Annamaria Bevivino

Soil Type and Maize Cultivar Affect the Genetic Diversity of Maize Root–Associated Burkholderia cepacia Populations

A bstractBurkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity. A total of 180 B. cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique). The genetic diversity among B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5. The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns. The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root–associated B. cepacia populations. In fact, the percentage of variation among populations was significantly higher between B. cepacia populations recovered from maize planted in different soils than between B. cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere. The analysis of the genetic relationships among B. cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure. The dendrograms were also in agreement with the AMOVA results. We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root–associated B. cepacia populations analyzed. On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure.

Influence of plant development, cultivar and soil type on microbial colonization of maize roots

Abstract An understanding of the environmental factors affecting size and composition of rhizosphere microbial populations is important when introducing exogenous microorganisms in the rhizosphere of crop plants for plant growth promotion. The influence of plant development, cultivar and soil characteristics on the total rhizosphere microbial population and community structure of maize plants was investigated using the concept of r/K strategy. During maize growth microbial population density did not vary significantly, whereas the microbial community structure changed markedly in the early stages of plant growth but afterwards remained stable. Comparisons of the rhizosphere microflora of several maize cultivars, showing differential susceptibility to Fusarium , revealed that different cultivars support similar numbers of indigenous bacteria. Moreover the bacterial community structures of different maize cultivars did not show any significant difference. On the contrary, soil type had a marked influence on the microbial population of maize rhizosphere. Indeed the rhizosphere microbial density and community structure varied significantly among the different sampling sites. In conclusion, plant development and soil type have a marked influence on the rhizosphere microflora of maize, whereas cultivar type does not have a role.

Burkholderia cepacia Complex Bacteria from Clinical and Environmental Sources in Italy: Genomovar Status and Distribution of Traits Related to Virulence and Transmissibility

ABSTRACT Sixty-eight Burkholderia cepacia complex isolates recovered from the sputum of 53 cystic fibrosis patients and 75 isolates collected from the maize rhizosphere were compared to each other to assess their genomovar status as well as some traits related to virulence such as antibiotic susceptibility, proteolytic and hemolytic activities, and transmissibility, in which transmissibility is determined by detection of the esmR and cblA genes. Among the clinical isolates, B. cepacia genomovar III comprised the majority of isolates examined and only a very few isolates were assigned to B. cepacia genomovar I, B. stabilis, and B. pyrrocinia; among the environmental isolates a prevalence of B. cepacia genomovar III and B. ambifaria was observed, whereas few environmental isolates belonging to B. cepacia genomovar I and B. pyrrocinia were found. Antibiotic resistance analysis revealed a certain degree of differentiation between clinical and environmental isolates. Proteolytic activity and onion tissue maceration ability were found to be spread equally among both clinical and environmental isolates, whereas larger percentages of environmental isolates than clinical isolates had hemolytic activity. The esmR gene was found exclusively among isolates belonging to B. cepacia genomovar III, with a marked prevalence in clinical isolates, whereas only one clinical isolate belonging to B. cepacia genomovar III was found to bear the cblA gene. In conclusion, the results of the present study show that the species compositions of the clinical and environmental B. cepacia complex populations examined are quite different and that some of the candidate determinants related to virulence and transmissibility are not confined solely to clinical isolates but are also spread among environmental isolates belonging to different species of the B. cepacia complex.

PHENOTYPIC COMPARISON BETWEEN RHIZOSPHERE AND CLINICAL ISOLATES OF BURKHOLDERIA-CEPACIA

The phenotypic characteristics of four Burkholderia cepacia strains isolated from the rhizosphere and the clinical environment were compared. Tests included optimum growth temperature, utilization of carbon sources, production of HCN, indole-3-acetic acid (IAA) and siderophores, proteolytic activity, nitrogen fixation, inhibition of some phytopathogenic fungi, adherence to human mucosal and plant root epithelia, and greenhouse-based plant-growth promotion experiments using cucumber (Cucumis sativus). Results indicated that the strains of B. cepacia isolated from the rhizosphere differ markedly from their clinical counterparts. Strains isolated from the rhizosphere grew over a wider temperature range, fixed nitrogen and produced IAA, did not produce proteases, displayed a wider antibiosis against the phytopathogenic fungi studied, did not adhere to human uroepithelial cells, promoted growth of C. sativus and only produced a hydroxamate-like siderophore. In contrast, clinical isolates could not fix nitrogen or produce IAA, produced proteases, adhered to human uroepithelial cells, did not promote the growth of C. sativus and, in addition to a hydroxamate-like siderophore, produced pyochelin and salicylate siderophores. All four isolates exhibited the ability to adhere to the root tissue of C. sativus and were unable to produce HCN.

Inoculation of Burkholderia cepacia, Pseudomonas fluorescens and Enterobacter sp. on Sorghum bicolor: Root colonization and plant growth promotion of dual strain inocula

Abstract Burkholderia cepacia strain PHP7 was tested for its ability to colonize roots and to promote the growth of Sorghum bicolor alone or in combination with Enterobacter sp. strain BB 23 T 4 d or Pseudomonas fluorescnes strain A 23 T 3 c . All three strains were able to colonize the root system of sorghum but only B. cepacia and P. fluorescens promoted plant growth in single strain inoculation tests. Dual strain inocula were no more effective than single ones. Moreover, in one case, the dual strain inoculum (B. cepacia and P. fluorescens) did not have any significant effect on plant growth in contrast to the separate inoculation of both strains. In dual strain tests, the B. cepacia population was significantly reduced in the presence of Enterobacter sp. but not of P. fluorescens. Establishment of large populations of bacterial inoculants on roots did not appear to be essential for plant growth promotion.

Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population.

A bstractThe influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium.

Epidemiology and Clinical Course of Burkholderia cepacia Complex Infections, Particularly Those Caused by Different Burkholderia cenocepacia Strains, among Patients Attending an Italian Cystic Fibrosis Center

ABSTRACT In this study, the epidemiology of Burkholderia cepacia complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Childrens Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by recA gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting. Burkholderia cenocepacia is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to B. cepacia genomovar I, Burkholderia stabilis, and Burkholderia pyrrocinia were found. Of the four recA lineages of B. cenocepacia, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with B. cenocepacia, in particular with strains belonging to recA lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of B. cenocepacia, except the case of a patient infected with B. cepacia genomovar I. Within B. cenocepacia, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either B. cenocepacia IIIA or B. cenocepacia IIID.

Efficacy of Burkholderia cepacia MCI 7 in disease suppression and growth promotion of maize

Abstract Repeated greenhouse experiments were performed to evaluate the ability of a maize-rhizosphere isolate of Burkholderia cepacia, applied as a seed coating, to promote maize growth in both uninfested soil and soil infested with a maize pathogenic strain of Fusarium moniliforme, and to displace or negatively affect the population of F. moniliforme throughout plant growth. Results demonstrated that the B. cepacia strain MCI 7 is a promising plant-growth-promoting inoculant for maize. In repeated greenhouse trials, bacterization of maize seeds with B. cepacia MCI 7 resulted in a significant (P<0.05) increase of maize plant growth in both uninfested soil and soil infested with F. moniliforme ITEM-504, as compared to uninoculated plants. Moreover, B. cepacia MCI 7 was able to negatively affect the rhizoplane colonization of F. moniliforme that showed significantly (P<0.05) lower values of population density throughout plant growth, as compared with respective values observed in the root system of uninoculated plants. The effect on plant growth following introduction of B. cepacia MCI 7 into the maize rhizosphere has also been investigated using two corn cultivars differing in their degree of resistance to Fusarium. Results showed that B. cepacia MCI 7 was able to determine an increased growth response (P<0.05) of the two corn cultivars in both uninfested soil and soil infested with F. moniliforme.

Modelling Co-Infection of the Cystic Fibrosis Lung by Pseudomonas aeruginosa and Burkholderia cenocepacia Reveals Influences on Biofilm Formation and Host Response

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy. To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response. Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.

Soil bacterial community response to differences in agricultural management along with seasonal changes in a Mediterranean region

Land-use change is considered likely to be one of main drivers of biodiversity changes in grassland ecosystems. To gain insight into the impact of land use on the underlying soil bacterial communities, we aimed at determining the effects of agricultural management, along with seasonal variations, on soil bacterial community in a Mediterranean ecosystem where different land-use and plant cover types led to the creation of a soil and vegetation gradient. A set of soils subjected to different anthropogenic impact in a typical Mediterranean landscape, dominated by Quercus suber L., was examined in spring and autumn: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards (ploughed and grass covered). Land uses affected the chemical and structural composition of the most stabilised fractions of soil organic matter and reduced soil C stocks and labile organic matter at both sampling season. A significant effect of land uses on bacterial community structure as well as an interaction effect between land uses and season was revealed by the EP index. Cluster analysis of culture-dependent DGGE patterns showed a different seasonal distribution of soil bacterial populations with subgroups associated to different land uses, in agreement with culture-independent T-RFLP results. Soils subjected to low human inputs (cork-oak forest and pasture) showed a more stable bacterial community than those with high human input (vineyards and managed meadow). Phylogenetic analysis revealed the predominance of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes phyla with differences in class composition across the site, suggesting that the microbial composition changes in response to land uses. Taken altogether, our data suggest that soil bacterial communities were seasonally distinct and exhibited compositional shifts that tracked with changes in land use and soil management. These findings may contribute to future searches for bacterial bio-indicators of soil health and sustainable productivity.