Clarisse Brígido

Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene

AimsThe main goal of the study reported herein was to assess the nodulation performance of a Mesorhizobium strain transformed with an exogenous ACC deaminase gene (acdS), and its subsequent ability to increase chickpea plant growth under normal and waterlogged conditions.MethodsThe Mesorhizobium ciceri strain LMS-1 was transformed with the acdS gene of Pseudomonas putida UW4 by triparental conjugation using plasmid pRKACC. A plant growth assay was conducted to verify the plant growth promotion ability of the LMS-1 (pRKACC) transformed strain under normal and waterlogging conditions. Bacterial ACC deaminase and nitrogenase activity was measured.ResultsBy expressing the exogenous acdS gene, the transformed strain LMS-1 showed a 127% increased ability to nodulate chickpea and a 125% promotion of the growth of chickpea compared to the wild-type strain, under normal conditions. Plants inoculated with the LMS-1 wild-type strain showed a higher nodule number under waterlogging stress than under control conditions, suggesting that waterlogging increases nodulation in chickpea. No significant relationship was found between ACC deaminase and nitrogenase activity.ConclusionsThe results obtained in this study show that the use of rhizobial strains with improved ACC deaminase activity might be very important for developing microbial inocula for agricultural purposes.

Survey of Chickpea Rhizobia Diversity in Portugal Reveals the Predominance of Species Distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum

Several Mesorhizobium species are able to induce effective nodules in chickpea, one of the most important legumes worldwide. Our aims were to examine the biogeography of chickpea rhizobia, to search for a predominant species, and to identify the most efficient microsymbiont, considering Portugal as a case study. One hundred and ten isolates were obtained from continental Portugal and Madeira Island. The 16S ribosomal RNA gene phylogeny revealed that isolates are highly diverse, grouping with most Mesorhizobium type strains, in four main clusters (A–D). Interestingly, only 33% of the isolates grouped with Mesorhizobium ciceri (cluster B) or Mesorhizobium mediterraneum (cluster D), the formerly described specific chickpea microsymbionts. Most isolates belong to cluster A, showing higher sequence similarity with Mesorhizobium huakuii and Mesorhizobium amorphae. The association found between the province of origin and species cluster of the isolates suggests biogeography patterns: most isolates from the north, center, and south belong to clusters B, A, and D, respectively. Most of the highly efficient isolates (symbiotic effectiveness >75%) belong to cluster B. A correlation was found between species cluster and origin soil pH of the isolates, suggesting that pH is a key environmental factor, which influences the species geographic distribution. To our knowledge, this is one of the few surveys on chickpea rhizobia and the first systematic assessment of indigenous rhizobia in Portugal.

Mesorhizobium ciceri LMS-1 expressing an exogenous 1-aminocyclopropane-1-carboxylate (ACC) deaminase increases its nodulation abilities and chickpea plant resistance to soil constraints.

Aims:  Our goal was to understand the symbiotic behaviour of a Mesorhizobium strain expressing an exogenous 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase, which was used as an inoculant of chickpea (Cicer arietinum) plants growing in soil.

ACC deaminase genes are conserved among Mesorhizobium species able to nodulate the same host plant

Rhizobia strains expressing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase have been reported to display an augmented symbiotic performance as a consequence of lowering the plant ethylene levels that inhibit the nodulation process. Genes encoding ACC deaminase (acdS) have been studied in Rhizobium spp.; however, not much is known about the presence of acdS genes in Mesorhizobium spp. The aim of this study was to assess the prevalence and phylogeny of acdS genes in Mesorhizobium strains including a collection of chickpea-nodulating mesorhizobia from Portugal. ACC deaminase genes were detected in 10 of 12 mesorhizobia type strains as well as in 18 of 18 chickpea Mesorhizobium isolates studied in this work. No ACC deaminase activity was detected in any Mesorhizobium strain tested under free-living conditions. Despite the lack of ACC deaminase activity, it was possible to demonstrate that in Mesorhizobium ciceri UPM-Ca7(T) , the acdS gene is transcribed under symbiotic conditions. Phylogenetic analysis indicates that strains belonging to different species of Mesorhizobium, but nodulating the same host plant, have similar acdS genes, suggesting that acdS genes are horizontally acquired by transfer of the symbiosis island. This data, together with analysis of the symbiosis islands from completely sequenced Mesorhizobium genomes, suggest the presence of the acdS gene in a Mesorhizobium common ancestor that possessed this gene in a unique symbiosis island.

Moderately acidophilic mesorhizobia isolated from chickpea

Aims:  Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea–rhizobia symbiosis.

Transcriptional analysis of major chaperone genes in salt-tolerant and salt-sensitive mesorhizobia

Salinity is an important abiotic stress that limits rhizobia-legume symbiosis, affecting plant growth, thus reducing crop productivity. Our aims were to evaluate the tolerance to salinity of native chickpea rhizobia as well as to investigate the expression of chaperone genes groEL, dnaKJ and clpB in both tolerant and sensitive isolates. One hundred and six native chickpea mesorhizobia were screened for salinity tolerance by measuring their growth with 1.5% and 3% NaCl. Most isolates were salt-sensitive, showing a growth below 20% compared to control. An association between salt tolerance and province of origin of the isolates was found. The transcriptional analysis by northern hybridization of chaperone genes was performed using tolerant and sensitive isolates belonging to different Mesorhizobium species. Upon salt shock, most isolates revealed a slight increase in the expression of the dnaK gene, whereas the groESL and clpB expression was unchanged or slightly repressed. No clear relationship was found between the chaperone genes induction and the level of salt tolerance of the isolates. This is the first report on transcriptional analysis of the major chaperones genes in chickpea mesorhizobia under salinity, which may contribute to a better understanding of the mechanisms that influence rhizobia salt tolerance.

A ClpB Chaperone Knockout Mutant of Mesorhizobium ciceri Shows a Delay in the Root Nodulation of Chickpea Plants

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

Survey of Plant Growth-Promoting Mechanisms in Native Portuguese Chickpea Mesorhizobium Isolates

Rhizobia may possess other plant growth-promoting mechanisms besides nitrogen fixation. These mechanisms and the tolerance to different environmental factors, such as metals, may contribute to the use of rhizobia inocula to establish a successful legume-rhizobia symbiosis. Our goal was to characterize a collection of native Portuguese chickpea Mesorhizobium isolates in terms of plant growth-promoting (PGP) traits and tolerance to different metals as well as to investigate whether these characteristics are related to the biogeography of the isolates. The occurrence of six PGP mechanisms and tolerance to five metals were evaluated in 61 chickpea Mesorhizobium isolates previously obtained from distinct provinces in Portugal and assigned to different species clusters. Chickpea microsymbionts show high diversity in terms of PGP traits as well as in their ability to tolerate different metals. All isolates synthesized indoleacetic acid, 50 isolates produced siderophores, 19 isolates solubilized phosphate, 12 isolates displayed acid phosphatase activity, and 22 exhibited cytokinin activity. Most isolates tolerated Zn or Pb but not Ni, Co, or Cu. Several associations between specific PGP mechanisms and the province of origin and species clusters of the isolates were found. Our data suggests that the isolate’s tolerance to metals and ability to solubilize inorganic phosphate and to produce IAA may be responsible for the persistence and distribution of the native Portuguese chickpea Mesorhizobium species. Furthermore, this study revealed several chickpea microsymbionts with potential as PGP rhizobacteria as well as for utilization in phytoremediation strategies.

The Role of Rhizobial ACC Deaminase in the Nodulation Process of Leguminous Plants

Symbiotic rhizobia-legumes associations are extremely important in terms of sustainable agricultural practices. This symbiosis involves a complex interaction between both partners, plant and bacterium, for bacterial infection and the formation of symbiotic N-fixing nodules. In this regard, the phytohormone ethylene plays a significant role in nodule formation, acting as an inhibitor of the nodulation process. Ethylene not only regulates nodule development but also regulates many other plant developmental cues, including various stress responses that inhibit overall plant growth. Some rhizobia produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, thus, being able to decrease ACC and, consequently, decrease deleterious ethylene levels that affect the nodulation process. This occurs because ACC is the immediate precursor of ethylene in all higher plants. Hence, rhizobia that express this enzyme have an increased symbiotic potential. In addition to the direct role that ACC deaminase plays in the nodulation process per se, in a limited number of instances, ACC deaminase can also modulate nodule persistence. This review focuses on the important role of rhizobial ACC deaminase during the nodulation process, emphasizing its significance to legume growth promotion.

Phytoremediation Using Rhizobia

The use of plants to clean up polluted sites, named phytoremediation, is an attractive methodology for the cleanup of metals and other soil contaminants. While some plants can hyperaccumulate metals from the environment or break down diverse organic contaminants, they typically do not produce a large amount of biomass, and many other plants are unable to grow under such conditions. Legumes are well known for their importance as both animal and human foods and for their role in maintaining soil fertility; they are also of great interest in sustainable agricultural systems. In this context, the use of rhizobia that form a symbiotic relationship with legumes to increase the productivity and yield of legume plants in metal-contaminated soils is a promising approach to metal phytoremediation. Thus, selection of particular rhizobial strains with some degree of tolerance to metals and possessing various plant growth-promoting activities is imperative. Recent studies have indicated that metal-resistant rhizobia possessing plant growth-promoting traits, including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore production and release, and synthesis of ACC deaminase may facilitate legume growth while lessening metal toxicity. This chapter discusses the possible application of rhizobia, as symbiotic nitrogen fixers and plant growth-promoting bacteria, which may improve the productivity and yield of legumes in metal-contaminated soils.