Volatilome modulation in Artemisia annua plants inoculated or not with soil beneficial microorganisms

Artemisia annua is a medicinal plant belonging to the Asteraceae family and known for the production of artemisinin, a bioactive compound used against malaria Plasmodium as a good alternative to conventional treatments. Many VOCs (Volatile Organic Compounds) are also released from its glandular trichomes, by determining the so-called plant volatilome. Since the artemisinin concentration in planta is quite low (0.1-1% DW) and extremely variable in plants obtained from seeds, in this study we have worked with micropropagated clones in order to rely on genetically uniform materials. It is well known from the literature that soil beneficial microorganisms, such as arbuscular mycorrhizal fungi (AMF) and plant growth promoting bacteria (PGPB) can affect plant growth and metabolism. Therefore, the aim of this work was to test different AMF and PGPB strains (alone or in combination) on two clones CL7 and CL26 under controlled conditions, in order to evaluate their effects on plant growth parameters, leaf pigment concentration and artemisinin production (HPLC analysis). Moreover, the volatilome composition was investigated by GC-MS in the CL26 clone. Plants were maintained in a growth chamber for 2 months and irrigated three times per week with Long Ashton nutrient solution. The obtained results showed that the two clones responded in different ways to the inoculation with the same microorganism and, conversely, different microorganisms differently affected the same clone, either in terms of plant shoot and root biomass production or chlorophyll and carotenoid concentration. Regarding the volatile compounds of A. annua, about 120 molecules were identified in the leaves of CL26 plants, a very high number compared to that reported in previous studies; in fact, some compounds were recorded for the first time in this work (e.g calarene, cedroxyde, hermophilon, thujopsenal and γ-muurolene). All the identified molecules were included in different chemical representative classes of the volatilome such as alkenes, alcohols, aldehydes, acids, ketones, ethers, esters, and "other compounds", whose percentage varied according to the used inocula; some molecules were unique to each treatment, and this diversity was due to the influence of each microorganism combination on the production of secondary metabolites, giving a specific particular odor to the plant of the same treatment. This odor is mainly due to various substances, with bioactive properties, composing the A. annua essential oil, that we found within the complex matrix of the leaf extract.