15N natural abundance technique does not reveal the presence of nitrogen from biological fixation in field grown carob (Ceratonia siliqua L.) trees

The presence in agroecosystems of plants able to develop symbiosis with nitrogen (N2) fixing microorganisms is encouraged as may decrease the need for mineral N fertilizers. Biological N2 fixation is widespread among Fabaceae, but legume species differ in their ability to develop symbiosis with N2 fixing microorganisms: for example, while this frequently occurs in the sub-family Papilionoideae, only about 5% of the species of the sub-family Cesalpinoideae have developed such symbiosis mechanism. Carob (Ceratonia siliqua L.) is an evergreen legume tree belonging to Cesalpinoideae, typical of the Mediterranean basin, used for animal feeding, but with a great potential as a crop for the food and cosmetic industries and medicin. The ability of carob trees to develop symbiosis with rhizobia is unclear: root nodules have not generally been found in nature but were formed on roots of carob trees inoculated with rhizobia. We have applied the 15N natural abundance technique to field grown carob trees in Sicily and Sardinia (Italy) to assess the potential contribution of atmospheric N2 for tree metabolism. As soil derived N is generally more enriched in 15N than atmospheric N2 (δ15N=0 ‰), if a tree uses atmospheric N2 through the symbiosis with N2 fixing microorganisms, then the 15N abundance in its tissues should be lower than that of a tree deriving all its N from the soil. Leaf sampling were performed in six sites in different periods of the year from mature carob trees and from adjacent trees either normally able (Spartium junceum or Robinia pseudoacacia) or unable (control trees, like Olea sativa, Olea oleaster, Arbutus unedo, Pistacia lentiscus) to develop efficient symbioses with rhizobia. Soil δ15N ranged from 5.1 to 8.4‰. While leaves of trees known to develop N2 fixing symbioses had lower δ15N than the others, leaves of carob trees had similar values of δ15N than control trees. Data from the study did not support the hypothesis that field grown carob trees used N derived from symbiotical fixation.