Biological investigations of essential oils extracted from three Juniperus species and evaluation of their antimicrobial, antioxidant and cytotoxic activities.

AIMS: To conduct biological investigations and to evaluate the antimicrobial and antioxidant activities of the essential oils (EOs) extracted from Juniperus communis, J. scopulorum and J. horizontalis; to screen their mechanisms of action by conducting the cell membrane permeability assay (CMP); and to determine the possible cytotoxicity of the three EOs against human neuroblastoma cells (SH-SY5Y). METHODS AND RESULTS: The antifungal activity was tested against four phytopathogenic fungi (Monilinia fructicola, Aspergillus niger, Penicillium expansum and Botrytis cinerea). The antibacterial activity was evaluated against two Gram-positive (G+ve) (Bacillus megaterium and Clavibacter michiganensis) and three Gram-negative (G-ve) bacterial strains (Pseudomonas fluorescens, P. syringae pv. phaseolicola and Xanthomonas campestris). Results showed that the three tested EOs have antifungal activity against M. fructicola and P. expansum and effective antibacterial activity against P. syringae pv. phaseolicola and B. megaterium. Moreover, the three EOs were evaluated for their ability to inhibit the growth of SH-SY5Y cells with MTT assay. J. communis EO was the more effective with an IC50 of 53·7 µg ml-1 . The antioxidant capacity of the three EO did not differ as measured by the DPPH assay. CONCLUSIONS: The three tested juniper EOs showed promising antimicrobial and antioxidant activity and cytotoxic effects against human neuroblastoma cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: The outfindings from this research showed promising antimicrobial effects of the three oils against the majority of the tested phytopathogens with a potential to utilize them as natural alternatives to synthetic drugs, the cause of global environmental problems, pathogen resistance and difficulty to control many post-harvest plant diseases.

Mycoremediation effect of Trichoderma harzianum strain T22 combined with ozonation in diesel-contaminated sand.

This study aimed to determine the ability of the fungus Trichoderma harzianum strain T22 (Th-T22) to utilize diesel fuel as a carbon source. The potential use of Th-T22 for diesel bioremediation in an artificial soil was tested by inoculating a diesel-sand mixture with a fungal mycelial suspension of Th-T22. Given the ability of ozone to degrade compounds with low biochemical reactivity, the effect of a pre- and post-ozonation was also evaluated. The survival, growth and sporulation of Th-T22 throughout the bioremediation trial were monitored in all the treatments. In the post-ozonation treatments, the biodegradation percentages of diesel removal were 70.16% and 88.35% in Th-T22-inoculated sand treated or untreated with the antibacterial streptomycin, respectively. The results showed that ozonation alone caused good removal efficiencies (41.9%) but it was much more effective if combined with Th-T22 in a post-ozonation regime, whereas pre-ozonation negatively affected the subsequent biodegradation, likely due to its disinfectant and oxidizing effect on Th-T22. The results obtained demonstrated the significant mycoremediation ability of Th-T22 in diesel-contaminated sand and its possible use as a bioremediation agent for diesel spills in polluted sites.

Effects of different heat treatments on lysozyme quantity and antimicrobial activity of jenny milk.

Thermal treatments are used to improve milk microbial safety, shelf life, and biological activity of some of its components. However, thermal treatments can reduce the nutritional quality of milk, affecting the molecular structure of milk proteins, such as lysozyme, which is a very important milk component due to its antimicrobial effect against gram-positive bacteria. Jenny milk is characterized by high lysozyme content. For this reason, in the last few years, it has been used as an antimicrobial additive in dairy products as an alternative to hen egg white lysozyme, which can cause allergic reactions. This study aimed to investigate the effect of pasteurization and condensation on the concentration and antimicrobial activity of lysozyme in jenny milk. Furthermore, lysozyme quantity and activity were tested in raw and pasteurized milk after condensation at 40 and 20% of the initial volume. Reversed-phase HPLC was performed under fluorescence detection to monitor lysozyme in milk samples. We evaluated the antimicrobial activity of the tested milk against Bacillus megaterium, Bacillus mojavensis, Clavibacter michiganensis, Clostridium tyrobutyricum, Xanthomonas campestris, and Escherichia coli. Condensation and pasteurization did not affect the concentration or antimicrobial activity of lysozyme in jenny milk, except for B. mojaventis, which showed resistance to lysozyme in milk samples subjected to heat treatments. Moreover, lysozyme in jenny milk showed antimicrobial activity similar to synthetic antibiotics versus some gram-positive strains and also versus the gram-negative strain X. campestris.

Fungal Planet description sheets: 214-280.

Novel species of microfungi described in the present study include the following from South Africa: Cercosporella dolichandrae from Dolichandra unguiscati, Seiridium podocarpi from Podocarpus latifolius, Pseudocercospora parapseudarthriae from Pseudarthria hookeri, Neodevriesia coryneliae from Corynelia uberata on leaves of Afrocarpus falcatus, Ramichloridium eucleae from Euclea undulata and Stachybotrys aloeticola from Aloe sp. (South Africa), as novel member of the Stachybotriaceae fam. nov. Several species were also described from Zambia, and these include Chaetomella zambiensis on unknown Fabaceae, Schizoparme pseudogranati from Terminalia stuhlmannii, Diaporthe isoberliniae from Isoberlinia angolensis, Peyronellaea combreti from Combretum mossambiciensis, Zasmidium rothmanniae and Phaeococcomyces rothmanniae from Rothmannia engleriana, Diaporthe vangueriae from Vangueria infausta and Diaporthe parapterocarpi from Pterocarpus brenanii. Novel species from the Netherlands include: Stagonospora trichophoricola, Keissleriella trichophoricola and Dinemasporium trichophoricola from Trichophorum cespitosum, Phaeosphaeria poae, Keissleriella poagena, Phaeosphaeria poagena, Parastagonospora poagena and Pyrenochaetopsis poae from Poa sp., Septoriella oudemansii from Phragmites australis and Dendryphion europaeum from Hedera helix (Germany) and Heracleum sphondylium (the Netherlands). Novel species from Australia include: Anungitea eucalyptorum from Eucalyptus leaf litter, Beltraniopsis neolitseae and Acrodontium neolitseae from Neolitsea australiensis, Beltraniella endiandrae from Endiandra introrsa, Phaeophleospora parsoniae from Parsonia straminea, Penicillifer martinii from Cynodon dactylon, Ochroconis macrozamiae from Macrozamia leaf litter, Triposporium cycadicola, Circinotrichum cycadis, Cladosporium cycadicola and Acrocalymma cycadis from Cycas spp. Furthermore, Vermiculariopsiella dichapetali is described from Dichapetalum rhodesicum (Botswana), Ophiognomonia acadiensis from Picea rubens (Canada), Setophoma vernoniae from Vernonia polyanthes and Penicillium restingae from soil (Brazil), Pseudolachnella guaviyunis from Myrcianthes pungens (Uruguay) and Pseudocercospora neriicola from Nerium oleander (Italy). Novelties from Spain include: Dendryphiella eucalyptorum from Eucalyptus globulus, Conioscypha minutispora from dead wood, Diplogelasinospora moalensis and Pseudoneurospora canariensis from soil and Inocybe lanatopurpurea from reforested woodland of Pinus spp. Novelties from France include: Kellermania triseptata from Agave angustifolia, Zetiasplozna acaciae from Acacia melanoxylon, Pyrenochaeta pinicola from Pinus sp. and Pseudonectria rusci from Ruscus aculeatus. New species from China include: Dematiocladium celtidicola from Celtis bungeana, Beltrania pseudorhombica, Chaetopsina beijingensis and Toxicocladosporium pini from Pinus spp. and Setophaeosphaeria badalingensis from Hemerocallis fulva. Novel genera of Ascomycetes include Alfaria from Cyperus esculentus (Spain), Rinaldiella from a contaminated human lesion (Georgia), Hyalocladosporiella from Tectona grandis (Brazil), Pseudoacremonium from Saccharum spontaneum and Melnikomyces from leaf litter (Vietnam), Annellosympodiella from Juniperus procera (Ethiopia), Neoceratosperma from Eucalyptus leaves (Thailand), Ramopenidiella from Cycas calcicola (Australia), Cephalotrichiella from air in the Netherlands, Neocamarosporium from Mesembryanthemum sp. and Acervuloseptoria from Ziziphus mucronata (South Africa) and Setophaeosphaeria from Hemerocallis fulva (China). Several novel combinations are also introduced, namely for Phaeosphaeria setosa as Setophaeosphaeria setosa, Phoma heteroderae as Peyronellaea heteroderae and Phyllosticta maydis as Peyronellaea maydis. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

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 manufacturer's 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.

Verticillium Wilt of Xanthium italicum Caused by Verticillium dahliae in Italy.

During the late summer of 2003, a wilt disease of the weed Italian cockleburr (Xanthium italicum Mor.) was observed in the Basilicata Region of southern Italy. Diseased plants were growing near an apricot orchard in which some trees were severely affected by Verticillium wilt. The most characteristic symptoms of the wilt disease affecting Italian cockleburr were yellowing, stunting, and gradual wilting. Also, diagonal and cross sections of stems revealed brown discoloration of their vascular tissues. To elucidate the etiology of the disease, we attempted detection and identification of the causal agents using traditional and polymerase chain reaction (PCR)-based methods. Small pieces of petiole and stem tissues from diseased and asymptomatic plants were surface disinfested in NaOCl solution, rinsed in sterile distilled water, blotted dry, and plated onto water agar (WA) medium. Following incubation at 22°C, the emerging colonies were transferred to potato dextrose agar (PDA). Verticillium dahliae (one isolate) was consistently identified on the basis of its morphological features according to the description of Smith (2). Using PCR assays with the primer pair ITS5/ITS4 (3), which are directed to fungal nuclear ribosomal DNA (rDNA) repeat sequences, an amplification product of approximately 560 bp was obtained by using total DNA extracted from wilt-affected Italian cockleburr plant tissues (five plants examined) as well as fresh mycelium from the V. dahliae-infected Italian cockleburr pure culture-maintained isolate mentioned above. No visible PCR products were obtained with total DNA from asymptomatic Italian cockleburr plants. Sequence analysis of the ITS5/ITS4 amplicons revealed no differences in their nucleotide positions. The obtained sequence of the V. dahliae-infected Italian cockleburr isolate (GenBank Accession No. AJ865691) was then used as query sequences in a BLAST 2.0 search (1). Sequence of the southern Italian isolate proved to be identical to that of the Greek strain "76 Greece" of V. dahliae (GenBank Accession no. AF104926). To prove Koch's postulates, 10 healthy Italian cockleburr seedlings were experimentally inoculated by dipping trimmed roots in a single-conidial suspension (1.5 × 106 CFU/ml) obtained from 10-day-old colonies of the V. dahliae-infected Italian cockleburr pure culture-maintained isolate. After 4 weeks, all inoculated Italian cockleburr plants showed symptoms identical to those of naturally infected field-grown plants. V. dahliae was consistently reisolated from inoculated plants. Additional inoculation experiments revealed that pepper and eggplant were also susceptible to the V. dahliae-infected Italian cockleburr isolate showing typical Verticillium wilt symptoms. To our knowledge, this is the first report of the occurrence of Verticillium wilt of X. italicum. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. C. Smith. N.Z. J. Agric. Res. 8:450, 1965. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.