Stefania Mirela Mang

An In Vitro Attempt for Controlling Severe Phytopathogens and Human Pathogens Using Essential Oils from Mediterranean Plants of Genus Schinus

Growing concerns about food safety and environmental protection enhanced the need for new and safe plant disease control strategies. The chemical composition of the three essential oils (EOs) extracted from leaves and fruits of Schinus terebinthifolius and leaves of Schinus molle, growing in Tunisia, was studied by GC and GC-MS. In all, 12 compounds were identified. The oils were mainly composed of terpene compounds. α-Pinene, α-phellandrene, and D-limonene were the major constituents. The aim of the current study was to evaluate the in vitro antimicrobial effectiveness of three EOs derived from plants of genus Schinus and extracted from leaves and fruits of S. terebinthifolius and leaves of S. molle. Both antifungal and antibacterial activities of the EOs were examined. The antifungal activity of the studied EOs was investigated against Colletotrichum acutatum and Botrytis cinerea in comparison with the systemic fungicide azoxystrobin used at 0.8 μL mL(-1). The antibacterial activity was evaluated against three strains of Gram-positive (G+ve) bacteria (Bacillus megaterium, Bacillus mojavensis and Clavibacter michiganensis) and four strains of Gram-negative (G-ve) bacteria (Escherichia coli, Xanthomonas campestris, Pseudomonas savastanoi, and Pseudomonas syringae pv. phaseolicola) compared with the synthetic antibiotic tetracycline at a concentration of 1600 μg mL(-1). The minimum inhibitory concentration of the studied EOs has been evaluated against the above microorganisms using the 96-well microplate method. Tested microorganisms exhibited different levels of sensitivity to each tested EO. All investigated EOs reduced the fungal mycelial growth when used at low concentrations from 250 to 1000 ppm and from 2000 to 8000 ppm against C. acutatum and B. cinerea, respectively. Higher concentrations of the same EOs exhibited a fungicidal effect against both mitosporic fungi. The EO extracted from leaves of S. terebinthifolius significantly inhibited the growth of tested bacterial strains. Nevertheless, E. coli showed a weak resistance toward the same EO and a high resistance toward the other two tested EOs. Finally, P. savastanoi and P. syringae pv. phaseolicola showed a high resistance toward all tested EOs.

Diversity Evaluation of Xylella fastidiosa from Infected Olive Trees in Apulia (Southern Italy).

Olive culture is very important in the Mediterranean Basin. A severe outbreak of Olive Quick Decline Syndrome (OQDS) caused by Xylella fastidiosa infection was first noticed in 2013 on olive trees in the southern part of Apulia region (Lecce province, southern Italy). Studies were carried out for detection and diversity evaluation of the Apulian strain of Xylella fastidiosa. The presence of the pathogen in olive samples was detected by PCR amplifying the 16S rDNA, gyrase B subunit (gyrB) and HL hypothetical protein genes and single nucleotide polymorphisms (SNPs) assessment was performed to genotype X. fastidiosa. Twelve SNPs were recorded over gyrB and six SNPs were found for HL gene. Less variations were detected on 16S rDNA gene. Only gyrB and HL provided sufficient information for dividing the Apulian X. fastidiosa olive strains into subspecies. Using HL nucleotide sequences was possible to separate X. fastidiosa into subspecies pauca and fastidiosa. Whereas, nucleotide variation present on gyrB gene allowed separation of X. fastidiosa subsp. pauca from the other subspecies multiplex and fastidiosa. The X. fastidiosa strain from Apulia region was included into the subspecies pauca based on three genes phylogenetic analyses.

USE OF VOLATILE METABOLITE PROFILES TO DISTINGUISH THREE MONILINIA SPECIES

SUMMARY Analyses of volatile organic compounds (VOCs) produced by three Monilinia species (M. laxa, M. fructigena and M. fructicola) grown on potato dextrose agar (PDA) were done by head space solid phase micro extraction combined with gas chromatography/mass spectrometry (HS-SPME-GS/MS). A total of 14 compounds of different chemical structure were identified, the most frequent of which were ethanol, dodecane and alpha-Muurolene. Specific VOCs were identified, which allowed the discrimination of the three Monilinia species. If coupled with the use of the electronic nose and upon validation under commercial conditions, the results of this study may have potential applications in postharvest for detecting and identifying diseases of stone and pome fruits, in relatively early stages of their development.

First Report of Albugo candida Causing White Rust on Lunaria annua in Italy

Money plant or annual honesty (Lunaria annua L.) is an ornamental landscape plant used in flower beds and borders and also in flower arrangements. It is a biennial plant with large, pointed, oval leaves. Plants of L. annua showing white-to-cream, blister-like lesions on leaves and siliques (2) were found in private gardens where approximately 800 plants of 1,000 (approximately 80 to 90%) that were observed showed symptoms. The disease was also found in two ornamental nurseries, although it was limited to a few mother plants because of extensive fungicide treatments. The gardens and ornamental nurseries were located in Potenza Province (Basilicata Region, southern Italy). Sporangiophores were mostly straight or arched and almost cylindrical with attenuated base and flat or rounded apex and measured 29.2 to 33.4 × 12.8 to 13.4 μm. Sporangia, produced in chains and joined by short connectives, exhibited a spherical or angular shape, were subhyaline, contained vacuoles, and had average maximum and minimum diameters ranging from 15.8 to 18.8 and 14 to 16 μm, respectively. The morphological characteristics closely resembled those reported for Albugo candida (Pers.) Kuntze (3). Sori were collected from naturally and artificially inoculated tissues of L. annua, with the aid of a stereomicroscope, and used to extract genomic DNA via a DNeasy Plant Mini DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturers directions. The extracted DNA was used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS4/DC6 (1,4) and sequenced. One sequence, GenBank Accession No. GQ328846, matched several sequences of A. candida (Pers). Kuntze (e.g., GenBank Accession Nos. GQ328837, GQ328836, GQ328835, GQ328834, and AF271231), showing 98% identity. Pathogenicity tests were performed and repeated twice. Leaves of 10 healthy seedlings of L. annua were surface cleaned during several washings with distilled water and then spray inoculated with a suspension of 103 sporangia/ml of A. candida. Five healthy seedlings were spray inoculated with the same volume of sterile water and served as controls. Inoculated seedlings were maintained in a moist chamber for 48 h at 20°C before being moved to a shaded glasshouse at 16 to 24°C and 90% relative humidity. White rust symptoms, similar to those observed in natural conditions, appeared on leaves of inoculated seedlings 10 to 14 days later, demonstrating that A. candida was the causal agent of the disease. Control plants remained symptomless. White rust has been reported on L. annua in Europe (Czech Republic, Germany, Poland, and the United Kingdom) and in the northwestern United States (3). To our knowledge, this is the first report of A. candida infecting annual honesty plant in Italy. References: (1) P. Bonants et al. Eur. J. Plant Pathol. 103:345, 1997. (2) D. Choi et al. Mycotaxon 53:261, 1995. (3) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2004-0317-01-HN. Plant Health Progress, 2004. (4) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Impact of airborne zinc pollution on the antimicrobial activity of olive oil and the microbial metabolic profiles of Zn-contaminated soils in an Italian olive orchard

The growing of microbial resistance leads to a great interest about some natural alternatives to synthetic compounds. This study was carried out in two olive orchards (Olea europaea L., cv. Coratina) South Italy (Basilicata region), one located in a polluted area near a fertilizers factory releasing Zn and the other in a control unpolluted site, both managed with similar cultivation techniques. Olive oil samples were studied from both areas during 2014 and 2015. The soil microbiological status of the polluted and unpolluted orchards has been characterized and the antimicrobial effects of olive oils extracted from polluted plants (PP) and control plants (CP) against some phytopathogens have been explored. Results showed that the antibacterial activity of PP oil was significantly higher than CP and this could be due to the high content of some phenolic compounds elicited by air and soil Zn pollution (especially in the layer 0-20 cm). There is no detectable antifungal activity of the studied oils. The metabolic activity (both total and for each carbon substrate group), diversity and evenness of PP soil bacterial communities were significantly different from CP soil, while the effects of soil depth was negligible. The same parameters measured on soil fungal communities are lower in PP soil at 0-20 cm soil depth. The current research clarified the impact of atmospheric Zn pollution on the antimicrobial activity of olive oil and the soil microbial metabolic profiles. The bioactive substances extracted from olive oils growing in Zn-polluted area might be used as antibiotics.

Biodiversity of Hypogeous Fungi in Basilicata

During the last two decades, systematic studies were carried out on biodiversity of hypogeous fungi in forestry territories of the two Basilicata (southern Italy) provinces, Matera and Potenza. Identification of fungus taxa found in the region was commonly accomplished on the basis of macro- and microscopic features, and only in a few instances, molecular analyses were utilized. Thanks to these investigations, Basilicata now occupies, among Italian regions, the first and fourth positions for number of Tuber species, varieties or forms and total number of hypogeous fungi (Ascomycota, Basidiomycota and Zygomycota) naturally growing in its woodlands and Mediterranean maquis areas. In fact, the last up-to-date acquirements on the topic bring up to 29 and 53 the number of Tuber taxa and that of the other hypogeous and semi-hypogeous (only three entities) fungi present in the region, respectively. In this chapter, the essential information regarding these fungi is given, so updating to 2014 the relative available knowledge. Among the Fungi, object of this review, the Ascomycota Pachyphloeus conglomeratus and Tuber malenconii, the Basidiomycota Hymenogaster decorus, H. hessey, H. rehsteineri, Schenella pityophilus and Myriostoma coliforme as well as the Zygomycota Youngiomyces multiplex deserve a particular mention because of their rarity.