Alessandra Turrini

Impact of Bt Corn on Rhizospheric and Soil Eubacterial Communities and on Beneficial Mycorrhizal Symbiosis in Experimental Microcosms

ABSTRACT A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.

Development of a model system to assess the impact of genetically modified corn and aubergine plants on arbuscular mycorrhizal fungi

We developed an experimental model system to monitor the impact of generically modified (GM) plants on arbuscular mycorrhizal (AM) fungi, a group of non-target soil microorganisms, fundamental for soil fertility and plant nutrition. The system allowed us to study the effects of root exudates of both commercial Bt corn and aubergine plants expressing Dm-AMP1 defensin on different stages of the life cycle of the AM fungal species G. mosseae. Root exudates of Bt 176 corn significantly reduced pre-symbiotic hyphal growth, compared to Bt 11 and non-transgenic plants. No differences were found in mycelial growth in the presence of Dm-AMP1 and control plant root exudates. Differential hyphal morphogenesis occurred irrespective of the plant line, suggesting that both exuded Bt toxin and defensin do not interfere with fungal host recognition mechanisms. Bt 176 affected the regular development of appressoria, 36% of which failed to produce viable infection pegs. Our experimental model system represents an easy assay for testing the impact of GM plants on non-target soil-borne AM fungi.

Establishment, persistence and effectiveness of arbuscular mycorrhizal fungal inoculants in the field revealed using molecular genetic tracing and measurement of yield components

• Inoculation of crop plants by non-native strains of arbuscular mycorrhizal (AM) fungi as bio-enhancers is promoted without clear evidence for symbiotic effectiveness and fungal persistence. To address such gaps, the forage legume Medicago sativa was inoculated in an agronomic field trial with two isolates of Funneliformis mosseae differing in their nuclear rDNA sequences from native strains. • The inoculants were traced by PCR with a novel combination of the universal fungal NS31 and Glomeromycota-specific LSUGlom1 primers which target the nuclear rDNA cistron. The amplicons were classified by restriction fragment length polymorphism and sequencing. • The two applied fungal inoculants were successfully traced and discriminated from native strains in roots sampled from the field up to 2 yr post inoculation. Moreover, field inoculation with inocula of non-native isolates of F. mosseae appeared to have stimulated root colonization and yield of M. sativa. • Proof of inoculation success and sustained positive effects on biomass production and quality of M. sativa crop plants hold promise for the role that AM fungal inoculants could play in agriculture.

Arbuscular mycorrhizal fungi in national parks, nature reserves and protected areas worldwide: a strategic perspective for their in situ conservation

Soil fungi play a crucial role in producing fundamental ecosystem services such as soil fertility, formation and maintenance, nutrient cycling and plant community dynamics. However, they have received little attention in the field of conservation biology. Arbuscular mycorrhizal fungi (AMF) are beneficial soil symbionts fulfilling a key function in the complex networks of belowground/aboveground biotic interactions as they live in association with the roots of most (80%) land plant families and influence not only soil fertility but also plant nutrition, diversity and productivity. The diversity of AMF communities can decline due to habitat loss and anthropogenic disturbance, especially in agro-ecosystems, and many valuable ecotypes could become extinct before they are even discovered. Consequently, long-term strategies are urgently needed to ensure their conservation in habitats where they naturally occur and have evolved. Protected areas, where living organisms are under the care of national and international authorities, represent an appropriate place for the in situ conservation of AMF, providing them with adapted situations together with established complex networks of interactions with different components within each specific ecosystem. Here, we review data available about the main present-day threats to AMF and the current state of knowledge about their occurrence in protected sites worldwide, providing a checklist of national parks and nature reserves where they have been reported. The aim was to offer a strategic perspective to increase awareness of the importance of conserving these beneficial plant symbionts and of preserving their biodiversity in the years to come.

Contrasting effects of cover crops on 'hot spot' arbuscular mycorrhizal fungal communities in organic tomato

Arbuscular mycorrhizal fungal (AMF) communities are fundamental in organic cropping systems where they provide essential agro-ecosystem services, improving soil fertility and sustaining crop production. They are affected by agronomic practices, but still, scanty information is available about the role of specific crops, crop rotations and the use of winter cover crops on the AMF community compositions at the field sites. A field experiment was conducted to elucidate the role of diversified cover crops and AMF inoculation on AMF diversity in organic tomato. Tomato, pre-inoculated at nursery with two AMF isolates, was grown following four cover crop treatments: Indian mustard, hairy vetch, a mixture of seven species and a fallow. Tomato root colonization at flowering was more affected by AMF pre-transplant inoculation than by the cover crop treatments. An enormous species richness was found by morphological spore identification: 58 AMF species belonging to 14 genera, with 46 and 53 species retrieved at the end of cover crop cycle and at tomato harvest, respectively. At both sampling times, AMF spore abundance was highest in hairy vetch, but after tomato harvest, AMF species richness and diversity were lower in hairy vetch than in the cover crop mixture and in the mustard treatments. A higher AMF diversity was found at tomato harvest, compared with the end of the cover crop cycle, independent of the cover crop and pre-transplant AMF inoculation. Our findings suggest that seasonal and environmental factors play a major role on AMF abundance and diversity than short-term agronomic practices, including AMF inoculation. The huge AMF diversity is explained by the field history and the Mediterranean environment, where species characteristic of temperate and sub-tropical climates co-occur.

The Impact of Genetically Modified Crops on Soil Microbial Communities

Genetically modified (GM) plants represent a potential benefit for environmentally friendly agriculture and human health. Though, poor knowledge is available on potential hazards posed by unintended modifications occurring during genetic manipulation. The increasing amount of reports on ecological risks and benefits of GM plants stresses the need for experimental works aimed at evaluating the impact of GM crops on natural and agro-ecosystems. Major environmental risks associated with GM crops include their potential impact on non-target soil microorganisms playing a fundamental role in crop residues degradation and in biogeochemical cycles. Recent works assessed the effects of GM crops on soil microbial communities on the basis of case-by-case studies, using multimodal experimental approaches involving different target and non-target organisms. Experimental evidences discussed in this review confirm that a precautionary approach should be adopted, by taking into account the risks associated with the unpredictability of transformation events, of their pleiotropic effects and of the fate of transgenes in natural and agro-ecosystems, weighing benefits against costs.

Mycorrhizal activity and diversity in a long-term organic Mediterranean agroecosystem

In organic agriculture, soil fertility and productivity rely on biological processes carried out by soil microbes, which represent the key elements of agroecosystem functioning. Arbuscular mycorrhizal fungi (AMF), fundamental microorganisms for soil fertility, plant nutrition and health, may play an important role in organic agriculture by compensating for the reduced use of fertilizers and pesticides. Though, AMF activity and diversity following conversion from conventional to organic farming are poorly investigated. Here we studied AMF abundance, diversity and activity in short- and long-term organically and conventionally managed Mediterranean arable agroecosystems. Our results show that both AMF population activity, as assessed by the mycorrhizal inoculum potential (MIP) assay, the percentage of colonized root length of the field crop (maize) and glomalin-related soil protein (GRSP) content were higher in organically managed fields and increased with time since transition to organic farming. Here, we showed an increase of GRSP content in arable organic systems and a strong correlation with soil MIP values. The analysis of AMF spores showed differences among communities of the three microagroecosystems in terms of species richness and composition as suggested by a multivariate analysis. All our data indicate that AMF respond positively to the transition to organic farming by a progressive enhancement of their activity that seems independent from the species richness of the AMF communities. Our study contributes to the understanding of the effects of agricultural managements on AMF, which represent a promising tool for the implementation of sustainable agriculture.

Belowground environmental effects of transgenic crops: a soil microbial perspective.

Experimental studies investigated the effects of transgenic crops on the structure, function and diversity of soil and rhizosphere microbial communities playing key roles in belowground environments. Here we review available data on direct, indirect and pleiotropic effects of engineered plants on soil microbiota, considering both the technology and the genetic construct utilized. Plants modified to express phytopathogen/phytoparasite resistance, or traits beneficial to food industries and consumers, differentially affected soil microorganisms depending on transformation events, experimental conditions and taxa analyzed. Future studies should address the development of harmonized methodologies by taking into account the complex interactions governing soil life.

Arbuscular mycorrhizal fungi of a Mediterranean island (Pianosa), within a UNESCO Biosphere Reserve

In this work we have determined the community composition of spore-forming arbuscular mycorrhizal fungi (AMF) in a maquis site on Pianosa island, a protected area within the Tuscan Islands UNESCO Biosphere Reserve, Italy. We have analysed rhizosphere soil of the dominant plant species Pistacia lentiscus, Smilax aspera, Rosmarinus officinalis and of the endemic plant Helichrysum litoreum. The AMF species recovered were: Scutellospora dipurpurescens, Glomus coronatum, Glomus mosseae, Glomus etunicatum, Glomus geosporum, Glomus viscosum, Entrophospora sp., Pacispora sp. and Glomus rubiforme. The identification of native S. dipurpurescens and G. coronatum was carried out on spores isolated from rhizosphere soil of H. litoreum, by combining morphological traits and 18S (SSU) and ITS rDNA sequences. Therefore, AMF species of Pianosa rhizosphere soils represent an important repository for the conservation and maintenance in their natural habitat of such beneficial symbionts, key microorganisms of soil fertility.

Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil

In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3′-hydroxylase isoform 1 (CS3′H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3′H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.