Evidence for calcium-mediated perception of plant symbiotic signals in aequorin-expressing Mesorhizobium loti

<p>Abstract</p> <p>Background</p> <p>During the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca²⁺changes are an integral part of the signalling mechanism. In rhizobia it is not yet known whether Ca²⁺can act as a transducer of symbiotic signals.</p> <p>Results</p> <p>A plasmid encoding the bioluminescent Ca²⁺probe aequorin was introduced into <it>Mesorhizobium loti </it>USDA 3147^Tstrain to investigate whether a Ca²⁺response is activated in rhizobia upon perception of plant root exudates. We find that <it>M. loti </it>cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca²⁺concentration. Only root exudates from the homologous host <it>Lotus japonicus </it>induce Ca²⁺signalling and downstream activation of nodulation genes. The extracellular Ca²⁺chelator EGTA inhibits both transient intracellular Ca²⁺increase and inducible <it>nod </it>gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible.</p> <p>Conclusion</p> <p>These findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis.</p

Camelina (Camelina sativa L. Crantz) a new oilseed crop for Mediterranean and Balkan European climates

Nowadays in Europe the new multipurpose oilseed crop, camelina (Camelina sativa L. Crantz), is not yet widely cultivated but in the last decade it has gained interest among farmers and other stakeholders in the value chain, in relation to its satisfactory yield, low input requirement, and suitability to different pedo-climates. If until now camelina has been grown as a spring crop in northern Europe, more recently southern environments in the Mediterranean basin and in the Balkan region have been targeted as suitable growing areas, either in autumn, winter or spring sowing. Nevertheless, the limited number of winter genotypes available, together with the typical winter season in those areas which is not very harsh, make the possibility to grow spring camelina genotypes with autumn cycle a feasible option. Aiming at defining the most suitable genotype and the optimal sowing date in the Mediterranean and Balkan regions a common trial has been established in autumn 2020 comparing four camelina genotypes (3 spring + 1 winter) and two sowing dates (early vs. late) across three locations in Italy (Bologna, 44° 30’ N, 11° 23’ E), Serbia (Novi Sad, 45° 15’ N, 19° 51’ E), and Spain (Lleida, 52° 10’ N, 4° 29’ E)

Monitoring a genetically modified Pseudomonas sp. released on pine leaves reveals concerted successional patterns of the bacterial phyllospheric community

The fate of a biocontrol agent released on pine phyllosphere in a greenhouse-confined trial was followed over 102 days. The microorganism used, a Pseudomonas sp., isolated from Pinus nigra, carries the cry9a toxin gene from Bacillus thuringiensis. In order to detect the GMM, specific primers were used, and a previously defined protocol for DNA isolation from bacteria colonizing pine needles was applied. The method, based on vortexing in a suspension of glass beads followed by microcolumn extractions, allowed sensitive PCR monitoring of the target transgenes. The presence of the released organism was recorded throughout the trial and compared with its entomocidal performance towards larvae of the pine processionary caterpillar Thaumetopoea pityocampa. At the same time the dynamics of the released Pseudomonas within the whole epiphytic bacterial community, was followed by amplifying 16S rDNA pools and comparing ARDRA profiles at seven sampling points. The resulting dendrogram allowed to follow the time-dependent progressive blending of the Pseudomonas profile into those of the resident biota. PCR-dominance of the released bacterium in the community was extended until 21 days from release while its activity against insect larvae lasted for over 3 months. The prokaryotic epiphytic population, irrespective of any particular impact from the released strain, showed no resilience but a general successional trend, which, remarkably, appeared synchronous on all trees tested, including non-inoculated controls. This observation suggests interesting patterns of concerted environmental shifts by phyllospheric microorganisms

Bioinsetticidi ricombinanti di natura microbica

Capitolo in libro di testo per studenti universitar

Methods and detection limits in tracking a genetically modified Pseudomonas sp released in the pine phyllosphere

A method suitable to detect the presence and follow the fate of specific bacteria released on the phyllosphere of conifer trees was devised, tested, and optimised. The procedure was set up using a biocontrol strain that had shown effectiveness and persistence in greenhouse trials against insect pests. The microorganism used is based on a Pseudomonas sp., originally isolated from Pinus nigra and carries the cry9a toxin gene from Bacillus thuringiensis. In order to assess its detectability, specific primers were designed, and the most suitable protocol for DNA isolation from bacteria colonising pine needles was defined upon an experimental comparison of various methods. Different conditions of physical pre-treatments and their combinations with commercially available kits protocols were tested. The most sensitive monitoring (about 102 released cells) was achieved by a procedure based on vortexing in a suspension of glass beads followed by the use of microcolumns designed for a soil DNA extraction kit. The application can be recommended in biosafety studies of released GMMs as well as in ecological surveys of phyllosphere microbiota

Extended plant protection by an epiphytic Pseudomonas sp derivative carrying the cry9Aa gene from Bacillus thuringiensis galleriae against the pine processionary moth Thaumetopoea pityocampa

A strain of Pseudomonas sp. isolated from the phyllosphere of Pinus nigra in northern Italy was used for the introduction and high expression level of the gene encoding the Cry9Aa entomocidal toxin from Bacillus thuringiensis spp. galleriae. Laboratory tests showed that the resulting bacterial construct was more efficacious in terms of LC50 when compared to the purified toxin alone, against the first instar larvae of the pine processionary moth (Thaumetopoea pityocampa), suggesting that the encapsulation of the toxin within the bacterial cell may prevent the degradation of the protein. When the efficacy of the strain was compared in a long-term greenhouse experiment (102 days) with that of a commercial preparation of Btk (Foray 48B), the latter was superior in terms of total mortality, but its effectiveness decreased with time at a faster rate than that of the cry9Aa-Pseudomonas. Overall data indicate that the expression of Bt toxins in heterologous epiphytic bacteria offers potential for more efficient and persistent delivery of toxins to the target insect pests

Susceptibility of adult Exorista larvarum to conventional and transgenic Bacillus thuringiensis galleriae toxin

Exorista larvarum (L.) (Diptera Tachinidae) is a polyphagous larval parasitoid of lepidopterans, including forest defoliators. Laboratory studies were conducted to investigate the side effects on adult parasitoid longevity and parasitization capacity of conventional and transgenic Bacillus thuringiensis golleriae (Big) toxins, active against the pine processionary moth Thaumetopoea pityocampa (Denis et Schiffermuller) and the wax moth Galleria mellonella (L.). The flies were fed on lump sugar soaked with the bacterial suspensions and were thus treated by direct ingestion. In a first experiment, the Cry9Aa entomocidal toxin from Big was administered at 3-times the close to which the target lepidopterous species previously proved to be highly susceptible. E. larvarum male and female longevity from emergence and parasitization capacity (expressed as eggs/female laid on G. mellonella larvae and percentages of eggs which gave puparia) were not significantly affected by the treatment with the Cry9Aa toxin compared to the commercial Bt preparation Foray 48B or to distilled water (control). No significant differences were also found between the two controls. In a second experiment, adult longevity and parasitization capacity were not significantly affected by the treatment with a suspension of the epiphytic bacterium Pseudomonas Clb01 carrying the cry9Aa Btg gene compared to wild type Pseudomonas or distilled water. These results indicate that E. larvarum adults were not affected either by the conventional or transgenic Big Cry9Aa toxin according to the parameters and under the conditions tested. To complement this study, future investigations will have to be performed in a more realistic scenario than in a laboratory situation