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Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius , respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus , respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus , respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Robust protocols for the regeneration of somatic embryos in vitro are essential for the efficient use of the most modern biotechnologies. Unfortunately, in perennial trees such as Citrus , plants regenerated from juvenile tissues usually exhibit strong, undesirable juvenile characters such as thorny habit and delayed flowering and fruit production. In this work, we tested whether the cell types (nucellar and stigma/style) used to regenerate Citrus plants through somatic embryogenesis affected the transition from the juvenile to mature phase. The results show that regenerants from nucellar cells presented persistent juvenile characters, whereas plants originating from stigma/style explants transited to the mature phase more rapidly. Our observations support the hypothesis that the totipotent cells originated from different cell types are not equivalent, possibly by maintaining memory of their previously differentiated state.

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called "Olive Quick Decline Syndrome" (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive.

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains AND from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

Mango (Mangifera indica L.) is an important fruit crop in many tropical and subtropical countries. This crop has been recently introduced in Italy, mainly in Sicily (southern Italy), where it proves to be a good commodity for the national market. However, the future of mango cultivation in Sicily is threatened by diverse biotic and abiotic factors, which may limit production and fruit quality. For this reason, an investigation into soilborne diseases of mango was carried out in summer 2009 and spring 2010 (1). During May 2009, typical symptoms of Verticillium wilt were observed in four young mango orchards, cv. Kensington Pride, in Catania Province. The symptoms observed included gradual leaf yellowing, marginal browning, slow growth, and dieback on one side of the shoots. The dead leaves remained attached to infected branches and no defoliation was observed. Cross sections of affected branches showed brown vascular discoloration. The incidence of infected trees was about 25% of 96 examined trees. Small pieces of brownish, vascular wood tissues were surface disinfested and placed onto potato dextrose agar (PDA). After 5 days of incubation at 25°C in the dark, the isolates were purified using the single-spore isolation method. Pathogen identification was initially based on morphological characteristics, and then confirmed by molecular methods. The colonies produced from all the tested isolates showed irregular shape, black microsclerotia, and hyaline, elliptical, single-celled conidia developed on verticillate conidiophores (2). For molecular identification, two specific primer pairs (Ver2-Ver3 and Vd7b-Vd10) of the intergenic spacer region (IGS) were used (3). A fragment of 339 bp was obtained by Ver2-Ver3 primer pair, which is a genus-specific primer, while a fragment of 139 bp was amplified by Vd7b-Vd10 primer pair specific for V. dahliae. To fulfil Koch's postulates, 10-month-old mango plants cv. Kensington Pride were inoculated by dipping the roots in Verticillium conidial suspension for 10 min. Conidial suspension was approximately 4 × 10 6 conidia/ml, prepared from Verticillium isolates Vd-1 and Vd-2 (10 plants for each isolate). Plants dipped in sterile water were used as controls. All plants were kept in a greenhouse at 25°C and 90 to 95% relative humidity. After 10 months, all inoculated plants showed symptoms identical to those observed in mango plants in the field; plants inoculated with water did not show symptoms. V. dahliae was consistently isolated from symptomatic plants but not from plants inoculated with water. To our knowledge, this is the first report of Verticillium wilt caused by V. dahliae on mango in Italy. Verticillium wilt is known to be a serious threat for the mango industry worldwide. The disease, still localized in Catania Province, may soon affects all Sicilian mango-growing provinces with serious economic consequences. References: (1) Y. M. Ahmed et al. J. Plant Pathol. 92:S4.71, 2010. (2) D. L. Hawksworth and P. W. Talboys. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 256. CAB International, Wallingford, UK, 1970. (3) L. Schena et al. Phytopathol. Mediterr. 43:273, 2004.

During spring-summer 2009, a survey was conducted to determine the species of Phytophthora present in citrus nurseries in Egypt. A total of 300 samples of soil and fibrous roots were collected from the rhizosphere of symptomatic Volkameriana lemon (Citrus volkameriana Tan. & Pasq.) plants growing in Delta (Benha-Qalyubia) and a desert (Cairo/Alexandria desert road) citrus nurseries. Plants showed various symptoms. Canopies of affected plants showed few and yellowish leaves, a general stunted growth, no new vegetation, and sometimes sudden desiccation; the root system showed few dark brown feeder roots, no new yellow-white apexes, and a fibrous appearance of the rootlets due to disintegration of the cortical bark but not of the xylem. Collected rootlets and soil were plated in Petri dishes containing a selective medium for the oomycete Phytophthora (2) and incubated for 3 to 6 days at 19 ± 1°C as described by Ippolito et al. (1). Pure cultures were obtained by single-hypha transfers and the isolates were identified as Phytophthora palmivora (Butler) Butler on the basis of morphological and cultural characteristics (3). Isolates formed stoloniferous colonies on potato dextrose agar (PDA) and grew between 10 and 30°C, with the optimum at 25°C. On V8 juice agar, they showed a highly fluffy pattern and produced terminal and intercalary globose chlamydospores. Sporangia were papillate, elliptical (45 to 51 × 29 to 35 μm; length/breadth ratio of 1.3:1.8), and were caducous with short pedicel. All isolates were A2 mating type, forming spherical oogonia and amphigynous antheridia in dual cultures with reference P. palmivora isolate of A1 mating type. Identification of the isolates was further confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region using the universal primers ITS4 and ITS6. BLASTn analysis of ITS sequences (GenBank Accession No. HE583183) showed 99% homology with P. palmivora isolates available in GenBank. Pathogenicity tests for P. palmivora were conducted by inoculating three groups of ten 6-month-old Volkameriana lemon plants, transplanted into 1.4 liter pots with growing medium artificially inoculated at the rate of 1% (v/v) of P. palmivora inoculum produced according to Yaseen (4). Ten uninoculated plants served as a control. Two months after the inoculation, plants were analyzed for canopy symptoms and the presence of pathogen in feeder roots. More than 50% of inoculated plants showed foliage symptoms and extensive decay of feeder roots. Colonies of Phytophthora were recovered from necrotic rootlets and identified as P. palmivora, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of P. palmivora as a pathogen to citrus plants in the Egyptian nurseries. P. palmivora should be considered a potential threat to the Egyptian citrus industry since it may negatively influence the nurseries and orchards production in the future. References: (1) A. Ippolito, V. De Cicco, and M. Salerno. Rivista di Patologia Vegetale 2:57, 1992. (2) H. Masago, M. Yoshikawa, M. Fukada, and N. Nakanishi. Phytopathology 67:425, 1977. (3) D. J. Stamps. Revised tabular key to the species of Phytophthora. CAB International Mycological Institute, Kew, Surrey, 1990. (4) T. Yaseen. Molecular diagnosis and biological control of Phytophthora-citrus root rot. PhD thesis. University of Bari, Italy, 2004.

During a survey for the sanitary status of stone fruits in southern Italy (Apulia region), symptoms of low vigor, sparse foliage, and chlorosis of leaves, frequently leading to decline or death of the plants, were observed on 3- to 5-year-old peach trees (Prunus persica) cvs. Tardi Belle, Zee Lady, and O'Henry grafted on GF677. Brown-to-black discolorations of the wood were observed in cross-sections of the trunks just below the graft union. Samples were collected from May to June 2010 from two symptomatic orchards in Brindisi and Foggia provinces. Small pieces of brownish, vascular wood and necrotic root tissues were surface disinfested, placed onto potato dextrose agar (PDA), and incubated for 7 days at 25°C in the dark. Single-conidial isolates were subsequently grown on PDA at 25°C for 10 days. Fungal colonies were presumptively identified as members of the genus Cylindrocarpon on the basis of their morphological and conidial characteristics. On PDA, the isolates developed abundant mycelium, which gradually became yellowish or partially brownish. Macroconidia were predominantly three septate, straight and cylindrical with both ends broadly rounded. Chlamydospores and ovoidal microconidia were observed on synthetic nutrient-poor agar (1). Sequence of the ribosomal internal transcribed spacer (ITS) region was obtained using universal primers (ITS6-ITS4) and deposited in GenBank (Accession No. HE577846). This sequence revealed 100% genetic identity with a sequence from Cylindrocarpon pauciseptatum Schroers & Crous (Accession No. EF607090), a recently described species (3). In nature, several species of the genus Cylindrocarpon affect a large number of woody plants, mainly grapevine, olive, and stone fruits, in which they attack the root surface (2). To verify Koch's postulates, the roots of 20 3-month-old peach seedlings (GF305) were dipped for 30 min in a spore suspension of the fungus (1 × 10 8 conidia ml -1 ). Seedlings were then transplanted in an artificial soil mix and held under controlled conditions in a greenhouse at 24°C. Typical black-foot symptoms developed on 92% of the inoculated plants within 3 months, whereas the control plants, whose roots had been dipped in distilled water, remained healthy. C. pauciseptatum was reisolated from infected tissues and internal vascular lesions of 45% of the inoculated plants, but none of the plants used as controls, fulfilling Koch's postulates. To our knowledge, this is the first report of this pathogen on peach in the Apulia Region of Italy. Currently, C. pauciseptatum is limited to a few orchards where presumably it was introduced with infected propagating material from extra-regional nurseries. C. pauciseptatum has the potential to negatively affect the stone fruit industry in Italy including reducing nursery production and productivity and vigor of trees in orchards, or even rapid death of young trees. References: (1) W. Gams et al. CBS Course of Mycology. 4th ed. Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, 1998. (2) M. E. S. Hernandez et al. Eur. J. Plant Pathol. 104:347, 1998. (3) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

During the spring of 2006, the main Syrian citrus-growing areas of Lattakia (Jableh, Aledyye, Eseelya, Siano, and Hresoon provinces) and Tartous (Almintar, Aljammase, Karto, Majdaloonelbahr, Yahmour, Amreet, Althawra, and Safita provinces) were surveyed to assess the presence of Citrus tristeza virus (CTV). Eight nurseries (approximately 130 plants per nursery), two budwood source fields (approximately 230 trees per field), and 19 groves (approximately 60 trees per grove) containing the main citrus varieties were visually inspected and sampled for serological assays. The hierarchical sampling method was carried out in each selected grove (2). Infected samples were collected from two nurseries, two budwood source fields, and six groves. Stems and leaf petioles from nursery trees and flower explants from the groves were collected and analyzed for CTV by direct tissue blot immunoassay (DTBIA) with the commercial kit from Plantprint (Valencia, Spain). Of 2,653 samples tested, 89 (4%) CTV-infected plants were detected. Five citrus varieties were found to be infected and Meyer lemon (Citrus limon 'Meyer') had the highest incidence at 16%. Numerous sweet orange varieties (Citrus sinensis L.) were found to be highly infected in the field, but only the Washington navel sweet orange was found to be infected in the nurseries. No clear CTV symptoms were observed during the survey. Samples that were positive for CTV by DTBIA were also positive by biological indexing on Mexican lime (C. aurantifolia) and immunocapture-reverse transcription-PCR as described by Nolasco et al. (3). Coat protein gene sequences obtained from five selected clones of a Syrian CTV isolate (GenBank Accession No. EU626555) showed more than 99 and 98% nucleotide sequence identity to a Jordanian CTV isolate (GenBank Accession No. AY550252) and the VT isolate (GenBank Accession No. U56902), respectively. Almost all infected samples induced moderate vein clearing symptoms when grafted to Mexican lime. Symptoms of vein clearing, leaf cupping, stunting, and stem pitting on Mexican lime were induced by graft transmission of CTV from one Valencia sample from the Tartous area. The viral inoculum is widely and randomly distributed in commercial groves, especially in the southern Tartous area and in some nurseries. To our knowledge, this is the first report of CTV in Syria. However, CTV was reported from the neighboring citrus-growing countries of Lebanon, Turkey, and Jordan (1), and the severe seedling yellows strain is present in this area, which poses a potential threat to Syrian citriculture. References: (1) G. H. Anfoka et al. Phytopathol. Mediterr. 44:17, 2005. (2) G. Hughes and T. R. Gottwald, Phytopathology 88:715, 1998. (3) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002.