Draft Genome Sequence of Pseudomonas mediterranea Strain CFBP 5447T, a Producer of Filmable Medium-Chain-Length Polyhydroxyalkanoates.

Pseudomonas mediterranea strain CFBP 5447(T) is a phytopathogenic bacterium isolated from tomato plants affected by pith necrosis disease. Moreover, its ability to produce medium-chain-length polyhydroxyalkanoates (mcl-PHAs) in culture from different carbon sources and valuable microbial products, such as cyclic lipopeptides, has been well documented. Here, we report the first draft genome sequence of this species.

Draft genome sequence of Pseudomonas corrugata, a phytopathogenic bacterium with potential industrial applications.

Pseudomonas corrugata was first described as the causal agent of a tomato disease called 'pith necrosis' yet it is considered as a biological resource in various fields such as biocontrol of plant diseases and production of industrially promising microbial biopolymers (mcl-PHA). Here we report the first draft genome sequence of this species.

A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily.

Aspergillus strains belonging to section Nigri isolated during a two year survey in eight Sicilian vineyards located on the slopes of Mount Etna (Sicily, Italy) were analysed analyzed in order to characterize species responsible for ochratoxin A (OTA) contamination of grapes. The polyphasic approach permitted analysis of biodiversity of Aspergillus isolates in relation to their morphology, ochratoxigenicity and genetic variability. We assessed OTA production by A. carbonarius, A. niger, A. tubingensis and A. japonicus using an enzyme-linked immunosorbent assay. A. carbonarius isolates were the strongest OTA producers. A subset of 66 representative strains was selected for further DNA-based characterization. PCR assays using species-specific primers discriminated between A. niger, A. carbonarius and A. japonicus on the basis of the target sequences for each species. The PCR-based methods matched morphological characterization in identifying all the black aspergilli (BA) isolates tested, whereas RFLP analysis with RsaI of isolates positive to PCRs with A. niger specific primers identified three A. tubingensis isolates. The identification of thirteen isolates was further confirmed by ITS analysis. By this method, each of the isolates was identified and assigned to an Aspergillus species. The fAFLP analysis of 40 isolates highlighted the power of this technique to discriminate different species and single strains, to verify the presence of mixed populations in the same vineyard, through homogeneous species clusters. No correlation was observed between the clusters and OTA production level or origin.

First Report of Leaf Spot and Blight Caused by Ralstonia pickettii on Bird of Paradise Tree in Italy.

Bird of Paradise tree (Strelitzia alba (L. f.) Skeels) is an ornamental perennial tropical plant grown in southern Italy. In the summer of 2006 and 2007, a widespread, severe leaf disease was observed on seedlings and 1- to 2-year-old plants in two glasshouses located in eastern Sicily. Disease incidence ranged from 10 to 25%. Symptoms on the leaves consisted of dark brown-to-black stripes of varying length and found between the lateral veins. Lesions sometimes coalesced into a large area of necrotic tissue. Symptomatic tissues were ground in a drop of sterile distilled water (SDW) with a scalpel. Suspensions were streaked on King's medium B (KB), nutrient agar, and yeast extract nutrient agar (2). Isolated strains were gram negative and oxidase positive, non-levan, negative in tobacco hypersensitivity test, white and nonmucoid on yeast dextrose calcium carbonate agar, did not produce fluorescent pigments on KB, and utilized glucose, mannitol, trehalose, arabinose, mannose, and N-acetylglucosamine. Bacterial strains were identified as Ralstonia pickettii by using the Biolog Identification System (MicroLogTM System Release 4.2; Biolog, Inc., Hayward, CA) with a similarity index ranging from 0.52 to 0.67. For an additional confirmation of identity, the small subunit rRNA gene (SSUrDNA) was amplified with primers 530F and Uni 1492R (1). The resulting nucleotide sequence was compared with sequences deposited in GenBank and showed the highest identity (99%) to sequences of R. pickettii strains. Pathogenicity tests were performed on 20 cm tall potted plants. Four S. alba plants were inoculated by infiltrating leaf veins with bacterial suspensions for each of the four isolates (107 CFU ml-1 in SDW) with a 25-gauge needle and syringe. Plants were placed in polyethylene bags 1 day before inoculation and maintained there for 3 days after inoculation. Four control plants were inoculated with SDW. Water-soaked areas in the lateral veins of leaves were observed in all inoculated plants 4 days after inoculation. Within 10 days, dark brown-to-black stripes that coalesced into dark necrotic areas were observed. All isolates induced similar symptoms. Control plants did not show any symptoms. The pathogen was reisolated from symptomatic tissue and identified as R. pickettii by Biolog. A similar disease on S. reginae caused by a Pseudomonas sp. was previously reported from Florida (3). To our knowledge, this is the first record in the world of leaf spot and blight caused by R. pickettii. References: (1) D. J. Lane. 16S/23S rRNA sequencing. Page 115 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley and Sons, NY, 1991. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001. (3) C. Wehlburg. Plant Dis. Rep. 55:447, 1971.

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 Thielaviopsis Trunk Rot of Date Palm in Italy.

As an alternative to Canary Island date palm (Phoenix canariensis Chabaud), date palm (P. dactylifera L.) is being planted in Sicilian landscapes. In January 2006, severe symptoms of trunk rot were noticed on 10 7- to 8-m high mature date palms in the central square of Aci Bonaccorsi (Catania) in eastern Sicily. In June 2004, these palms were transplanted directly from Egypt. In 4 of 13 planted date palms, the canopy suddenly fell off the trunk. The canopy of all palms appeared normal and healthy with no stem bleeding observed before trunk collapse. Cross sections of affected date palms revealed a brown rot of nonlignified or lightly lignified tissues along with a strong odor of fermented fruit (amyl acetate) associated with the presence of Geotrichum candidum Link ex Pers. Brown rot was not detected in three of the date palms examined. Symptoms were not detected on lignified and external fibers. Internal tissue adjacent to the rotted areas was placed on carrot agar amended with 500 µl of streptomycin sulfate and acidified (lactic acid; pH = 3.6) potato dextrose agar. Large sections (10 to 18 cm) of affected palm tissues were maintained in a moist chamber for 8 days. Microscopic examinations of five single-conidia isolates on media and sporulation from affected tissues yielded Thielaviopsis paradoxa De Seyn. (Höhn) (1). Endoconidia measuring 3 to 5.5 × 7 to 11 µm were cylindrical to somewhat oval when mature, hyaline to mid brown and smooth walled. Endoconiodophores were usually straight, colorless to pale brown, as much as 150 µm long, with a terminal spore-bearing cell through which spores are born. Chlamydospores were smooth, thick walled, brown, in chains, and were 7.5 to 13 µm width × 10 to 18 µm length (values referred to 50 examined chlamydospores). T. paradoxa is a pathogen that can infect any part of a palm and its pathogenicity to date palm is well documented (3). T. paradoxa is endemic in northern Italy (Ligurian Riviera) on P. canariensis where it causes a disease known as bud rot (2). To our knowledge, this is the first record of trunk rot caused by T. paradoxa on date palm in Italy and is the first report of the fungus in Sicily. It is recommended to avoid the foreign trade of mature date palms from known infected areas because of the symptomless infections by this pathogen. References: (1) A. R. Chase and T. K. Broschat, eds. Diseases and Disorders of Ornamental Palms. The American Phytopathological Society, St. Paul, MN, 1991. (2) A. Garibaldi et al., eds. Malattie Delle Piante Ornamentali. Calderini Edagricole, Bologna, Italy, 2000. (3) P. Suleman et al. Plant Dis. 85:80, 2001.

Polygala myrtifolia as a New Natural Host of Cucumber mosaic virus.

Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) are among the most important viral pathogens of ornamental plants (1). Polygala myrtifolia L. (myrtle-leaf milkwort), originating from South Africa, and a member of the Polygalaceae, was recently introduced in Italy as a cultivated ornamental shrub for its fast and attractive free-flowering growth and drought-resistant characteristics. It can become an invasive plant and is now considered a serious problem in coastal areas of Australia where it was introduced as a garden plant. In Italy, P. myrtifolia is propagated by cuttings in commercial nurseries during the summer. In the winter of 2002, plants of P. myrtifolia growing in pots in an ornamental nursery in Sicily showed virus-like mosaic and malformation of leaves that appeared lanceolate with a lack of flowering. Leaf tissue was analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antisera to CMV, TSWV (Lettuce type), and INSV. Positive ELISA results were obtained only with the CMV polyclonal antisera. Complete remission of symptoms was observed on new flushes after pruning and incubation of infected plants at warm temperatures (30 and 20°C, day and night, respectively). This evidence led to the hypothesis that the disease or virus was disseminated by transportation and propagation of plants without visible symptoms during the hot season. A survey was also performed in two historical gardens of Catania (Sicily) where a group of apparently healthy P. myrtifolia plants, from the previously mentioned ornamental nursery in Sicily, were introduced as a single specimen or to form a hedge. These plants showed the same leaf malformations and mosaic symptoms observed in the nursery. DAS-ELISA confirmed the presence of CMV but not TSWV and INSV. To our knowledge, this is the first report of CMV on P. myrtifolia and it adds a new host to over 1,000 species (85 plant families) infected by this virus. Reference: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997.

First Report of Bacterial Stem Rot Caused by Pectobacterium carotovorum subsp. carotovorum and P. carotovorum subsp. atrosepticum on Grafted Eggplant in Italy.

In December 1999, widespread dieback of eggplant (Solanum melongena L., hybrid Mission Bell), grafted onto the interspecific hybrid Beaufort (Lycopersicon esculentum × Lycopersicon hirsutum) and on tomato hybrid Energy, was observed during four rootstock evaluation trials in greenhouses in Sicily, Italy. Dark brown to black, firm but sometimes fissured lesions, 1 to 20 cm long, were observed just above the grafting point. Water-soaked, soft, dark green lesions that turned brown with age were observed on the upper stem. Extensive discoloration of vascular tissues and, in some cases, breakdown of the pith and stem hollowness occurred. Eggplant seedlings, present in the same experimental trials, did not show any symptoms. From symptomatic tissues, numerous bacterial colonies were obtained on nutrient dextrose agar. Bacteria from purified colonies were gram-negative, oxidase-negative, facultatively anaerobic pectolitic on crystal violet pectate agar, which is nonfluorescent on King's B medium. On the basis of biochemical and physiological tests (1), seven of 10 isolates were identified as Pectobacterium carotovorum subsp. carotovorum (Jones 1901) Hauben et al. 1999, comb. nov. (2), and the remaining three were identified as P. carotovorum subsp. atrosepticum (van Hall 1902) Hauben et al. 1999, comb. nov. (2). Four days after prick inoculation of the stems of eggplant and tomato plants, all isolates caused extensive collapse of stems and internal brown discoloration and hollowness, respectively. In a second pathogenicity test, basil leaves of grafted eggplants and eggplant seedlings were either removed or left intact. These plants were then sprayed with bacterial suspensions (108 CFU/ml) of one isolate of each pathogen. Plants were held at 100% relative humidity or in a low-moisture incubator. After 6 days incubation, stem lesions and vascular discoloration, typical of symptoms observed in greenhouses, were observed on plants held at 100% relative humidity. No symptoms were observed in unwounded plants or plants maintained in low relative humidity. Similar symptoms on grafted eggplant have been attributed to physiological disorders (3). To our knowledge, only P. carotovorum subsp. carotovorum has been reported on eggplant as causing fruit rot. References: (1) N. W. Schaad, ed. 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 2nd ed. American Phytopathological Society, St. Paul, MN. (2) L. Hauben et al. Int. J. Syst. Bacteriol. 41:582, 1999. (3) G. Ginoux and H. Laterrot. Rev. Horticole (Paris) 321:49, 1991.

First Report of Occurrence of Verticillium Wilt on Some Ornamental Trees in Sicily.

In July 1998, a widespread lethal collapse of butterfly tree (Bauhinia purpurea L.), puka tree (Meryta sinclairii Hook.), Indian laurel (Ficus microcarpa L.), golden rain tree (Koelreuteria paniculata Laxm.), and Brazilian pepper tree (Schinus terebinthifolius Raddi.) has been observed in an ornamental growing area near Catania in eastern Sicily. The diseased plants showed yellowing of the leaves and twig dieback. In cross sections of infected trunks, roots, twigs or shoots, characteristic discoloration of xylem vessels was observed, resembling a vascular wilt disease. The disease was chronic with a lingering decline progressing for years. The isolate that was consistently recovered from diseased tissues on PDA was identified as Verticillium dahliae Kleb., based on verticillate conidiophores with phialides and conidia and melanized microsclerotia. Pathogenicity tests were performed on 4- to 6-month-old seedlings of all five ornamental plants by inoculating a suspension of the fungus (1). Symptoms were observed after 5 to 7 months, and V. dahliae was reisolated, confirming Koch's postulates. To our knowledge this is the first report of V. dahliae on Indian laurel, golden rain tree, and Brazilian pepper tree in Italy, and the first report of the disease on butterfly tree and puka tree. Reference: H. A. Melouk and C. E. Horner. Phytopathology 65:767, 1975.

First Report of Leaf Spot Caused by Cylindrocladium pauciramosum on Acacia retinodes, Arbutus unedo, Feijoa sellowiana, and Dodonaea viscosa in Southern Italy.

In October 1997, severe leaf spotting was observed on several ornamental plants growing in different nurseries in southern Italy. These symptoms were detected for the first time on strawberry tree (Arbutus unedo L.) and water wattle (Acacia retinodes Schltdl.). In the latter species, diseased plants showed also stem canker and shoot blight symptoms. Cylindrocladium pauciramosum C.L. Schoch & Crous (teleomorph Calonectria pauciramosa) were found associated with these symptoms (2). More recently, leaf spots were found also on feijoa (Feijoa sellowiana Berg.) and on Florida hopbush (Dodonaea viscosa (L.) Jacq.) in two other nurseries located in Sicily. Infected tissues were surface-sterilized and plated on potato dextrose agar. Plates were incubated at room temperature under fluorescent cool white lights on a 12-h light/dark regime. A Cylindrocladium species was consistently associated with diseased tissues. The isolates of Cylindrocladium collected from feijoa and Florida hopbush were identified on carnation leaf agar as C. pauciramosum on the basis of their obpyriform to broadly ellipsoidal terminal vesicles, conidiophore branching pattern, and conidium morphology, as well as mating type studies with tester strains of C. pauciramosum (1,3). Koch's postulates were fulfilled by inoculating 6-month-old seedlings of the four ornamental plants with a spore suspension of the fungus (104 conidia per ml). Following inoculation, all plants were maintained in a glasshouse where the temperature was 15 to 25° C and relative humidity was 80 to 90%. After 6 to 8 days, symptoms resembling those seen in the nurseries were apparent. The Cylindrocladium species was reisolated from lesions on inoculated plants, thus confirming it to be the causal organism of these diseases. This is apparently the first report of C. pauciramosum leaf spot on these hosts. References: (1) P. W. Crous and M. J. Wingfield. Mycotaxon 51: 341, 1994. (2) G. Polizzi and P.W. Crous. Eur. J. Plant Pathol. 105:407, 1999. (3) C. L. Schoch et al. Mycologia 91:286, 1999.

Extreme Susceptibility of Primosole Mandarin to Alternaria Fruit Rot in Italy.

Primosole mandarin is a promising mandarin-like hybrid of Satsuma Miho and Carvalhais mandarin that ripens very early, at the beginning of October, in southern Italy (2). During August and September 1999 and 2000 in Sicily, widespread fruit rot, affecting from 80 to 95% of the total production, was observed in a 4-year-old Primosole mandarin orchard. The fruits developed color prematurely and light brown-to-black discoloration of the rind at the stylar end. Internal symptoms consisted of a black rot of the fruit core. Sometimes the exterior of the fruits appeared healthy. No premature fruit drop was observed, and infected fruits became mummified and remained attached on the trees. Alternaria citri Ellis & N. Pierce in N. Pierce was consistently isolated from infected tissues, and the identification of the fungus was based on morphological characteristics of the conidia (1). Pathogenicity tests of single-spore isolates were carried out on surface-sterilized Primosole fruits and were repeated twice. Either a conidial suspension (2 × 104 conidia per ml) was injected into the core of fruits, or fruits were pricked at the stylar end near or through growth cracks in poorly formed navels, and the conidial suspension was placed on the wound. Thirty fruits were used per treatment, and thirty noninoculated fruits were used for controls. Following inoculation, the fruits were placed in plastic bags and kept at 30°C for 15 days. No external symptoms were observed on any of the fruits, but when cut in half, decay of the core was observed in all inoculated fruits. A. citri was reisolated from inoculated fruits, fulfilling Koch's postulates. No symptoms were observed on fruits used as controls. We believe that infection is facilitated by growth cracks at the stylar end and the sensitivity to sunburn of Primosole mandarin. To our knowledge, this is the first report of the extreme susceptibility of Primosole mandarin to Alternaria fruit rot. References: (1) G. E. Brown and J. W. Eckert. Postharvest fungal diseases. Page 37 in: Compendium of Citrus Diseases, 2nd ed. L. W. Timmer, S. M. Garnsey, and J. H. Graham, eds. The American Phytopathological Society, St. Paul, MN, 2000. (2) E. Tribulato and G. La Rosa. Italus Hortus 1:21, 1993.