of crude phenolics extracted from the chickpea roots was tested in vitro on PDA amended with various concentrations of these extracts and inoculated with Foc. Crude phenolics significantly reduced fungal growth and caused considerable morphological changes in the mycelium, including marked cellular disorganization