’Has Ceratonia siliqua formed a joint venture with mycorrhizal and nitrogen fixing symbioses? Potential for juvenile tree growth and orchard establishment’

’Among the legumes, three subfamilies are recognized: Papilionoideae, Mimosoideae and Caesalpinioideae. If the first two have overwhelmingly nodulated species, among Caesalpinioideae nodulation concern only 5% of genera. Among the different clades of Caesalpinioideae, the closest to Mimosoideae include the genus Ceratonia. The carob tree, Ceratonia siliqua, is cultivated in the Mediterranean area mainly for its fruits. It is not nodulated but mycorrhizal (arbuscular mycorrhizae). The objective of our work is to study the diversity of bacteria associated with the roots of this species in relation to mycorrhization.
Our approach comprised three steps:
(1) In different field sites, we collected leaves of carob, adjacent non-leguminous species and legumes to analyze the behavior of the carob tree with respect to biological nitrogen fixation by analysis of natural isotopic abundance of 15N. The analyses were carried out by mass spectrometry in UMR BPMP. The results showed a strong geographic variability with sometimes contradictory trends. A second campaign of sampling and analyses showed a particular behavior of the carob tree, different from that of non-legume and nodulated legumes. However, the variability in the analyses performed require to further increase the number of samples studied by multiplying the crop sites and considering well-distributed stands all around the Mediterranean sea, including the area of origin of the carob tree (especially Lebanon). It is in this context that the Mycamed project (ANR Jeune Chercheur and Apège INEE were submitted, and that a PHC Cèdre (Lebanon) project will be prepared.
(2) Roots and soil in carob were also collected to assess the presence of arbuscular mycorrhizal symbioses and to analyze the microbial communities associated with natural carob. Structure and diversity of bacterial communities and arbuscular mycorrhizal were revealed by PCR-DGGE of 16S and 28S rDNA. Carob showed a relatively high diversity of mycorrhizal compared to associated crops (barley, corn) and compared to the argan tree. Cloning / sequencing analyses reveal the presence of several species of the families Glomeraceae and Claroideoglomeraceae. Bacterial communities were also analyzed from the roots of carob (more than twenty field collected plants) to identify bands found in all or virtually all carob analyzed. These bands are currently under sequencing to identify the bacterial taxa constently associated to carob root system.
Microscopic observations revealed the presence of arbuscular mycorrhizal structures in roots and also root cells invaded by bacteria.
(3) From surface sterilized root fragments, bacterial isolation trials were performed. The isolates were selected on their ability to induce a phenotype of monoxenic nodulation or swelling of root portions in two legume with broad nodulation spectrum: Macroptilium atropurpureum and Mimosa pudica. Bacterial strains inducing one or the other of these phenotypes were characterized by their 16S rDNA sequencing. Bacterial taxa included the genera Rhizobium and Pseudomonas.’