Environmental stresses are main factors limiting crop production worldwide. Water stress is a primary cause of crop losses, reducing average yields for most of the major crops by more than 50%, particularly in arid and semi-arid regions. Moreover, the increasing frequency of dry periods in many regions of the world, as a consequence of the global warming, frequently results in saline soils with low agricultural potential. Bacteria are among the first colonizers of soil habitats at different stages of development, largely contributing in determining soil structure and composition. In this context, understanding the diversity of soil- and plant-associated bacteria and their roles in soil structure maintenance, protection of plant health and development under harsh conditions is crucial, assuming that these associations could be manipulated to increase the productivity and sustainability of agro-ecosystems. A new emerging approach to improve crop production without extending agriculture surfaces and agro-chemicals use, is the exploitation of the natural microbiota contribute in soil structure determination and of the association of plants with Plant Growth Promoting Bacteria (PGPB). During the last couple of decades, the use of PGPB for sustainable agriculture was increased in different parts of the world resulting in significant improvement in growth and yield of agronomical important crops, also in arid land. These beneficial bacteria can colonize the rhizosphere soil or the root system, and even thrive in the plant tissues as endophytic populations. Bacterial mechanisms of plant growth promotion include biological nitrogen fixation, synthesis of phytormones, stress reduction or protection from pathogens. In addition, plant-associated communities also increase availability of nutrients such as phosphorous, iron and other elements, playing key roles in ecosystem processes such as nutrient cycling and conservation of soil structure. To remove the erratic performance of microbial inoculation, the selection of native bacteria acclimatized to harsh environmental condition has been advocated. Recent reports documented the efficiency of PGP bacteria isolated from plants growing in arid and semi-arid regions. This PhD thesis aimed to explore the microbial contribution in soil neogenesis in deglaciating environments where a soil fertility gradient can be envisaged. Besides, this thesis has the proposal to exploit the PGP properties of endophytes and rhizobacteria adapted to harsh conditions to develop an eco-friendly approach for the improvement of crop productivity in arid environments. For this purpose, a large collection of 5000 bacteria associated to plants grown in arid areas was established. Bacteria were isolated from the root tissues and soil fraction at increasing distance from the plant: the rhizosphere, the root surrounding soil and the bulk soil. The selected plants were Olea europea (olive tree), Vitis vinifera (grapevine) and Capsicum annuum (pepper) growing in arid and semi-arid regions of Tunisia, Egypt and Italy. These agricultural crops have a relevant economic interest in the Mediterranean basin and are known to be highly adapted to extreme stressful conditions of arid land, thus allowing to improve the chances to select drought resistant bacteria. Analysis of the abundance and diversity of the cultivable bacterial fraction from root tissues and rhizosphere showed a difference in the spatial distribution of bacterial genera associated to the plants. In olive tree we observed higher bacterial densities in the rhizosphere compared to endophytic populations, due to the release of exudates in the rhizosphere from living roots. 2 Besides, the low phylogenetic diversity observed in the endophyte fraction indicated that plant tissues may select specific bacterial colonizers. Particularly, the endophytes were dominated by the sporeformer Bacillales order represented by the genera Bacillus, Paenibacillus and Brevibacillus. The dominant order cultured from soil fractions was Actinobacteria, with dominant genera such as Arthrobacter and Streptomyces, frequently isolated from dry soils and desert crusts. Differently, in pepper plants sampled in Egypt and Italy the endophytic isolates were affiliated exclusively to the Bacilli class. Differently, isolates belonging to the gamma subgroup of the class Proteobacteria were predominant in rhizosphere, root surrounding soil and bulk soil of pepper plants from Egypt, with many of these isolates assigned to Enterobacteriaceae family, in particular to Klebsiella, Citrobacter, Raoultella genera and to the genus Pseudomonas. The family Enterobacteraceae comprises many species with enteric habitat, which origin could be attributable to the use of irrigation water of low hygienic quality. The isolates from the different plant models were tested in vitro for their plant growth promotion (PGP) activities allowing to identify polyvalent isolates, i.e. those capable of expressing multiple activities against drought and salinity stresses. The results showed that 90% of the isolated bacteria presented multiple and differentiated PGP activities. All the isolates were osmotolerant, but a differential distribution of the PGP activities was observed among the different root system fractions sampled, with enhanced abilities for rhizobacteria in phosphate solubilisation, siderophore and ammonia production. Moreover, the results showed that bacteria belonging to the Bacillus genus often presented all the PGP activities tested and were able to tolerate harsh abiotic stresses, suggesting their possible use as inocula for sustaining agriculture in arid land. Hence, the research was focused on bacteria endowed with 1-aminocyclopropane-1-carboxylate deaminase (ACCd) activity, able to hydrolyze ACC, the immediate biosynthetic precursor of ethylene. Ethylene is the stress hormone synthesized by plants as a direct consequence of stressful condition, causing a reduction of in plant growth. The expression of ACCd by plant associated bacteria could decrease the concentration of ethylene, releasing the plant from stress and preserving normal growth. The most active ACCd endophyte and rhizobacteria strains with multiple PGP activities isolated from herbaceous, Capsicum annuum L, and arboreal, Vitis vinifera cv. Barbera, plants were further assayed in a in vivo assay on the plants of origin. Experiments in microcosms subjected to different levels of water stress were performed using non sterilized soil in order to evaluate PGP inocula interaction with the autochthonous microbiota. The results showed different efficiency in plant growth promotion potential, nevertheless some strains displayed the distinctive attribute to sustain pepper and grapevine growth under drought. In pepper, strains PACC-R01, -R08 and -R10 increased both the fresh and dry root biomass by a 40-60% range, whereas two consortia of grapevine strains were able to improve the biomass of the aerial plant portion in grapevine plantlets. To unravel the mechanism involved in plant growth promotion and the inner relationship between plant and bacteria, we focused our attention on endophytic bacteria isolated from pepper plant in arid Italian soils endowed with ACCd activity. Pepper plants grown hydroponically were inoculated with two strains, E1 and E3 belonging to Bacillus and Paenibacillus genera, respectively, in a nutrient solution with the addition of 3 polyetilenglycole (PEG) to induce water stress. The strains were rhizo-competent and actively colonized different plant organs, as observed by microscopy analysis of gfp-cells and by re-isolation experiments. Gfp-E1 cells were able to tightly adhere on root surface and to envelop root hairs. Confocal microscopy showed the presence of gfp-cells in the outer cortex, confirming the bacteria ability to penetrate plant tissue. Re-isolation of rifampicin resistant (RifR) mutant strains revealed that endophytes were able to colonize the root system and also stems and leaves. The promotion of plant growth during drought was assessed at the leaf level by gas exchange measurements, determining net photosynthesis (Pn), evaporation/ transpiration (E) and stomatal conductance (Gs). After PEG addition, inoculated plants showed values of Pn, E and Gs parameters significantly higher than non inoculated plants. These data were reconfirmed also in a soil system in which plants experienced 12 days of water deprivation. At the root level, bacterial strains directly affected Na+ and K+ content: in the presence of PEG their concentration increased in inoculated plants respect to the control ones. The involvement of the vacuolar proton pumps H+-PPase and ATPase was investigated. An increase in H+-PPase expression in inoculated plants respect to control plants was observed both by Western Blot analysis and immuno-histochemical experiments. H+-PPase protein levels increased up to 51% in E1 and 96% in E3 inoculated plants. A similar increase in H+-PPase activity was observed. The proposed mechanism is that the increased expression and activity of the pump determines a decrease in the vacuolar potential, thus requiring an enhanced flux of water from the soil. The collected data suggest that the pump activation is mainly driven by the endophytes interaction. The data obtained within this thesis project emphasize that PGP bacteria represent a potential eco-friendly solution to optimize soil stability, maximize plant survival and productivity under adverse environmental conditions. Besides, we provided a fine tuning of the experimental design to enhance the chances to select bacteria with the distinctive trait of stimulating plant growth during drought, using a combined approach based on the selection of site and plant of origin. A large microbial collection has been established and besides several strains showed (i) high levels of plant colonisation ability, (ii) plant growth promotion, and (iii) osmotic tolerance, that could be further exploited in field trials in the regions and countries suffering desertification.
'MORE CROP PER DROP': THE CONTRIBUTE OF PLANT GROWTH PROMOTING BACTERIA / R. Marasco ; supervisor: D. Daffonchio ; coordinator: D. Daffonchio. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Jan 26. ((24. ciclo, Anno Accademico 2011.
|Titolo:||'MORE CROP PER DROP': THE CONTRIBUTE OF PLANT GROWTH PROMOTING BACTERIA|
|Supervisori e coordinatori interni:||DAFFONCHIO, DANIELE GIUSEPPE|
|Data di pubblicazione:||26-gen-2012|
|Parole Chiave:||PLANT GROWTH PROMOTING BACTERIA ; DROUGHT ; ARID LAND ; MICROBIAL DIVERSITY|
|Settore Scientifico Disciplinare:||Settore AGR/16 - Microbiologia Agraria|
|Citazione:||'MORE CROP PER DROP': THE CONTRIBUTE OF PLANT GROWTH PROMOTING BACTERIA / R. Marasco ; supervisor: D. Daffonchio ; coordinator: D. Daffonchio. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Jan 26. ((24. ciclo, Anno Accademico 2011.|
|Appare nelle tipologie:||Tesi di dottorato|