Microbial Community Dynamics and Response to Plant Growth-Promoting Microorganisms in the Rhizosphere of Four Common Food Crops Cultivated in Hydroponics.

Plant growth promoting microorganisms (PGPMs) of the plant root zone microbiome have received limited attention in hydroponic cultivation systems. In the framework of a project aimed at the development of a biological life support system for manned missions in space, we investigated the effects of PGPMs on four common food crops (durum and bread wheat, potato and soybean) cultivated in recirculating hydroponic systems for a whole life cycle. Each crop was inoculated with a commercial PGPM mixture and the composition of the microbial communities associated with their root rhizosphere, rhizoplane/endosphere and with the recirculating nutrient solution was characterised through 16S- and ITS-targeted Illumina MiSeq sequencing. PGPM addition was shown to induce changes in the composition of these communities, though these changes varied both between crops and over time. Microbial communities of PGPM-treated plants were shown to be more stable over time. Though additional development is required, this study highlights the potential benefits that PGPMs may confer to plants grown in hydroponic systems, particularly when cultivated in extreme environments such as space.

Intravarietal polymorphisms reveal possible common ancestor of native Schinus terebinthifolius Raddi populations in Brazil.

Schinus terebinthifolius Raddi is a perennial native from Atlantic forest. It is of high ecological plasticity and is used in traditional medicine. Based on promising reports concerning its bioactivity, it was included as a species of great interest for distribution through the National Health System. A number of agronomic studies to guide its crop production are therefore underway. This study examined diversity and phylogenetic relationships among native S. terebinthifolius populations from different Brazilian ecosystems: Cerrado; sandbanks; dense rainforest; and deciduous forest. The intergenic regions rpl20-5'rps12, trnH-psbA, and trnS-trnG were sequenced from cpDNA and aligned using BLASTn. There were few fragments for comparison in GenBank and so only region trnS-trnG was informative. There were variations among and within populations with intravarietal polymorphisms and three distinct haplotypes (HpSM, HpDDO, HpNE), once populations from NE (sandbanks and rainforest) clustered together. Sequences from HpSM, HpNE, and HpDDO returned greater similarity to haplotypes A (AY928398.1), B (AY928399.1), and C (AY928400.1), respectively. A network, built by median-joining among native haplotypes and 10 available on GenBank, revealed HpSM as the origin of all other haplogroups. HpDDO showed the most mutations and was closely related to haplogroups from Argentina. While this could indicate hybridization, we believe that the polymorphisms resulted from adaptation to events such as deforestation, fire, rising temperature, and seasonal drought during the transition from Atlantic forest to Cerrado. While more detailed phylogeographical studies are needed, these results indicate eligible groups for distinct climates as an important step for pre-breeding programs before field propagation.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.

The nodD locus from Azorhizobium caulinodans is flanked by two repetitive elements.

The sequence surrounding the Azorhizobium caulinodans (Ac) regulatory nodD gene was analyzed. Upstream from nodD and in the opposite orientation, two small open reading frames were identified (ORF1 and ORF2). The DNA sequence corresponding to ORF1, termed epsilon 1, is similar to a part of the insertion element IS51 from Pseudomonas savastanoi. Immediately downstream from nodD, a repeated element, delta 1, has been described [Goethals et al., Mol. Plant-Microbe Interact. 5 (1992) 405-411]. The elements epsilon 1 and delta 1 form the borders of a shift in GC content between nodD and its surrounding sequences. delta 1 and the ORF1+ORF2 sequence both occur as two copies in the Ac genome. Based on these observations, we postulate that the repeated elements played a role in the horizontal transfer of nodD during evolution. Insertion mutations in epsilon 1 and delta 1 did not influence the induction of the nodulation operon, nodABCSUIJ, and had no effect on the nodulation behavior on Sesbania rostrata. lacZ fusion studies suggested that nodD is constitutively transcribed and that the promoter driving nodD expression overlaps with the ORF1 sequence. In contrast, promoter activity in the direction of ORF1 and ORF2 was not observed. In the nodD-ORF1-intervening sequence, a nod box-related motif was recognized that deviates from active nod boxes by the absence of an ATC-9-bp-GAT palindrome, i.e., a sequence involved in NodD-mediated transcription stimulation [Goethals et al., Proc. Natl. Acad. Sci. USA 89 (1992) 1646-1650].

Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endoglucanase (Egl) activity. A clone carrying the gene responsible for this phenotype was isolated via introduction of a genomic library into the wild-type strain and screening for transconjugants with enhanced Egl activity. By subcloning and expression in Escherichia coli, the Egl phenotype was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence analysis showed the egl gene to be much larger, consisting of an open reading frame of 1,836 amino acids. Within the deduced polypeptide, three kinds of putative domains were identified: a catalytic domain, two cellulose-binding domains, and an eightfold reiterated motif. The catalytic domain belongs to the family A of cellulases. A C-terminal stretch of 100 amino acids was similar to family II cellulose-binding domains. A second copy of this domain occurred near the middle of the polypeptide, flanked by reiterated motifs. ORS571 mutants carrying a Tn5 insertion in the egl gene had lost the Egl activity. These mutants as well as Egl-overproducing strains showed a normal nodulation behavior, indistinguishable from wild-type nodulation on Sesbania rostrata under laboratory conditions.

Identification of a new inducible nodulation gene in Azorhizobium caulinodans.

The narrow host range bacterial strain Azorhizobium caulinodans ORS571 induces the formation of nitrogen-fixing nodules on the root and stem of the tropical legume Sesbania rostrata. Here, a new flavonoid-inducible locus of ORS571 is described, locus 4. The locus was identified and isolated via the occurrence of particular sequences, the gamma and delta elements. These elements are reiterated in the ORS571 genome, linked to symbiotic loci. Sequencing of locus 4 showed the presence of an open reading frame (ORF6) that is flanked downstream by a gamma element and upstream by a delta element. The gamma element is approximately 180 bp in size, and shows homology to the insertion element ISRm3, an insertion sequence belonging to a distinct class of IS elements. The delta element is about 300 bp in size and has homology with repeated sequences found in other Rhizobiaceae. The ORF6 gene product shows a low, but significant homology to the mouse mastocytoma antigen P35B (Szikora et al., EMBO J. 9: 1041-1050, 1990) and to a class of NAD/NADP-binding sugar epimerase/dehydrogenases (Pissowotzki et al., Mol. Gen. Genet. 231: 113-213, 1991). Immediately upstream from ORF6, a nod box-related sequence is present, the arrangement of which is fully consistent with a recently presented model for the nod box structure (Goethals et al., Proc. Natl. Acad. Sci. USA 89: 1646-1650, 1992). Insertional inactivation of ORF6 did not affect the nodulation and fixation performance on S. rostrata. However, on S. formosa roots the nodulation kinetics of such a mutant was clearly affected (about 5 days delay). We propose to call this new symbiotic gene nolK.