Annita Toffanin

Enhanced micropropagation response and biocontrol effect of Azospirillum brasilense Sp245 on Prunus cerasifera L. clone Mr.S 2/5 plants

Inoculation with Azospirillum brasilense Sp245 exerts beneficial effects on micropropagated plants of Prunus cerasifera L. clone Mr.S 2/5, as seen in the results of a comparative analysis of inoculated and non-inoculated explants, during both the rooting and acclimatation phases. The presence of Azospirillum brasilense Sp245 increased root system, root hair biomass production and apical activity. Although the presence of the bacteria had a positive effect on rooting, the addition of indolebutyric acid (IBA) to Murashige and Skoog (MS) medium was seen as indispensable in order to promote the rooting of explants. Aside from the promotion of plant growth, A. brasilense Sp245 protects plants against pathogen attacks, such as Rhizoctonia spp., with a plant survival rate of nearly 100% vs. 0% as seen in the negative control. The biocontrol effect of A. brasilense Sp245 on the fungal rhizospheric community has been confirmed by denaturing gradient gel electrophoresis (DGGE) profiles of the rhizospheric microbial community. This study indicates that A. brasilense Sp245 could be employed as a tool in plant biotechnology.

Identification of Two New Sets of Genes for Dibenzothiophene Transformation in Burkholderia sp. DBT1

A novel genotype for the initial steps of the oxidative degradation of dibenzothiophene(DBT) is described in a Burkholderia sp. strain isolated from a drain receiving oil refinery wastewater. The strain is capable of transforming DBT with significant efficiency when compared to other microorganisms. Its genotype was discovered by investigating insertional mutants of genes involved in DBT degradation by the Kodama pathway. The cloned dbt genes show a novel genomic organization when compared to previously described genes capable of DBT catabolism in that they constitute two distinct operons and are not clustered in a single transcript. Sequence analysis suggests the presence of a σ54-dependent positive transcriptional regulator that may be involved in the control of the transcription of the two operons, both activated by DBT. The achieved results suggest the possibility of novel features of DBT biotransformation in nature.

Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants

Plant growth-promoting rhizobacteria and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere. The role of different strains of Azospirillum on AM fungi development was evaluated by measuring the percentage of AM colonisation of the root system in durum wheat and maize plants, grown under both greenhouse and field conditions. The effect of wild-type Azospirillum brasilense strain Sp245 and genetically modified (GM) derivatives overproducing indole-3-acetic acid was assessed at greenhouse level in (1) three different cultivars of durum wheat, in the presence of indigenous AM fungi and (2) maize plants artificially inoculated with Glomus mosseae and Glomus macrocarpum. In addition, the establishment of natural AM fungal symbiosis was evaluated using Azospirillum lipoferum CRT1 in maize plants at field level. Despite the stimulatory effect of the different Azospirillum inocula on root growth, no significant differences in AM colonisation were found, independently of the AM fungus involved, either in wheat or in maize plants. Similarly, GM A. brasilense, which strongly stimulates root development, did not affect AM formation. Although these results were obtained in conditions in which the mycorrhization rate was moderate (15–30%), overall considered they indicate that the use of wild-type or GM Azospirillum phytostimulators does not alter mycorrhization.

Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of 'Malvasia delle Lipari'

Aims:  Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of ‘Malvasia delle Lipari’.

Development of a strain‐specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.

Improving micropropagation: effect of Azospirillum brasilense Sp245 on acclimatization of rootstocks of fruit tree

Abstract The effect of Azospirillum brasilense Sp245 on the micropropagation of three fruit rootstocks: Mr.S 2/5 plum (Prunus cerasifera×P. spinosa), GF 677 hybrid (Prunus persica×P. amigdalus), and MM 106 apple (Northen Spy×M1) was assessed. Rooted shoots were treated with 3×107 of Sp245 cells during transplantation from in vitro cultures to the acclimatization phase. After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of Azospirillum can significantly contribute to optimize plant performance during the phase of adaptation of plants to post-vitrum conditions.

Introduction of Rhizobium “hedysari” in alkaline clay‐loam soil by different inoculation techniques

Abstract Seed inoculation is widely used in agriculture, especially for grain legumes. However, it may be suitable for forage legumes too, particularly for the introduction of a new crop or for soil reclamation in marginal areas. This is the case of the present study, aimed at evaluating some strains of Rhizobium sp. (Rhizobium “hedy‐sari “) to inoculate Hedysarum coronarium plants used for reclamation of marginal areas in Tuscany. Strain CC1335, able to produce growth inhibitors in vitro, was able to nodulate the roots both in pot and in all the field inoculation trials, while strain RH19st3 did not enter root system by any inoculation techniques employed. Strains HCNT1 and HCNA, showing detectable competition activity in vitro, produced good nodulation only by using calcium sulphate with the peat‐arabic gum inoculant. The buffering effect of lime played evidently a determinant role in the performance of the inoculant. The results reported here suggest that improved inoculation methods will make possible...

Plant Beneficial Microbes and Their Application in Plant Biotechnology

Anna Russo1, Gian Pietro Carrozza4, Lorenzo Vettori2, Cristiana Felici4, Fabrizio Cinelli3 and Annita Toffanin4 1Department of Biological and Environmental Sciences and Technologies, University of Salento 2Department of Agriculture Biotechnology, University of Florence 3Department of Fruit Science and Plant Protection of Woody Species ‘G. Scaramuzzi’, University of Pisa 4Department of Crop Plant Biology, University of Pisa Italy

Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii

Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P. tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis. This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil. However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w. Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.

Investigation into the role of the truncated denitrification chain in Rhizobium sullae strain HCNT1

Most denitrifying bacteria reduce nitrate to the inert gases nitrous oxide or nitrogen. A remarkable exception to this is Rhizobium sullae strain HCNT1, which catalyses only a single step in the denitrification pathway, the reduction of nitrite to the reactive molecule nitric oxide. Further study demonstrated that HCNT1 does not encode the genes for NO reductase. Prolonged incubation of HCNT1 under anoxic conditions revealed that the cells had reduced culturability but not viability when nitrite was present. This may indicate an adaptation to anoxic conditions to provide resistance to environmental stresses. A closely related strain of R. sullae, strain CC1335, which is unable to denitrify, was found to lose culturability but not viability irrespective of the presence of nitrite. When the gene for nitrite reductase was mobilized into CC1335, this increased culturability with or without nitrite. These results indicate that the presence of nitrite reductase can influence the long-term survival of R. sullae strains and may provide an explanation as to why HCNT1 possesses this unusual truncation of its denitrification electron transport chain.