First Report of Web Blight on Rose Campion (Lychnis coronaria) Caused by Rhizoctonia solani AG 1-IB in Italy.

Lychnis coronaria (syn. Silene coronaria), rose campion, is a perennial in the Caryophyllaceae used in gardens. In the summer of 2014, a web blight was observed in a private garden located near Biella (northern Italy), approximately 45°39'N 8°00'E, on 40% of 100 5-month-old plants grown in sandy soil. In the days preceding the outbreak of the disease, daytime temperatures ranged from 18 to 24°C and relative humidity from 45 to 83%. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Under conditions of high relative humidity, a scant amount of whitish mycelium developed on leaves of about 50% of diseased plants. Eventually, infected plants died about 10 days after symptoms appeared. Symptomatic tissues of stems and leaves were disinfected for 10 s in 1% NaOCl, rinsed in sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates were paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 4, AG 7, and AG 11) (2) and examined microscopically. The Lychnis isolates anastomosed only with the AG 1 tester strain, with low fusion frequency. The anastomosis point was obvious: the hyphal diameter at the point of anastomosis was reduced and death of adjacent cells was observed, indicating an anastomosis reaction (1). Mycelium maintained on PDA at 23 ± 1°C was coarse and reddish brown. After 5 days of growth, mycelium started differentiating numerous sclerotia, often aggregated. Mature sclerotia were dark, spheroidal, with diameters ranging from 0.2 to 1.6 (average 0.6) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 609-bp amplicon (GenBank Accession No. KM596899) showed a 98% homology with the sequence of the R. solani isolate FJ746937 obtained from Zoysiagrass. On the basis of molecular and cultural characteristics and anastomosis tests, the isolates from L. coronaria were identified as R. solani AG 1-IB (4). For pathogenicity tests performed in August, mycelial disks (8 mm diam.) from 10-day-old PDA cultures of an isolate of the fungus were placed on four healthy 6-month-old L. coronaria plants (four stem and six leaf disks per plant). Four plants inoculated with disks of PDA served as controls. Plants were covered with plastic bags for 4 days and maintained in a garden located in the same area in which the disease appeared, at field temperatures ranging from 15 to 28°C. The first symptoms developed 4 days after inoculation, and 15 days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated from the stem of symptomatic plants, whereas no colonies developed from controls, which all remained healthy. This is the first report of blight of L. coronaria caused by R. solani in Italy or anywhere else in the world. The impact of this disease may become a significant problem for L. coronaria, a very common species in Italian gardens. References: (1) D. E. Carling. Page 37 in: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, The Netherlands, 1996. (2) A. Ogoshi. Ann. Rev. Phytopathol. 25:125, 1987. (3) B. Sneh et al. Identification of Rhizoctonia species. APS Press, St. Paul, MN, 1991. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

Phytophthora cryptoea on Common Sage (Salvia officinalis L.) in Italy.

During the spring of 2013, many plants of common sage (Salvia officinalis L.), grown as potted plants in a commercial farm at Albenga (northern Italy) showed extensive symptoms of foliar wilt and root rot. The first symptoms developed with temperatures ranging between 8 and 26.5°C, average 17°C, and consisted of leaf chlorosis, wilting, and collapse. Severe root and crown rot were also observed, leading to sudden collapse of approximately 60% of the 6,000 plants within 60 days from transplant. Symptomatic tissues from the root and collar of infected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in water, and placed on a selective medium for oomycetes (3). A Phytophthora-like organism (1) was consistently isolated and was transferred to carrot agar. Mycelial disks of the isolate DB13GIU02 were floated in petri plates containing soil extract (1), under continuous fluorescent light at room temperature. Hyphal swelling was abundant in such aqueous medium, measuring 6.4 to 20.1 (13.1 average) µm. Sporangia were obpyriform, persistent, and nonpapillate, measuring 25.3 to 55.1 × 17.9 to 37.1 (average 42.8 to 27.9) µm. Oospores and chlamydospores were absent. The same isolate was tested with two isolates of P. cryptogea from Quercus ilex (PH050, mating type A1) and from Pistacia lentiscus (PH017, mating type A2) on carrot agar, at 23 ± 1°C in the dark. Only the paring of DB13GIU02 with PH017 was successful and produced oogonia with diameter of 28.3 to 34.6 (average 31.7) µm, oospores with diameter of 28.0 to 32.2 (average 29.2) µm, and anphigynous antheridia of 10.5 to 15.1 × 11.6 to 15.1 (average 13.5 × 13.3) µm. DNA of the three isolates was extracted by using the Nucleospin Plant kit (Macherey Nagel). PCR of DNA amplified with primers Cryp 1 and Cryp 2 (4) from all P. cryptogea isolates produced a specific amplicon. The internal transcribed spacer (ITS) region of rDNA of the isolate DB13GIU02 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis of the 845-bp segment (GenBank Accession No. KM458193) showed a 99% homology with the sequence of P. cryptogea GU111631. Pathogenicity tests were performed on healthy common sage 60-day-old plants by using one strain of P. cryptogea grown on a mixture of 2:1 wheat/hemp kernels. Infested kernels (10 g/liter of substrate) were mixed into a steam-disinfested substrate based on sphagnum peat/pomix/pine bark/clay (50:20:20:10 v/v). Control plants were treated with uninoculated wheat/hemp kernels mixed into the steam-disinfested soil. The trial was repeated once. Fifteen plants per treatment were used. All plants were kept in a growth chamber at 20 ± 1°C. Inoculated plants became chlorotic 7 days after inoculation, and root and crown rot developed 15 days after inoculation. P. cryptogea was consistently reisolated from inoculated plants. No colonies were isolated on the selective medium from control plants that remained symptomless. P. cryptogea has been reported on S. officinalis in the United States (2), while in Italy the same pathogen has been observed on S. leucantha. This is the first report of P. cryptogea on S. officinalis in Italy. The economic importance of the disease can increase due to the expanding use of this plant both as an aromatic for culinary purposes and for landscaping. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN, 1996. (2) S. T. Koike et al. Plant Dis. 81:959, 1997. (3) H. Masago et al. Phytopathology 67:25, 1977. (4) D. Minerdi et al. Eur. J. Plant Pathol. 122:227, 2008.

Root Rot of Spinach in Southern Italy Caused by Pythium aphanidermatum.

During the spring of 2014, spinach (Spinacia oleracea L.) plants of the cv. Crocodile (Rijk Zwaan, De Lier, The Netherlands), grown in a clay loam soil under commercial greenhouse conditions near Salerno (southern Italy), showed stunting, extensive chlorosis, and root rot. Plants were irrigated by overhead sprinklers using well water. Symptoms first developed 20 days after sowing, at air temperatures of 23 to 30°C, and 35% of plants (approximately 15 million plants in 10 ha) were affected. Roots were severely affected, appeared water-soaked and brown, and were characterized by a soft rot. Eventually, affected plants wilted and collapsed. Fifty fragments, each 1 mm2, were excised from symptomatic roots of 10 plants, dipped in a solution containing 1% sodium hypochlorite, rinsed in sterilized water, dried on sterilized paper towel, and plated on both potato dextrose agar (PDA) and the medium BNPRA, which is semi-selective for oomycetes (3). After 5 days of incubation under constant fluorescent light at 22 ± 1°C, 80% of the root sections developed oomycete colonies. One representative isolate, grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup; 15 g agar; 0.5 g CaCO3; 1 liter distilled water) and observed with a light microscope, showed aseptate hyphae 3.3 to 6.5 (mean 5.5) µm wide. Oogonia were globose, smooth, and 22.2 to 31.0 (average 26.3) µm in diameter. Antheridia were barrel-shaped, while oospores were globose and 17.3 to 22.6 (mean 20.9) µm in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of this isolate was amplified using ITS1/ITS4 primers and sequenced. BLAST analysis (1) of the 647-bp segment showed 100% homology with ITS sequences of Pythium aphanidermatum in GenBank (Accession Nos. KJ755088.1, KJ162355.1, KF840479, and KF561235.1). The nucleotide sequence for the Italian spinach isolate was assigned No. KM111256. Pathogenicity tests were performed twice on 20-day-old spinach plants of the cv. Merlo (L'Ortolano, Cesena, Italy), grown in 2-liter pots in a steam-disinfested organic peat substrate (black peat, pH 6.5 to 6.8, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) moistened to field capacity, and infested with wheat and hemp kernels colonized with isolate Py 1-14 of P. aphanidermatum at 1 g/liter. Five plants were transplanted into each of four pots filled with infested peat, while the same number of plants was grown in non-infested substrate as a control. Plants were kept in two growth chambers, each with 12 h of light per day at 20 or 30°C, and were irrigated daily to maintain the potting medium at field capacity. Symptoms first developed 5 days after the spinach was transplanted into infested potting medium in the growth chamber maintained at 30°C. After 10 days, all plants in this growth chamber were dead, while only 5% of the plants growing in infested potting medium in the 20°C growth chamber were affected. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in infested medium. No fungi were re-isolated from the asymptomatic control plants grown in non-infested substrate. To our knowledge, this is the first report of P. aphanidermatum causing root rot on S. oleracea in Italy. The same pathogen has been reported to cause root rot of spinach in other countries, including the United States (2). The disease is, at present, limited to the affected greenhouses observed in this study. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. L. Bates and M. E. Stanghellini. Plant Dis. 68:989, 1984. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, FL., 2002.

First Report of Dry and Soft Rot of Cereus marginatus var. cristata Caused by Fusarium oxysporum in Italy.

During the winter of 2013, 50% of 20,000 plants of Cereus marginatus var. cristata, Cactaceae family, grown in a commercial farm located in Liguria (northern Italy) showed symptoms of a dry or soft rot. In the case of dry rot, affected plants showed on the stem superficial necrosis and dry rot, irregularly shaped, 1 to 10 mm, while epidermal and cortical tissues were wounded. Affected plants survived but they lost ornamental value. In the case of soft rot, associated with conditions of higher relative humidity, rots on the stem extended as far as 4 cm in width. The internal part of bark, cambium, and xylem tissues as far as about 3 cm in depth was rotted. Vascular tissues were not discolored. Plants died in about 20 days. A Fusarium sp. was consistently isolated from symptomatic tissue on Komada selective medium (2) from plants showing soft rot. The isolates were purified and subcultured on potato dextrose agar (PDA). On PDA, the cultures produced a thick and soft growth of white to light pink mycelium and pale pink pigments in the agar. On Spezieller Nährstoffarmer agar (SNA), cultures produced short monophialides with unicellular, ovoid-elliptical microconidia measuring 3.7 to 8.2 × 1.7 to 3.5 (average 5.4 × 2.5) µm. On carnation leaf-piece agar (CLA), chlamydospores were abundant, terminal or intercalary, single or paired, but frequently also aggregated. On the same medium, at temperatures ranging from 20 to 24°C (14 h daylight, 10 h dark), cultures produced light orange sporodochia with macroconidia. These were 3 to 4 (sometimes 5) septate, nearly straight with a foot-shaped basal cell and a short apical cell, and measured 28.5 to 41.4 × 3.3 to 4.9 (average 35.0 × 4.0) µm. Such characteristics are typical of Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen (3). Amplification of the internal transcribed spacer (ITS) of the rDNA using primers ITS1/ITS4 yielded a 504-bp amplicon (GenBank Accession No. KJ909935). Sequencing and BLASTn analysis of this amplicon showed a 100% homology with the sequence of F. oxysporum KC304802. To confirm pathogenicity, two Fusarium isolates were tested. For each isolate, three 2-year-old healthy plants of C. marginatus were inoculated by introducing into lesions (4 lesions/plant) artificially produced on the stem sterile needles contaminated with the pathogen (4). Inoculum was obtained from pure cultures grown on PDA. Control plants were punctured with sterile needles without inoculum. All the plants were placed in a greenhouse, at temperatures ranging between 16 and 24°C. For both tested strains, the first necrosis of stem tissues developed around the needles 7 days after the artificial inoculation, while non-inoculated plants remained healthy. Then, necrosis extended causing soft rot on plants maintained at relative humidity ranging from 55 to 65%. F. oxysporum identified by morphological characteristics was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. F. oxysporum has been reported on Cereus sp. in the United States and on C. peruvianus monstruosus in Italy (1). Currently, this disease is present in a few commercial nurseries in Liguria, although it could spread further and cause important economic losses. References: (1) A. Garibaldi et al. Plant Dis. 95:877, 2011. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (4) V. Talgø and A. Stensvand. OEPP/EPPO Bulletin 43:276, 2013.

First Report of Web Blight on Rebutia perplexa Caused by Rhizoctonia solani AG 2-2-IIIB in Italy.

Rebutia perplexa, Cactaceae family, is a clumping fine thorny cactus, producing several flushes of pink flowers. In the spring of 2013, a blight was observed in a farm located near Imperia (northern Italy) on 2% of 2,000 3-year-old plants, grown in plastic pots. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Flowers also rotted and wilted. In the presence of high relative humidity, a rare, whitish mycelium developed on the surface of the substrate. Eventually, infected plants died. Symptomatic tissues of the stem were taken from 10 plants and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates obtained from affected plants were successfully paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 2-2-IV, AG 4, AG 7, AG 11) (2) and examined microscopically. Three replicated pairings were made for each tester strain. The Rebutia isolates anastomosed only with AG 2-2-IIIB tester strain with high hyphal fusion frequency. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed, indicating anastomosis reactions (1). Tests were performed twice. Mycelium of 15-day-old isolates maintained at 27 to 30°C, appeared whitish or pale buff in color, coarse, with a concentric zonation, scarce aerial mycelium, and without sclerotia. The optimum temperature for mycelium growth was 30°C (daily growth rate: 24.6 mm) and isolates grew also at 35°C. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 523-bp amplicon (GenBank Accession No. KF719318) showed a 99% homology with the sequence of the R. solani AG 2-2-IIIB isolate GU811672. The nucleotide sequence has been assigned the GenBank Accession No. KF719318. Therefore, on the basis of molecular characteristics, anastomosis tests, temperature growth, and cultural characteristics, the isolates from R. perplexa were identified as R. solani AG 2-2-IIIB. For pathogenicity tests, 3 g of colonized wheat kernel from 10-day-old cultures of a representative isolate of the fungus was added per 1 l of substrate in 12 potted healthy plants of R. perplexa. The inoculum was prepared by inoculating wheat kernels with the mycelium of 10-day-old cultures of the fungus and incubating at 25 ± 1°C (12 h fluorescent light, 12 h dark). Twelve plants inoculated with non-infested wheat kernels served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C. The first symptoms, similar to those observed in the farm, developed 5 days after inoculation. Fifteen days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated only from the stems of symptomatic plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. This is, to our knowledge, the first report of blight of R. perplexa caused by R. solani in Italy as well as worldwide. References: (1) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease control. Kluwer Academic Publishers, The Netherlands, pp. 37-47, 1996. (2) A. Ogoshi. Ann. Rev. Phytopathol. 25:125, 1987. (3) B. Sneh et al. Identification of Rhizoctonia species. APS Press, St Paul, MN, 1991.

First Report of Root Rot Caused by Pythium aphanidermatum on Bell Pepper (Capsicum annuum L.) in Italy.

During July 2012, symptoms of root rot were observed on bell pepper (Capsicum annuum) grown in 2,000 m2 of commercial greenhouses near Cuneo in northern Italy. Symptoms first developed 30 to 40 days after transplanting, when greenhouse temperatures ranged from 25 to 30°C, and 10% of the plants were affected. Affected plants were stunted with leaf chlorosis, reduced growth, and sudden wilting. Roots were severely affected with a brown discoloration, water-soaking, and soft rot. Eventually, affected plants collapsed. Tissue fragments of 1 mm2 were excised from symptomatic roots, dipped in a 1% sodium hypochlorite solution, and placed on potato dextrose agar (PDA) and an agar medium selective for oomycetes (3). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. An isolate grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup, 15 g agar, 0.5 g CaCO3, and 1 liter distilled water) showed aseptate hyphae that were 3.5 to 6.3 µm (avg. 5.2 µm) wide. Oogonia were globose, smooth, and 24.3 to 29.0 (avg. 25.1) µm in diameter. Antheridia were barrel-shaped, while oospores were globose, and 17.3 to 23.5 µm (avg. 21.2 µm) in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The ITS region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 781-bp segment (GenBank Accession KF840479) showed 100% homology with the ITS sequence of an isolate of Pythium aphanidermatum in GenBank (AY598622.2). Pathogenicity tests were performed twice on 30-day-old plants of C. annuum cv. Cuneo grown in 2-L pots (4 plants/pot), containing a steam-disinfested, organic peat substrate (70% black peat and 30% white peat, pH 5.5 to 6.0, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) that was infested with wheat and hemp kernels colonized by the isolate of P. aphanidermatum, at a rate of 1 g colonized kernels/liter potting medium. The inoculum was prepared by autoclaving at 121°C for 30 min a mixture of wheat-hemp kernels (2:1 v/v) in a 1-liter flask, to which the bell pepper isolate of P. aphanidermatum was added in the form of colonized agar medium selective for oomycetes plugs. Before use, the inoculated flask was incubated for 10 days at 22°C in the dark. Four plants/pot were transplanted into each of four pots filled with the infested medium/growth chamber, while the same number of plants were grown in non-infested substrate in pots in each growth chamber. Plants were kept in two growth chambers, one set at 20°C and the other at 28°C. Symptoms first developed 7 days after inoculation. After 30 days, 50% of inoculated plants showed brown roots and died in the growth chamber set at 28°C, while only 10% of the plants were symptomatic at 20°C. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in the infested soil by using the same protocol as the original isolations, while no fungal colonies were obtained from asymptomatic roots of the non-inoculated control plants. To our knowledge, this is the first report of the presence of P. aphanidermatum on C. annuum in Italy. The same disease was reported in the United States (2). The importance of the disease, although limited in distribution at present to the greenhouses surveyed in northern Italy, could increase in areas where sweet pepper is grown intensively. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. O. Chellemi et al. Plant Dis. 84:1271, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Boca Raton, FL, 2002.

First Report of Damping-off Caused by Pythium aphanidermatum on Leaf Beet (Beta vulgaris subsp. vulgaris) in Italy.

During July 2010, symptoms of crown and root rot were observed on leaf beet (Beta vulgaris L. subsp. vulgaris) grown in a commercial field near Torino (northern Italy). The first symptoms developed 25 days after sowing with temperatures ranging from 25 to 30°C, and 20% of plants were affected. Affected plants were stunted and leaves showed chlorosis and suddenly wilted. The collar and young stems were affected first and appeared brown, water-soaked, and were characterized by a soft rot. Eventually, all affected plants collapsed. Thin aerial mycelia were visible on the surface of the infected plants if maintained at a high relative humidity. Tissue fragments of 1 mm2 were excised from the margins of the lesions, dipped in a solution containing 1% sodium hypochlorite, and plated on potato dextrose agar (PDA) and on a medium selective for oomycetes (2). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. Five isolates, grown on V8 medium (vegetable mix 300 g; agar 15 g; CaCO3 1.5 g; distilled water 1 liter) and observed under light microscope showed the morphological characters of Pythium aphanidermatum (3). This result was confirmed by PCR and sequence analysis. The internal transcribed spacer (ITS) region of rDNA of a single isolate (Py 7/10) was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 815 bp segment showed a 99% homology with the sequence of P. aphanidermatum (GenBank Accession JN695786). The nucleotide sequence has been assigned to the GenBank Accession JX462954. Pathogenicity tests were performed twice on B. vulgaris subsp. vulgaris grown in 2-liter pots, containing a steam disinfested organic peat substrate (70% black peat, 30% white peat, pH 5.5 to 6, N 110 to 190 mg L-1, P2O5 140 to 230 mg L-1, K2O 170 to 280 mg L-1), infested with wheat and hemp kernels colonized with a strain of P. aphanidermatum at a rate of 1 g L-1. Ten seeds per pot were sown in four pots filled with the infested medium, while the same number of seeds were sown in non-infested substrate. Plants were kept in two growth chambers, at 20 and 27°C. The first symptoms developed 7 days after the artificial inoculation. After 20 days, 70% of plants were infected at 27°C, while 10% were affected at 20°C. Control plants remained healthy at both temperatures. P. aphanidermatum was consistently reisolated from the lesions. To our knowledge, this is the first report of damping off of B. vulgaris subsp. vulgaris caused by P. aphanidermatum in Italy. The importance of the disease, at present limited, could increase in areas where leaf beet is intensively grown. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Florida, 2002.

First Report of a New Leaf Spot Caused by Plectosphaerella cucumerina on Field Grown Endive (Cichorium endivia) in Italy.

During summer 2012, symptoms of a new leaf spot disease were observed in several commercial fields in Treviglio (Bergamo, northern Italy) on plants of curly (Cichorium endivia var. crispum) and Bavarian (C. endivia var. latifolium) endive (Asteraceae). This crop is widely grown in the region for fresh market. The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, up to 10 to 15 mm diameter. Eventually, spots were surrounded by a yellow halo. Particularly, affected tissues rotted quickly under high moisture. Disease severity was greatest at 75 to 90% RH and air temperature between 23 and 30°C, where affected tissues rotted quickly. This disease resulted in severe production losses. On one farm in particular, three different fields totaling 2 ha, 5 to 13% of the plants were affected. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/liter of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 23°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, rarely septate, measuring 5.0 to 9.0 × 1.7 to 3.9 (average 6.0 × 2.9) µm. Conidia were born on phialides, single, clavate, and 2.8 × 1.4 µm. Such characteristics are typical of Plectosphaerella sp. (1,2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 530-bp segment obtained from C. endivia var. crispum isolate PLC28 and of the 527-bp from C. endivia var. latifolium isolate PLC 30, respectively, showed 99% similarity with the sequence of Plectosphaerella cucumerina (anamorph Plectosporium tabacinum), GenBank EU5945566. The nucleotide sequences of isolates PLC 28 and PLC 30 have been assigned the GenBank accession numbers KC293994 and KC293993, respectively. To confirm pathogenicity, tests were conducted on 30-day-old C. endivia plants. C. endivia var. crispum cv Myrna and C. endivia var. latifolium cv. Sardana plants, grown in 2-liter pots (1 plant per pot, 10 plants per treatment) were inoculated by spraying a 106 CFU/ml conidial suspension of the two isolates of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 25 ± 1°C and 90% RH for 5 days. Non-inoculated plants, only sprayed with water, served as controls. All plants inoculated with the two isolates, showed typical leaf spots 7 days after the artificial inoculation, similar to those observed in the field. Later, spots enlarged and leaves rotted. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity tests were conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on endive n Italy, as well as worldwide. Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) M. E. Palm et al. Mycologia 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Southern Blight Caused by Sclerotium rolfsii on Common Bean (Phaseolus vulgaris) in Italy.

Common bean (Phaseolus vulgaris L.) is grown worldwide for consumption of dry or green beans. During late spring of 2012, yellowing and wilting symptoms were observed in a commercial bean field cv. Lingua di fuoco in Cagliari Province (Sardinia, southern Italy) on 30% of plants 4 to 5 months after sowing. The first symptoms developed in May, when temperatures reached 18 to 30°C. Affected plants showed crown rot, necrosis of the cortex, and foliar chlorosis. As disease progressed, plants collapsed. In the presence of abundant moisture, white mycelium developed on the senescent tissue along with light to dark brown sclerotia (3.0 to 4.8 mm in diameter). Symptomatic tissue was disinfested for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 25 mg streptomycin sulfate/liter. The fungus that was isolated consistently from symptomatic plants onto PDA at 23°C grew rapidly in culture with silky-white, sterile mycelium, formed light to dark brown sclerotia (each 1.8 to 3.2 mm in diameter) after 7 days, and readily produced aerial hyphae. These morphological features are typical of Sclerotium rolfsii (2). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified for one isolate using ITS1/ITS4 primers (4), and sequenced (GenBank Accession No. KF002510). BLASTn analysis (1) of the 656-bp segment showed 87% homology with the ITS sequence of an S. rolfsii isolate (JF819727). Pathogenicity of one isolate was confirmed by inoculating healthy P. vulgaris plants cv. Lingua di fuoco grown in 2-liter pots in a steamed potting mix containing 50% Tecno2 (70% white peat and 30% clay) and 50% Tiesse 3 (60% white peat, 20% clay, and 20% perlite) (Turco Silvestro terricci, Bastia d'Albenga, SV, Italy). Inoculum consisting of mycelium and sclerotia of the pathogen produced from 10-day-old cultures on PDA was mixed in the soil at 0.5 g/liter substrate. Four 7-day-old plants per pot, with three replicate pots, were used for inoculation. The same number of control plants grown in the same substrate were inoculated with non-colonized PDA as a negative control treatment. The pathogenicity test was repeated. Plants were kept in a growth chamber at 30°C and 85% RH. Inoculated plants developed symptoms of leaf yellowing within 10 days, followed by crown rot, appearance of white mycelium and sclerotia, and eventual wilting. Control plants remained asymptomatic. Isolations from inoculated plants demonstrated the absence of latent infections by the fungus S. rolfsii, but the fungus was not reisolated from non-inoculated control plants. To our knowledge, this is the first report of S. rolfsii infecting P. vulgaris in Italy. Southern blight has been reported on common bean in sub-tropical and tropical areas of the world (3), where it can cause severe crop losses. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) J. E. M. Mordue. CMI Descriptions of Pathogenic Fungi and Bacteria No. 410, 1974. (3) H. F. Schwartz et al. Page 20 in: Compendium of Bean Diseases. American Phytopathological Society Press, St. Paul, MN, 2005. (4) T. J. White et al. PCR Protocols. Page 315 in: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Leaf Spot of Lettuce (Lactuca sativa L.) Caused by Phoma tropica in Italy.

Lettuce (Lactuca sativa L.) is widely grown in Italy, with the production for the preparation of ready-to-eat salads becoming increasingly important. During the spring of 2011, a previously unknown leaf spot was observed on L. sativa plants, cv Rubia, grown in several plastic tunnels in Lumbardy (northern Italy), 20 to 25 days after sowing. Thirty to forty per cent of leaves of the plants growing in the part of the tunnel with the highest relative humidity were affected. Leaves of infected plants showed extensive, irregular, dark brown, necrotic lesions with a chlorotic halo. Lesions initially ranged from 0.5 to 3 mm, then eventually coalesced, reaching 2 to 3 cm, showing a well-defined, dark brown border. Affected leaves senesced and withered. The crown was not affected by the disease. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then cultured on potato dextrose agar (PDA), amended with 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed, producing a greenish grey mycelium with a white border when incubated under 12 h/day of fluorescent light at 21 to 23°C. In order to favor the production of conidia, the fungus was transferred on malt extract agar (MA) and maintained under 12 h/day of fluorescent light at 22°C. After 15 days, black pycnidia, 175 to 225 µm, developed, with hyaline, elliptical, unicellular conidia, measuring 3.21 to 6.7 × 1.08 to 3.2 (average 5.5 × 1.9) µm. On the basis of these morphological characteristics, the fungal causal agent of the disease could be related to the genus Phoma (2). The internal transcribed spacer (ITS) region of rDNA of the isolate PHT30 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 466-bp segment showed a 99% similarity with the sequence of Phoma tropica (GenBank Accession No. JF923820.1). The nucleotide sequence has been assigned the GenBank Accession No. JQ954396. Pathogenicity tests were performed by spraying healthy 20-day-old lettuce plants, cv Rubia, with a spore suspension (1 × 105 conidia/ml) prepared from 14-day-old colonies of the strain PHT30 grown on MA cultures. Plants inoculated with water alone served as controls. Ten plants per isolate were used. Plants were covered with plastic bags for 5 days after inoculation and maintained in a growth chamber at 20°C and 80% relative humidity. The first foliar lesions, similar to those occurring on the naturally infected plants, developed on leaves 12 days after inoculation. Control plants remained healthy. The pathogen was consistently reisolated from leaf lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of P. tropica on lettuce in Italy as well as worldwide. In the United States, the presence of P. exigua was reported in 2006 (3). The economic importance of the disease at present is limited, probably also because symptoms can be confused with those caused by Botrytis cinerea. However, P. tropica could become a more significant problem because of the importance of the crop. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (3) S. Y. Koike. Plant Dis. 90:1268, 2006.

Phytophthora capsici: A Soilborne Pathogen Dangerous on Grafted Tomato (Solanum lycopersicum × S. hirsutum) in Italy.

During an extensive survey carried out in Piedmont (northern Italy) aimed at identifying the emerging soilborne diseases affecting tomato in commercial fields where alternatives to methyl bromide have been implemented in response to national and international regulations, sudden collapse of tomato plants, cv. Tomahawk, grafted on cv. Beaufort, were repeatedly observed in a commercial plastic tunnel operation. Affected plants suddenly collapsed 60 days after transplant during the month of May 2010. Symptoms included chlorosis, stunting, and severe root and crown rot, leading to sudden collapse of approximately 25% of the plants within 60 days of transplant. Symptomatic tissues from the root and collar of infected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in water, and submerged in selective medium based on corn meal agar. A Phytophthora-like organism (2) with characteristic coenocytic hyphae was consistently isolated and transferred to V8 agar. The sporangia were spherical to ovoid, papillate, and 40 to 77 × 23 to 34 (average 55.1 × 30.3) µm. Oospores were globose and 22.2 to 30.8 µm. The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 750-bp segment showed a 100% homology with the sequence of Phytophthora capsici JN382543.1. The nucleotide sequence has been assigned the GenBank Accession No. JX090306. Pathogenicity tests were performed on healthy 30-day-old tomato plants cv. Beaufort by using one strain of P. capsici grown for 15 days at 22 to 25°C on a mixture of 2:1 wheat/hemp kernels, and then 1 g per L of the inoculum was mixed into a substrate based on peat blonde/peat black (15:85 v/v). Two plants were transplanted into 3-L pots, with five replicates. Ten non-inoculated plants represented the control treatment; the trial was repeated once. All plants were kept in a greenhouse at temperatures ranging from 22 to 25°C. Inoculated plants became chlorotic 7 days after inoculation and root and crown rot developed 30 days after inoculation. Control plants remained symptomless. P. capsici consistently was reisolated from inoculated plants. In Italy, the presence of P. nicotianae on hybrids of Solanum lycopersicum × S. hirsutum is known (3), while, to the best of our knowledge, this is the first report of P. capsici on the hybrid S. lycopersicum × S. hirsutum in Italy. The economic importance of the disease can increase due to the expanding use of grafted tomato plants. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St Paul, MN, 1996. (3) A. Garibaldi and M. L. Gullino. Acta Hortic. 833:35, 2010. (4) H. M. Masago et al. Phytopathology 67:425, 1977.

First Report of Plectosphaerella cucumerina on Greenhouse Cultured Wild Rocket (Diplotaxis tenuifolia) in Italy.

During spring 2012, symptoms of an unusual leaf spot disease were observed in several commercial greenhouses near Salerno (southern Italy) on plants of Diplotaxis tenuifolia (cv Selvatica). The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, 1 cm in diameter. Spots were surrounded by a yellow halo, and were mostly located on the foliar limb, rib, and petiole. Affected leaves were often distorted, appearing hook-like. The disease was severe under 75 to 90% RH, at air temperature of 20 to 26°C, and caused severe production losses on about 50 ha. Particularly, affected tissues rotted quickly after packaging and during transit and commercialization of processed rocket. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 22°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, sometimes one-septate, measuring 4.5 to 9.2 × 1.7 to 3.5 (average 6.8 × 2.6) µm. Conidia were born on phialides, measuring 6.8 to 20.2 × 1.3 to 3.1 (average 16.5 × 2.1) µm. Such characteristics are typical of Plectosphaerella sp. (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 519-bp segment showed a 98% similarity with the sequence of Plectosphaerella cucumerina (GenBank Accession No. AB469880). The nucleotide sequence has been assigned the GenBank Accession JX185769. To confirm pathogenicity, tests were conducted on 45-day-old D. tenuifolia plants. Plants (21/treatment), grown in 15 liter pots (7 plants/pot) were inoculated by spraying a 1 × 106 CFU/ml conidial suspension of one isolate of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 23 ± 1°C, at 90% RH for 4 days. Non-inoculated plants served as control. Inoculated plants showed the typical first leaf spots 6 days after the artificial inoculation. Four days later, spots enlarged and leaves became distorted, showing chlorosis. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity test was conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on D. tenuifolia in Italy as well as worldwide. P. cucumerina has been described as associated with root and collar rots of other horticultural crops in southern Italy (1). Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al., Persoonia, 28:34, 2012. (2) M. E. Palm et al. Mycologia, 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Rust Caused by Pucciniastrum circaeae on Fuchsia × hybrida in Italy.

Fuchsia is a genus of flowering plants that is native to South America and New Zealand and belongs to the family Onagraceae. In September 2011, 2-year-old potted plants of Fuchsia × hybrida, cv. Citation, in a garden located near Biella (northern Italy) showed signs and symptoms of a previously unknown disease. Typically, infected plants showed leaf chlorosis followed by the appearance of necrosis on the adaxial leaf surfaces, while the abaxial surfaces showed orange uredinia irregularly distributed. As the disease progressed, infected leaves turned yellow and wilted. Affected plants showed a progressive phylloptosis and also flowering was negatively affected. Urediniospores were globose, yellow to orange, and measured 14.6 to 25.9 (average 19.6) µm. Teliospores were not observed. Morphological characteristics of the fungus corresponded to those of the genus Pucciniastrum. DNA extraction and PCR amplification were carried out with Terra PCR Direct Polymerase Mix (Clontech, Saint Germain-en-Laye, France) and primers ITS1/ITS4 (4). A 700-bp PCR product was sequenced and a BLASTn search (1) confirmed that the sequence corresponded with a 96% identity to Pucciniastrum circaeae. The nucleotide sequence has been assigned the GenBank Accession No. JQ029688. Pathogenicity tests were performed by spraying leaves of healthy 1-year-old potted Fuchsia × hybrida plants with an aqueous suspension of 1 × 103 urediniospores ml-1. The inoculum was obtained from infected leaves. Plants sprayed only with water served as controls. Three plants were used for each treatment. Plants were covered with plastic bags for 4 days after inoculation and maintained outdoors at temperatures ranging between 18 and 25°C. Lesions developed on leaves 20 days after inoculation with the urediniospore suspension, showing the same symptoms as the original plants, whereas control plants remained healthy. The organism that was recovered from the lesions after inoculation was the same as the one obtained from the diseased plants. The pathogenicity test was carried out twice with similar results. The presence of P. fuchsiae, later identified as P. epilobii, was repeatedly reported in the United States (3). P. epilobii and P. circaeae have closely related hosts and morphologically similar urediniospores. These species were reported to form a single group in molecular phylogenetic trees (2). This is, to our knowledge, the first report of P. circaeae on Fuchsia × hybrida in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) Y. M. Liang et al. Mycoscience 47:137, 2006. (3) L. B. Loring and L. F. Roth. Plant Dis. Rep. 48:99, 1964. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

The synthesis of [26,27-11C]dihydroxyvitamin D(3), a tracer for positron emission tomography (PET).

1Alpha,25-dihydroxyvitamin D(3), an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with 11C for use in biological experiments. The radionuclide was incorporated via the reaction of [11C]methyllithium on a methyl ketone precursor in tetrahydrofuran at -10 degrees C. Deprotection of the labeled intermediate yielded 2.5-3 GBq [26,27-11C]1alpha,25-dihydroxyvitamin D(3) [11C-1,25(OH)(2) D(3)] with specific radioactivity averaging 100 GBq/micromol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; 11C-1,25(OH)(2) D(3) is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.

Potential (18)F-labeled biomarkers for epidermal growth factor receptor tyrosine kinase.

As PET candidate tracers for EGFr-TK, five 4-(anilino)quinazoline derivatives, each fluorinated in the aniline moiety, were prepared. Each was tested in vitro for inhibition of EGFr autophosphorylation in A431 cell line. The leading compounds were then radiolabeled with (18)F and cell binding experiments, biodistribution and PET studies in A431 tumor-bearing mice were performed. Metabolic studies were carried out in a mice control group. From our results, we concluded that while in vitro experiments indicates efficacy of 4-(anilino)quinazoline compounds, kinetic factors and rapid blood clearance make them unsuitable as tracers for nuclear medicine imaging of EGFr-TK.

Evaluation of [O-methyl-(11)C]fluvoxamine as a tracer for serotonin re-uptake sites.

We evaluated [(11)C]fluvoxamine as a tracer for the serotonin re-uptake site. Studies of the distribution of the tracer in rat and primate brain showed adequate uptake of [(11)C]fluvoxamine, but failed to reveal regions with known high density of serotoninergic re-uptake sites. Pretreatment with unlabeled fluvoxamine did not substantially change the distribution. In rat brain tissue, nearly all radioactivity represented intact [(11)C]fluvoxamine. [(11)C]Fluvoxamine does not function as a tracer for serotonin re-uptake sites, owing to high nonspecific binding in the brain.

Age-related variations in hepatic biosynthesis of phosphatidylcholine: a study of choline metabolism with perfused rat liver.

This study investigated the age-related increase in phospholipid secretion previously described in perfused rat livers. The hypothesis of this study was that the increased secretion is mainly due to an enhanced hepatic biosynthesis of phosphatidylcholine (PC). Specifically, we evaluated the contribution to this increase of the major hepatic pathway of phosphatidylcholine formation (i.e., the conversion of choline into phosphatidylcholine via cytidine diphosphate [CDP]-choline). The measurements of [3H]choline incorporation into phosphatidylcholine and its precursors in liver and bile throughout the 2-hr duration of the experiments showed significant differences in the amount of newly synthesized labeled PC secreted in the bile produced by adult and young rat livers. However, the present findings do not support the idea that the age-related increase in phosphatidylcholine hepatic secretion was due only to a strong increase in phosphatidylcholine synthesis by via CDP-choline. Conversely, they suggest that future research should be directed towards the mechanisms regulating the diacylglycerol metabolism in the hepatocytes, as the alteration of the splitting ratio of hepatic diacylglycerol flow could lead to an age-related increase in conversion of diacylglycerol into phosphatidylcholine, rather than into triacylglycerol. This, in turn, may decrease the availability of triacylglycerol for hepatic very low density lipoprotein (VLDL) assembly and contribute to altered VLDL synthesis, as previously observed in the aging process.

Probucol reduces hepatic cholesterol secretion in hyperlipidemic Yoshida rats.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

Comparison of the hepatic uptake and processing of cholesterol from chylomicrons of different fatty acid composition in the rat in vivo.

The effect of the fatty acid composition of chylomicrons on the uptake and processing of the cholesterol they carry was investigated in the rat in vivo. Rats kept on a standard low fat pellet diet and tube fed a single dose of palm, olive, corn or fish oil (rich in saturated, n-9 monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids, respectively) were used to prepare [3H]cholesterol-labelled chylomicrons of different fatty acid composition. These were then injected intravenously into rats (kept on the standard diet), and the clearance of radioactivity from the blood, distribution in the plasma lipoprotein density fractions, uptake by the liver and appearance in the bile were studied. [3H]Cholesterol from fish and corn oil chylomicrons was cleared from the blood more rapidly than that from palm and olive oil chylomicrons. After 180 min the proportion of the radioactivity present in the plasma in high density lipoprotein (HDL) was less when the chylomicrons were derived from palm oil as compared to any of the other oils. Approx. 40% of the administered label was recovered in the liver after 180 min in all experiments. The percentage of the injected radioactivity secreted into bile during 180 min was significantly higher with corn and fish oil chylomicrons than with palm oil chylomicrons, with chylomicrons from olive oil in an intermediate position, and these differences were most pronounced between 60 and 120 min after administration of the label. These studies clearly demonstrate that the fatty acid composition of chylomicrons has important effects on the hepatic uptake and processing of the cholesterol they carry, with enrichment with polyunsaturated fatty acids leading to an increased rate of uptake and more rapid removal from the body via the bile as compared to enrichment with saturated or monounsaturated fatty acids.