Secondary metabolites produced by four Colletotrichum species in vitro and on fruits of diverse olive cultivars.

This study was aimed to characterize the secondary metabolites produced by four Colletotrichum species, C. acutatum, C. gloeosporioides, C. godetiae and C. karsti, both in vitro, on potato dextrose agar (PDA) and oatmeal agar (OA), and during the infection process of fruits of four olive cultivars differing in susceptibility to anthracnose, 'Coratina' and 'Ottobratica', both susceptible, 'Frantoio' and 'Leccino', both resistant. The metabolites were extracted from axenic cultures after seven days incubation and from olives inoculated singularly with each Colletotrichum species, at three different times, 1, 3 and 7 days post inoculation (dpi). They were identified using the UHPLC-QTOF-MS analysis method. In total, as many as 45 diverse metabolites were identified. Only 10 metabolites were present in both fruits and axenic cultures while 19 were found exclusively on olives and 16 exclusively in axenic cultures. The identified metabolites comprised fatty acid, phenolics, pyrones, sterols, terpenes and miscellaneous compounds. Each Colletotrichum species produced a different spectrum of metabolites depending on the type of matrices. On artificially inoculated olives the severity of symptoms, the amount of fungal secondary metabolites and their number peaked 7 dpi irrespective of the cultivar susceptibility and the virulence of the Colletotrichum species.

First Report of Root Rot Caused by Phytophthora bilorbang on Olea europaea in Italy.

Leaf chlorosis, severe defoliation and wilt associated with root rot were observed on mature olive trees cv. Nera di Gonnos in an experimental orchard at Mirto Crosia (Calabria, southern Italy). An oomycete was consistently isolated from rotten roots of symptomatic olive trees. It was identified as Phytophthora bilorbang by morphological characters and sequencing of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA). Pathogenicity was verified by inoculating potted two-month-old rooted cuttings of Olea europaea var. Nera di Gonnos in a soil infestation trial. P. bilorbang was re-isolated from roots of symptomatic, artificially inoculated olive cuttings to fulfill Koch's postulates. This is the first report of P. bilorbang on O. europaea L. and on a species of the Oleaceae family worldwide.

Effect of arbuscular mycorrhizal fungi on growth and on micronutrient and macronutrient uptake and allocation in olive plantlets growing under high total Mn levels.

The reported work was designed to increase knowledge about the role of arbuscular mycorrhizal fungi (AMF) on the phytoavailability and allocation of some of the principal macroelements and microelements in young potted olive plants growing in a soil presenting high levels of manganese (Mn), taken from an experimental olive field. A greenhouse trial was performed using self-rooted cuttings of Ascolana tenera, Nocellara del Belice and Carolea cultivars inoculated or not with two mycorrhizal inocula (commercial vs native). Molecular characterization of the indigenous AMF indicated that the species found in the experimental soil were different from those present in the commercial inoculum. The important incidence of AMF on P uptake was confirmed with generally double the concentration in mycorrhizal olive plants as compared to non-mycorrhizal controls, irrespective of genotype and inocula. Furthermore, apart from promoting plant growth (from 1.7- to 5-fold), the symbiosis reduced Mn concentrations from 43 to 83%. The observed differences depended on the cultivar and the inoculum, with native AMF being more effective probably as a result of their adaptation to the experimental soil. No clear direct relationship was found between AMF inoculation and other elements analysed.

Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR.

BACKGROUND: Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta. RESULTS: Specific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi were designed to be used in End Point and Real-Time PCR, both with SYBR Green or TaqMan chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants. CONCLUSIONS: Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of P. savastanoi on olive and oleander propagation materials.