Explorando associações benéficas em grão-de-bico : o papel de bactérias endofíticas não-rizobianas na simbiose rizóbio-leguminosa

Objectives:

This project aimed to investigate: 1) how non-rhizobial endophytic bacteria (NREB) can promote the growth of chickpea and how this association benefits from the presence of mesorhizobia and vice-versa; 2) the hypothesis that NREB may help rhizobia to nodulate non-specific hosts and to determine the mode of entry and colonization process of NREB with chickpea; 3) the plant growth promoting traits involved in these interactions under control and different stress conditions, to validate the synergistic effects using soil and to evaluate the possible impacts in other subsequent crops.

Activities and Results:

Isolation, identification and characterization of culturable NREB from chickpea (Cicer arietinum L.) roots grown in different soils were performed. In addition, the effects of rhizobial inoculation, soil and stress on the functionality of those culturable NREB communities were also investigated. Phylogenetic analysis based on partial 16S rRNA gene sequences revealed that the NREB isolated belong to the phyla Proteobacteria, Firmicutes and Actinobacteria, with Enterobacter and Pseudomonas being the most frequently observed genera. Production of indoleacetic acid and ammonia were the most widespread plant growth-promoting features while antifungal activity was relatively rare among the isolates. Although the majority of NREB were salt- and Mn-tolerant, the NREB obtained from soil with Mn toxicity were generally more Mn-tolerant than those obtained from the same soil amended with dolomitic limestone. Our data suggest that soil strongly impacts the tolerance to Mn of NREB communities present in chickpea roots while rhizobial inoculation induces significant changes in terms of isolates' plant growth-promoting abilities. In parallel, the diversity and functionality of NREB strains from internal root tissues of native legume species grown in two distinct sites in South of Portugal were assessed. 122 NREB isolates were obtained from 12 different native legume species. Most of these NREB possess at least one of the plant growth-promoting features tested in vitro, with indole acetic acid production being the most common feature among the isolates followed by the production of siderophores and inorganic phosphate solubilization. Altogether, NREB from native legume species are a reservoir of plant growth-promoting bacteria, that are also tolerant to salinity and to toxic levels of Mn. At the end, a collection of about 180 NREB isolates was obtained from several legume plant species grown in different soils in this task.

A group of NREB with potential to promote plant growth from task 1 was selected to conduct plant growth trials with chickpea plants inoculated with different consortia were performed under control, salt and manganese stress conditions. Each consortium consisted in a combination of one of the two different Mesorhizobium strains, namely M. ciceri LMS-1 or M. mediterraneum UPM-Ca36T, and one of the 4 NREB isolates (Q1, D4, MH5 or L13). The results of in planta experiments revealed that co-inoculation of chickpea plants with specific NREB along with N2-fixing symbionts significantly improved the total biomass of chickpea plants, particularly when these plants were grown under saline conditions.

The construction of knockout mutants has been performed. Two single knockout mutants of Pseudomonas sp. Q1 were obtained, where the genes coding for the ACC deaminase (acdS) and a membrane-bound sensor hybrid histidine kinase (gacS) were deleted from the mutant’s genome. Three single knockout mutants of Kosakonia sp. MH5 were also obtained, the qseG (encodes for quorum-sensing regulator protein G), nifH (encodes a dinitrogenase reductase component of the iron-molybdenum nitrogenase) and trpAB (encode the alpha and beta chain of tryptophan synthase) genes were deleted from the mutant’s genome. To evaluate the role of ACC deaminase produced by strain Q1 in the tripartite NREB - legume - rhizobium interaction, plant trials with three legume species, Cicer arietinum (chickpea), Trifolium subterraneum (subterranean clover), and Medicago polymorpha (burr medic) were performed under non-stressed and Mn-stress. The Q1 strain only promoted the symbiotic performance of Rhizobium leguminosarum bv. trifolii ATCC 14480T and Ensifer meliloti ATCC 9930T, leading to an increase of the growth of their hosts in both conditions. Notably, the acdS gene disruption of strain Q1 abolished the beneficial effect of this bacterium as well as causing this mutant strain to act deleteriously in those specific symbioses. Our results suggest that the addition of non-rhizobia with functional ACC deaminase may be a strategy to improve the pasture legume-rhizobial symbioses, particularly when the use of rhizobial strains alone does not yield the expected results due to their difficulty in competing with native strains or in adapting to inhibitory soil conditions. Similarly, the role of QseG protein of a plant-associated enterobacterial bacterium in the beneficial plant-bacteria interactions was evaluated. Our data shows that knockout of the qseG gene in MH5 strain resulted in decreased expression of genes related to host immune system evasion, reducing chickpea roots colonization. Furthermore, inoculation of chickpea seedlings with the qseG knockout mutant increase the expression of the defence-related gene CaRBOH-like in the roots. Moreover, co-inoculation of chickpea plants with Kosakonia sp. MH5 ∆nifH mutant and strain M. ciceri LMS-1 revealed that this gene is important to the tripartite interaction while Q1 ∆gacS mutant revealed that gacS gene mutation altered the capability of strain Q1 to provide iron to subclover plants cultivated under iron deficiency conditions. Kosakonia sp. MH5 ∆trpAB mutant revealed that these genes are essential for the grow in medium without tryptophan, but disruption of trpAB genes did not affect the synthesis of IAA by MH5 strain. From these results, 1 peer-reviewed article was published (see A3) and three others are in advanced state of preparation (see A4, A5, A6).

The examination of the mode of entry of NREB in several plant species, namely was performed using a confocal laser scanning microscopy. Our results showed that all NREB strains tested were able to colonize the interior or plant tissues independently of the plant species. Differences on the mode of entry were observed, MH5 strain was able to enter through root hairs while Q1 or L13 strains enter to root cells throughout crack entry. Moreover, our results also showed that after 5 or 14 days after inoculation, legume plants when co-inoculated with R. leguminosarum or E. melliloti and MH5 strain, presented a higher number of rhizobial bacteria attached to the root hairs compared to the plants single inoculated with rhizobia. Our results also showed that QseG protein of MH5 strain is involved in the ability of this strain to internally colonize root tissues (A4).

To test the hypothesis that NREB may help the rhizobia to form nodules with non-specific hosts, deletion of three genes with unknown function was performed in M. ciceri LMS-1 in order to obtain rhizobial derivatives with deficiency on nodulation or impaired nitrogen-fixation. The symbiotic performance of these three knockout mutants was compared to the wild-type LMS-1 strain and all showed lower ability to form nodules or to fix nitrogen compared to their wild-type strain.

We also performed high-throughput proteomic analyses of two NREB (Q1 and MH5) to analyze their cellular proteins and secretome when exposed to chickpea root exudates. Proteins involved in metabolism, cell envelope biosynthetic process, stress response, defense against oxidative stress, chemotaxis, nitrogen metabolic process, type IV secretion system and transmembrane transport were identified and suggesting that they could be implicated on plant root colonization by these NREB. This comparative high-throughput proteomics analysis contributed to unveil genetic factors involved in the NREB lifestyle as well as a better understanding of the functioning of NREB (A7, A8).

Plant growth trials were performed to evaluate the effect of different NREB consortia in promoting the growth of chickpea or increasing the symbiotic performance of a specific Mesorhizobium isolate, under salinity and manganese conditions. Only one NREB consortium improved the symbiotic performance of the Mesorhizobial strain and legume growth under salinity.

A two-phase factorial experiment was performed to test the effects of NREB consortium on legume and on the subsequent crop, wheat, using pot experiments containing unsterilized soil known for its Mn toxicity under greenhouse conditions. Since we did not obtain promising results with the chickpea under Mn toxicity, a mixture of Trifolium and Medicago was used in this pot trial as usually used in Alentejo pastures. The best results in soil with Mn toxicity were obtained in plants co-inoculated with rhizobium and Q1, while in the treatments containing soil amendment with dolomitic limestone, the best performance was obtained with plants co-inoculated with rhizobia and Q1 + L13.