Monica Agnolucci

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis.

ABSTRACT We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.

Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis

The effect of different sulphur dioxide concentrations on culturability and viability of seven strains of Brettanomyces bruxellensis was tested in a synthetic wine medium (SWM) and a different response to molecular SO(2) among strains was detected. Sulphur dioxide induced a viable but non culturable (VBNC) state in all the strains. The greater percentage of VBNC cells were identified for five strains at molecular SO(2) concentrations of 0.2mg/L and for two strains at the concentration of 0.4mg/L. Vinyl phenols were detected in media containing VBNC or not viable B. bruxellensis, suggesting that its spoilage metabolism could be maintained during wine storage. Overall, this study indicates that SO(2) is a chemical stressor inducing VBNC state in B. bruxellensis grown in synthetic wine medium. Further studies are needed to evaluate the effects of SO(2) on the metabolism of this yeast in wine spoilage.

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l(-1)), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.

Microbiological and Chemical Characterisation of Composts at Different Levels of Maturity, with Evaluation of Phytotoxicity and Enzymatic Activities

Composts from different vegetable residues as well as pig and horse manure at different levels of maturity were used in the present study. In order to characterise the different composts, microbial populations (namely, total aerobic bacteria, actinomycetes, filamentous eumycetes, aerobic cellulolytic fungi and bacteria), physicochemical and chemical parameters (moisture, organic matter, total nitrogen, ammonium nitrogen, C:N ratio, pH, electrical conductivity), self-heating capacity and phytotoxicity (measured by means of the germination test with Lepidium sativum),as well as enzymatic activities [cellulase, lipase (C10), protease], were evaluated. The research was designed to focus on a possible correlation between the enzymatic activities and the maturity level of composts. The results showed evidence that the protease:cellulase activity ratio was significantly correlated to the compost phytotoxicity.

Diverse bacterial communities are recruited on spores of different arbuscular mycorrhizal fungal isolates

Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbioses with the roots of most food crops, playing a key role in soil fertility and plant nutrition and health. The beneficial activity of AMF may be positively affected by bacterial communities living associated with mycorrhizal roots, spores and extraradical hyphae. Here, we investigated the diversity of bacterial communities associated with the spores of six AMF isolates, belonging to different genera and species and maintained for several generations in pot cultures with the same host plant, under the same environmental conditions and with the same soil. The occurrence of large bacterial communities intimately associated with spores of the AMF isolates was revealed by PCR denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of DGGE bands. Cluster and canonical correspondence analysis showed that the six AMF isolates displayed diverse bacterial community profiles unrelated with their taxonomic position, suggesting that each AMF isolate recruits on its spores a different microbiota. The 48 sequenced fragments were affiliated with Actinomycetales, Bacillales, Pseudomonadales, Burkholderiales, Rhizobiales and with Mollicutes-related endobacteria (Mre). For the first time, we report the occurrence of Mre in Funneliformis coronatum and Rhizophagus intraradices and sequences related to endobacteria of Mortierella elongata in F. coronatum and Funneliformis mosseae. The bacterial species identified are known to possess diverse and specific physiological characteristics and may play multifunctional roles affecting the differential performance of AMF isolates, in terms of infectivity and efficiency.

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’.

Microbially-enhanced composting of wet olive husks

The production of a compost from olive wet husks is described. The process is enhanced through the use of starters prepared with virgin husks enriched with selected microbial cultures. This approach, with respect to composting without the use of starters, allows to achieve faster start of the process (10 vs. 45 days), deeper humification (humification rate 19.2 vs. 12.2), shorter maturation time (2 vs. 4-5 months) and better detoxification of the starting material. Furthermore, the compost produced can effectively substitute for turf as a cultivation substrate in horticulture at greenhouse level, with beneficial effects on nutraceutical traits of tomato fruits.

Microbially-enhanced composting of olive mill solid waste (wet husk): Bacterial and fungal community dynamics at industrial pilot and farm level.

Bacterial and fungal community dynamics during microbially-enhanced composting of olive mill solid waste (wet husk), used as a sole raw material, were analysed in a process carried out at industrial pilot and at farm level by the PCR-DGGE profiling of the 16 and 26S rRNA genes. The use of microbial starters enhanced the biotransformation process leading to an earlier and increased level of bacterial diversity. The bacterial community showed a change within 15 days during the first phases of composting. Without microbial starters bacterial biodiversity increased within 60 days. Moreover, the thermophilic phase was characterized by the highest bacterial biodiversity. By contrast, the biodiversity of fungal communities in the piles composted with the starters decreased during the thermophilic phase. The biodiversity of the microbial populations, along with physico-chemical traits, evolved similarly at industrial pilot and farm level, showing different maturation times.

Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices

Arbuscular Mycorrhizal Fungi (AMF) live in symbiosis with most crop plants and represent essential elements of soil fertility and plant nutrition and productivity, facilitating soil mineral nutrient uptake and protecting plants from biotic and abiotic stresses. These beneficial services may be mediated by the dense and active spore-associated bacterial communities, which sustain diverse functions, such as the promotion of mycorrhizal activity, biological control of soilborne diseases, nitrogen fixation, and the supply of nutrients and growth factors. In this work, we utilised culture-dependent methods to isolate and functionally characterize the microbiota strictly associated to Rhizophagus intraradices spores, and molecularly identified the strains with best potential plant growth promoting (PGP) activities by 16S rDNA sequence analysis. We isolated in pure culture 374 bacterial strains belonging to different functional groups-actinobacteria, spore-forming, chitinolytic and N2-fixing bacteria-and screened 122 strains for their potential PGP activities. The most common PGP trait was represented by P solubilization from phytate (69.7%), followed by siderophore production (65.6%), mineral P solubilization (49.2%) and IAA production (42.6%). About 76% of actinobacteria and 65% of chitinolytic bacteria displayed multiple PGP activities. Nineteen strains with best potential PGP activities, assigned to Sinorhizobium meliloti, Streptomyces spp., Arthrobacter phenanthrenivorans, Nocardiodes albus, Bacillus sp. pumilus group, Fictibacillus barbaricus and Lysinibacillus fusiformis, showed the ability to produce IAA and siderophores and to solubilize P from mineral phosphate and phytate, representing suitable candidates as biocontrol agents, biofertilisers and bioenhancers, in the perspective of targeted management of beneficial symbionts and their associated bacteria in sustainable food production systems.

Effects of Humic Acids Extracted from Mined Lignite or Composted Vegetable Residues on Plant Growth and Soil Microbial Populations

Growing interest in the market for humic substances with agricultural applications has led to the development of new potential sources of these compounds other than fossil matrices (e.g. different kinds of lignite), which, until now, have represented the main raw material for the extraction of humus-like products. High quality compost (green compost) obtained through the aerobic biostabilization of selected organic residues, such as vegetable waste from source-collection at garden-produce markets, may be considered for this purpose. Beyond the primary need to develop technically and economically reliable procedures for the extraction of humic substances from compost at the industrial scale, importance must be placed on controlling the influence of such compounds on soil-plant systems. Humates from leonardite, representative of the active agents among humus-based commercial preparations, have been compared in pot trials with humic acids, potassium salts, from green compost in order to evaluate their respec...