Feasibility study of tomato fruit characterization by fast XRF analysis for quality assessment and food traceability.

Food product nutritional and sensory characteristics are often deeply linked to its territory of origin; therefore, its authentication by means of elemental composition becomes crucial for traceability and fighting food fraud. This study aims to establish a fast and reproducible procedure for origin and quality assessment of Sicilian tomato fruits, including PGI "Pomodoro di Pachino", by using the X-ray fluorescence (XRF) technique. Measurements were performed on different parts of PGI Pachino tomatoes belonging to the same production lot. Principal Component and Cluster Analyses show that the samples cluster accordingly with the production lot, disentangling the different parts of the fruit. This procedure, which uses XRF yield elemental pattern and statistical analysis, establishes a solid basis for characterizing elemental profiles by a fast XRF in-situ campaign, supporting the traceability system. The reliability of XRF results was confirmed by comparing elemental concentrations with ICP-MS measurements, performed for comparison, and tomato literature values.

First Report of Verticillium Wilt of Mango (Mangifera indica) Caused by Verticillium dahliae in Italy.

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 × 106 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.

First Report of Sclerotinia Stem and Twig Blight Caused by Sclerotinia sclerotiorum on Citrus volkameriana Rootstock in Italy.

Volkamer lemon (Citrus volkameriana Ten. & Pasq., Rutaceae family) is the most commonly used rootstock for some ornamental citrus (oval kumquat and calamondin), improving the aesthetic quality of the plants and their marketable value. During the winter of 2011, symptoms of stem blight were observed on approximately 10% of 12,000 1-year-old potted C. volkameriana seedlings grown in different blocks in a commercial nursery near Catania (eastern Sicily, Italy). In the same nursery, only 1% of 15,000 older seedlings (2-year-old) showed disease symptoms. Initial symptoms included gray lesions on stems and occasionally on twigs. Later, buff lesions and gum exude appeared. Symptomatic stems and twigs were usually girdled and killed. In the lesions, irregular, dark gray sclerotia (1.0 to 5 × 1.0 to 7.0 mm, average 2.5 × 3.9 mm) were produced. In high relative humidity, cottony, white mycelia on the bark surface of infected tissues were also observed. Isolations were performed by transferring approximately 300 fragments of symptomatic tissues from 15 C volkameriana seedlings, surface-sterilized with 1% NaClO for 1 min, on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary was recovered from all infected plants. Colony type, morphology, and dimensions of sclerotia were examined on PDA at 22 ± 1°C after 10 days in the dark. Sclerotia produced on PDA measured 2.0 to 7.0 × 1.5 to 4.0 mm (average 5.6 × 2.6 mm). DNA isolation was performed with the DNA Purification Kit (Puragene-Gentra, Minneapolis, MN) following the manufacturer's instructions. Amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA was performed with primers ITS1/ITS4 (2). BLAST analysis of the 550-bp segment showed a 98% homology with S. sclerotiorum strain ms85 (GenBank Accession No HQ833450.1), thus confirming identification based on morphology. Koch's postulates were fulfilled by pathogenicity tests carried out on 20 1-year-old potted C. volkameriana seedlings. Each seedling was inoculated with five mycelial agar plugs (6 mm in diameter) and five sclerotia from the edge of 10-day-old colonies on PDA and placed in wounds made with a sterile blade in the bark of stem and twigs. Inoculated wounds (10 for each plant) were wrapped with Parafilm. The same number of control plants were wounded and inoculated with sterile PDA plugs. All inoculated plants were incubated in a growth chamber at 22°C with 80 to 90% relative humidity for 14 days. Blight symptoms and lesions on the stem and twigs identical to those observed in the nursery developed on all plants with both types of inoculum. Noninoculated control plants remained symptomless. S. sclerotiorum was reisolated from all symptomatic tissues and identified by morphology as previously described, completing Koch's postulates. To our knowledge, this is the first report of S. sclerotiorum stem and twig blight on C. volkameriana. Worldwide, Sclerotinia stem and twig blight is considered a minor disease on citrus (1), but this evidence suggests that in eastern Sicily, S. sclerotiorum may be an important pathogen of young C. volkameriana seedlings in nurseries. References: (1) J. A. Menge. Page 35 in: Compendium of Citrus Diseases. 2nd ed. The American Phytopathological Society, St. Paul, MN, 2000. (2) T. J.White et al. Page 315in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Pink Ipê (Tabebuia impetiginosa) in Italy.

Pink ipê or pink lapacho (Tabebuia impetiginosa Martius ex DC., family Bignoniaceae) is one of the most attractive blooming trees in the world. In Europe, pink ipê is widely used as an ornamental tree in landscaped gardens and public areas. In August 2010, a widespread damping-off was observed in a stock of approximately 100,000 potted 2-month-old seedlings in a nursery in eastern Sicily (Italy). The seedlings were being watered with overhead irrigation. More than 5% of the seedlings showed disease symptoms. Initial symptoms were black lesions at the seedling crown that expanded rapidly to girdle the stem. On infected seedlings, leaves turned black and gradually died. Black extended stem lesions were followed by death of the entire seedling in a few days. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently isolated from crown and stem lesions when plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 µg/ml. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Mycelium was branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells removed from cultures grown at 25°C on 2% water agar were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11 on 2% water agar in petri plates (4). Anastomosis was observed only with tester isolates of AG-4, giving both C2 and C3 reactions (2). Pathogenicity tests were performed on container-grown, healthy, 3-month-old seedlings. Forty seedlings of T. impetiginosa were inoculated near the base of the stem with two 1-cm2 PDA plugs from 5-day-old mycelial cultures. The same number of plants only inoculated with PDA plugs served as controls. Plants were incubated in a growth chamber and maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Crown and stem lesions identical to those observed in the nursery appeared 5 days after inoculation and all plants died within 25 days. No disease was observed on control plants. R. solani AG-4 was reisolated from symptomatic tissues and identified as previously described. R. solani AG-4 was previously detected in the same nursery on Chamaerops humilis (3). To our knowledge, this is the first report of R. solani causing damping-off on T. impetiginosa. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (3) G. Polizzi et al. Plant Dis. 94:125, 2010. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction.

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, whereas the linear quantification range was within 100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfully detected the fungus in woody samples of naturally infected lemon and artificially inoculated sour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold more sensitive than conventional PCR, and preliminary results indicate that the former technique achieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtain suitable DNA samples from fungal cultures and citrus woody samples for PCR assays enable diagnosis to be completed in a short time.

First Report of Leaf Spot and Blight of Strelitzia reginae Caused by Burkholderia gladioli in Italy.

During the summer of 2005, a new disease of bird of paradise (Strelitzia reginae Aiton) was observed on young seedlings (2 to 3 months old) in a nursery located in Giarre (Catania) in eastern Sicily. Symptoms included brown, water-soaked leaf spots that first appeared after seedling emergence and then gradually enlarged and became necrotic. Occasionally, during wet conditions, seedlings were completely blighted, resulting in total loss. The disease was observed on 10% of the 3,000 plants present in one nursery. A single bacterial colony was consistently isolated on King's medium B (KB) supplemented with 0.01% cycloeximide from surface-sterilized, brownish lesions and water-soaked leaf tissues. The isolates were purified on nutrient agar (NA). Three bacterial strains isolated from three different symptomatic plants were used for pathogenicity and identification tests on S. reginae plants. Five plants were inoculated per bacterial strain by spraying the leaves with a buffer phosphate suspension (0.1 M) at 106 CFU/ml prepared from KB plates incubated for 24 h at 28°C and wounding the leaves (four wounds per leaf) with a sterile needle. The same number of noninoculated plants was used as control. All plants were covered with plastic bags and maintained in a greenhouse at 25 ± 1°C with 95 to 100% relative humidity until symptoms occurred 3 to 4 days later. All three bacterial strains tested were virulent and caused symptoms identical to those observed in the nursery. No symptoms were observed in control plants. Koch's postulates were fulfilled by the reisolation of the three strains from inoculated plants. The strains were gram-negative, aerobic rods, grew aerobically, were white and nonmucoid on yeast dextrose calcium carbonate agar, nonfluorescent on KB, produced diffusible nonfluorescent pigment on KB, and were oxidase and urease negative. All strains utilized glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate, caprate, malate, citrate, and phenyl acetate and none of the strains produced indole or acidified glucose. Using the API 20NE test strips (bioMérieux, Marcy l'Etoile, France) incubated at 28°C for 24 to 48 h, all strains were initially identified as Burkholderia cepacia. On the basis of the nutrient profiles revealed by the BIOLOG system (Microlog System Release 4.2, Hayward, CA), the strains were identified as B. gladioli (Severini 1913) Yabuuchi et al. 1993. The index of probability was 100% and the index of similarity was 0.75%. For molecular identification of strains, 16S rDNA was amplified by using species-specific primers Eub-16-1 and Gl-16-2457, obtaining a polymerase chain reaction (PCR) product of 463 bp (1). PCR analysis indicated that the strains belong to B. gladioli. Other bacteria have been previously reported in Italy as pathogens of Strelitzia spp. (2,3). To our knowledge, this is the first outbreak of leaf spot and blight caused by B. gladioli on S. reginae. References: (1) A. Bauernfeind et al. J. Clin. Microbiol. 37:1335, 1999. (2) P. Bella et al. J. Plant Pathol. 82:159, 2000; (3) G. Polizzi et al. Plant Dis. 89:1010, 2005.

First Report of Bacterial Blight of Strelitzia augusta Caused by Pseudomonas syringae pv. lachrymans.

White bird of paradise tree (Strelitzia augusta Thunb.), originally from South Africa, is a tender perennial cultivated as an ornamental plant and is used in gardens in Italy. During February of 2004, a new blight disease was noticed on potted S. augusta at different ages (6 months to 4 years) in several commercial nurseries of eastern Sicily. Field inspections revealed disease incidences as high as 40%. Initial symptoms were small, water-soaked leaf spots that expanded throughout the veins in dark brown streaks. Stem cross sections revealed browning of the vascular tissues, which might involve the entire stem. In some cases, the necrosis extended to the apical bud, causing death of the plant. Thirty explants from infected tissues were washed in sterile water and plated on plate count agar (PCA) from which two types of bacterial colonies were consistently isolated. Pathogenicity tests were performed on S. augusta plants. Twenty-four plants were inoculated (12 per bacterial isolate) using two different procedures: spray with a bacterial suspension (106 CFU/ml) and wounding with an infected needle on the midribs. The same number of noninoculated plants was used as controls. All plants were maintained at 24 to 26°C with 95 to 100% relative humidity until symptoms occurred 4 days later. Just one of the two tested bacterial types was pathogenic. The symptoms were similar to those previously observed in the field. No symptoms were observed in the plants spray inoculated with the bacterial suspension, proving that the bacteria were unable to infect in the absence of a wound. The controls showed no symptoms. Koch's postulates were fulfilled by the reisolation of the infective strain which was sent to the CBS (Centraalbu-reau voor Schimmelcultures) and identified as Pseudomonas syringae pv. Lachrymans/pisi using the Biolog MicroLog3 4.01C program (Biolog Inc., Hayward, CA). Further pathogenicity tests have been carried out on zucchini and pea pods to characterize the pathovar using 48SR1 of P. syringae pv. syringae and B4 of P. syringae pv. pisi as reference strains. Necrotic, sunken, water-soaked spots surrounded by a chlorotic halo, reported in the literature as typical symptoms of P. syringae pv. lachry-mans (Smith & Bryan) Young, Dye & Wilkie (1), were observed on zucchini when inoculated with our strain. Our P. syringae strain did not cause the typical symptoms of P. syringae pv. pisi on inoculated pea pods. The results of the pathogenicity tests and the inability of the P. syringae strain isolated from S. augusta to utilize homoserine, used to discriminate pv. pisi from other pathovars of P. syringae, allowed us to identify the strain as P. syringae pv. lachrymans. Low temperature damage and late transplant may have promoted the spread of the disease in the nurseries. Under these conditions, the economic importance of this disease for the crop can be considered high. To our knowledge, this is first report of P. syringae pv. lachrymans on S. augusta. Reference: (1) K. Pohronezny et al. Plant Dis. Rep. 62:306, 1978.

Cloning cassettes containing the reporter gene xylE.

Two pUC-derived vectors containing the promoterless xylE gene (encoding catechol 2,3-dioxygenase) of Pseudomonas putida mt-2 were constructed. The t(o) transcriptional terminator of phage lambda was placed downstream from the stop codon of xylE. The new vectors, pXT1 and pXT2, contain xylE and the t(o) terminator within a cloning cassette which can be excised with several endonucleases. When inserted into a transcribed sequence, this xylE cassette reports promoter activity and interrupts downstream transcription of the target sequence.