Phytochemical effects of phytoplasma infections on essential oil of Monarda fistulosa L.

Monarda fistulosa L. (“wild bergamot”) is an annual or perennial medicinal plant known for the strong thera-peutic effects: its essential oil is characterized by high antibacterial, antimycotic, and anti-inflammatory activi-ties and, for this reason, has been recently proposed for the treatment of seborrhoea (Zhilyakova et al., 2009). During a survey carried out in 2009 in Italy, wild ber-gamot showing yellows, stunting, virescence and flower bud proliferation was found for the first time infected by a phytoplasma belonging to ribosomal subgroup 16SrXII-A “stolbur” (Bellardi et al., 2011). In 2010, a study to verify the correlations between phytoplasma presence and symptom expression, and the effects of these prokaryotes on essential oil composition was car-ried out. During Summer 2010, molecular tests to confirm phy-toplasma presence were carried out in M. fistulosa plants belonging to the same crop surveyed in 2009, (Herb Garden, Casola Valsenio, Italy). Starting from July, increasing percentages of plants showing phyto-plasma symptoms (more than 80%) were observed. Symptomless (=SLP) and symptomatic plants (=SP) (Fig. 1) were labelled by visual inspection of their aerial parts and tested at blooming stage to verify phytoplasma presence and to determine their identity. After a chloro-form/phenol extraction (Prince et al., 1993), plants were tested by direct PCR with primers P1/P7 followed by nested PCR with primers R16F2/R2 . RFLP analyses were performed with TruI and Tsp509I for 16 hours at 65°C and with HhaI for 16 hours at 37°C and analysed on 5% polyacrilamide gels after ethidium bromide staining. About 900 gr fresh aerial part material of SP and about 120 gr of SLP were collected in August at the end of flowering, and hydrodistilled; the two oils were separated from water and kept in tightly closed amber vials before analyses. Identification of the compounds was made by combined gas chromatography mass spec-trometry (GC/MS) and by comparing retention times of M. fistulosa components with those of a control oil from plants grown in Trentino Region (Northern Italy) While in 2009, only 50% of the plants showed phyto-plasma symptoms and was found to be infected only by stolbur, in 2010, amplifications resulted from both symptomless and symptomatic plants (any phyto-plasma-free plant was detected). Both, direct and nested PCR, as well as RFLP analyses on 16Sr DNA gene, confirmed that in all wild bergamot plants, stolbur phy-toplasma was present in single infection in SLP sam-ples, and in mixed infection with phytoplasma belong-ing to ribosomal subgroup 16SrI-B (Aster yellows, ‘Candidatus Phytoplasma asteris’: AY) in SP samples. Considering that every single plant, with or without symptoms, was phytoplasma-infected, no phytoplasma-free material has been collected to obtain control oil from healthy plants. The increasing of phytoplasma in-fection (100%) inside the Herb Garden occurred as a consequence of the presence of leafhoppers, weeds and other medicinal phytoplasma-infected plants, such as Echinacea purpurea Moench, Digitalis lutea L., D. lanata Ehrh., Grindelia robusta L., recently reported as “new natural host” of AY and/or stolbur phytoplasmas (Bellardi et al., 2007). Differences in compositions between the oils from SP and SLP were observed. As shown in table 1, in which the percentage presence of 18 components is listed ac-cording to their elution order, the oil from SP showed an increase in the quantity of some monoterpenes like -thujene, - and -pinene, and -phellandrene as well as in the content of -caryophyllene and a marked de-crease in the content of thymol. On the other hand, oil from SLP yielded only a significant increase in thymol concentration as only relevant modification and a marked decrease in monoterpene compounds. Composition of the oils obtained by distillation varies significantly among different M. fistulosa hybrids (Mazza and Marshall, 1992); thy...