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.

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 Stem Rot on Cereus peruvianus monstruosus Caused by Bipolaris cactivora (Petr.) Alcorn in Italy.

Cereus peruvianus monstruosus, known as "monster cactus," family Cactaceae, is grown as a potted plant. In the winter of 2013, a stem rot was observed on a farm located near Ventimiglia (northern Italy) on 80% of 4,000 9-month-old plants grown in trays in a peat substrate. Symptoms consisted of a rapid rot of the upper portion of the stem. Affected stems at first showed yellowish spots that became brown irregular necrotic lesions with well-defined margins. The tissues below the affected areas were blackened and dry but became soft in the presence of high relative humidity. Fungal sporulation on rotted tissues consisted of caespitose, non-branched, septate conidiophores, olivaceous to brown at the base, paler above, measuring 89.0 to 196.9 × 6.2 to 8.7 (average 124.8 × 7.0) µm. Single conidia were borne on terminal cells. At maturity, conidia with 2 to 5 (average 3) septa were brownish-olivaceous, varying in shape from obclavate, fusiform, ellipsoid or sometimes furcate, and measuring 23.4 to 48.6 × 8.0 to 12.6 (average 38.8 × 10.3) µm. Symptomatic tissues were immersed in 1% sodium hypochlorite for 2 to 3 s and rinsed in sterile distilled water, then fragments excised from the margin of internal lesions were cultured on potato dextrose agar (PDA) medium amended with 25 mg/l of streptomycin sulfate and incubated at 20 to 23°C under alternating daylight and darkness (10 h light, 14 h dark). A fungus that was consistently isolated was subcultured on PDA. At maturity, dark green floccose colonies comprised of light brown septate hyphae, 4.2 to 8.1 (average 5.6) µm in width, produced non-branched, pale to dark brown, septate conidiophores, measuring 99.6 to 176.1 × 4.5 to 6.5 (average 146.7 × 5.4) µm. The conidia produced on PDA were similar to those observed on infected tissues and measured 20.6 to 40.7 × 7.5 to 11.4 (average 32.0 × 9.7) µm, with 1 to 3 septa (average 2). On the basis of the morphological characteristics, the fungus was identified as Bipolaris cactivora (Petr.) Alcorn [Syn.: Drechslera cactivora (Petr.) M. B. Ellis] (4). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was amplified for one isolate using ITS1/ITS4 primers and sequenced (GenBank Accession No. KF041822). BLASTn analysis (1) of the 557-bp segment showed a 99% similarity with the ITS sequence of Bipolaris cactivora HM598679. For pathogenicity tests, 8 mm diameter mycelial disks removed from 15-day-old PDA cultures of the fungus were placed at the wounded stem apexes of three 7-month-old healthy plants (three disks per plant). Three plants inoculated with non-inoculated PDA disks served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 23 ± 1°C with 12 h light/dark. By 8 days after inoculation, all the inoculated stems were rotted and 10 colonies of B. cactivora were re-isolated from infected tissues. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Several hosts are listed for B. cactivora including C. peruvianus, and the pathogen has been reported in the United States (2) and in South Korea (3). To our knowledge, this is the first report of B. cactivora on C. peruvianus monstruosus in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. APS Press, St Paul, MN, 1989. (3) I. H. Hyun et al. Res. Plant Dis. 7:56, 2001. (4) A. Sivanesan. Mycopathologia 111:125, 1990.

First Report of Crown and Stem Rot of Crested Molded Wax Agave (Echeveria agavoides) caused by Fusarium oxysporum in Italy.

During March 2012, 95% of 24-month-old plants of crested molded wax agave (Echeveria agavoides), a succulent plant belonging to the Crassulaceae family, showed symptoms of a basal stem and leaf rot in a commercial farm near Ventimiglia (northern Italy). Affected plants showed extensive chlorosis from the crown level to the stem apex, followed by yellowing and by the appearance of a water-soaked aspect of stem and leaf tissues. As disease progressed, leaves became brown, wilted, and rotted. Wilting was at first unilateral and later affected the entire plant. Brown discoloration was observed in the vascular system of cut stems and leaves. In some cases, leaves were covered by a whitish-orange mycelium. This produced 3-septate, slightly curved macroconidia with a foot-shaped basal cell and a short apical cell, measuring 27.4 to 39.6 × 3.0 to 4.1 (average 34.2 × 3.7) µm and unicellular, ovoid to elliptical microconidia measuring 4.8 to 11.6 × 1.5 to 3.7 (avg. 7.2 × 2.7) µm. A fungus was consistently isolated from discolored vascular leaf tissues on Komada selective medium. Cultures on potato dextrose agar (PDA) and carnation leaf-piece agar (CLA) were incubated at 24 to 29°C. On PDA, a thin growth of whitish mycelium without pigments in the agar was observed. On CLA, sparse macroconidia, 18.9 to 30.7 × 3.0 to 4.2 (avg. 23.9 × 3.6) µm, microconidia, 4.7 to 7.7 × 1.7 to 3.1 (avg. 6.0 × 2.4) µm, and abundant chlamydospores that were single or paired, terminal and intercalary, rough walled, and 6.8 to 9.5 (avg. 7.7) in diameter were produced. Such characteristics are typical of Fusarium oxysporum (2). Amplification of the internal transcribed spacer (ITS) of the rDNA using primers ITS1/ITS4 (3) yielded a 486-bp band (GenBank Accession No. JX441893). Sequencing and BLASTn analysis of this band showed 100% identity and an E-value of 0.0 with the ITS sequence of F. oxysporum (JN232163). To confirm pathogenicity, five 3-month-old healthy plants of E. agavoides were inoculated by dipping unwounded roots in a conidial suspension (1.0 × 107 CFU/ml) of one isolate of F. oxysporum obtained from affected plants, grown for 10 days in potato dextrose broth. Plants were transplanted into pots filled with steam-sterilized substrate (sphagnum peat-perlite-pine bark-clay 50:20:20:10) and maintained in a glasshouse at 28 to 33°C. Five non-inoculated plants served as a control. Chlorosis and yellowing developed on the inoculated plants 15 days after the inoculation. Basal stem rot and vascular discoloration in the crown and stem developed within 30 days on inoculated plants. A whitish-orange mycelium producing macroconidia covered the affected leaves. Non-inoculated plants remained healthy. F. oxysporum was consistently reisolated from symptomatic plants. The pathogenicity test was conducted twice. A Fusarium sp. has been reported as the causal agent of a stem rot on Echeveria sp. in the U.S. (1). To our knowledge, this is the first report of F. oxysporum on E. agavoides in Italy. The disease is currently present in few nurseries, although it could spread, causing significant economic losses due to the increasing cultivation of E. agavoides in Italy. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. APS Press, St. Paul, MN, 1989. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (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 Leaf Spot of Saponaria officinalis Caused by Alternaria nobilis in Italy.

Saponaria officinalis (Vize) Simmons (common name bouncingbet) is a low maintenance perennial plant belonging to the Caryophyllaceae family, typically grown in parks and gardens. During the summers of 2011 and 2012, extensive necrosis were observed on leaves of plants grown in private gardens, near Biella (northern Italy). The disease affected 90% of 1- to 2-year-old plants. The first symptoms were usually pale brown lesions 1 to 5 mm in diameter and sometimes coalesced. Lesions were circular to irregular with a dark purple halo, with infected leaves eventually turning chlorotic. The conidia observed on infected leaves were olivaceous brown and obclavate, with a beak. Conidia showed 8 to 15 (average 12) transverse and 4 to 14 (average 11) longitudinal septa, with slight constrictions connected with septa, and were 78.3 to 177.7 (average 135.5) × 19.0 to 34.3 (average 26.5) µm. The beak was 20.0 to 62.2 (average 33.7) µm in length, with 0 to 6 (average 3) transverse septa and no longitudinal septa. The fungus was consistently isolated from infected leaves on potato dextrose agar (PDA). The isolate, grown for 14 days at 20 to 24°C with 10 h of darkness and 14 h of light on sterilized host leaves plated on PDA, produced conidiophores single, unbranched, flexuous, septate with conidia in short chains, similar to those observed on the leaves and previously described. On the basis of its morphological characteristics, the pathogen was identified as Alternaria sp. (3). DNA was extracted using Nucleospin Plant Kit (Macherey Nagel) and PCR carried out using ITS 1/ITS 4 primer (4). A 542-bp PCR product was sequenced and a BLASTn search confirmed that the sequence corresponded to A. dianthi (AY154702), recently renamed A. nobilis (2). The nucleotide sequence has been assigned the GenBank Accession No. JX647848. Pathogenicity tests were performed by spraying leaves of healthy 3-month-old plants of S. officinalis with an aqueous 2 × 105 spore/ml suspension. The inoculum was obtained from cultures of the fungus grown on PDA amended with host leaves for 14 days, in light-dark, at 22 ± 1°C. Plants sprayed only with water served as controls. Four pots (1 plant/pot) were used for each treatment. Plants were covered with plastic bags for 4 days after inoculation and maintained in a glasshouse at 21 ± 1 °C. Lesions developed on leaves 9 days after inoculation with the spore suspension, whereas control plants remained healthy. A. nobilis was consistently reisolated from these lesions. The pathogenicity test was carried out twice. The presence of A. dianthi was reported on S. officinalis in Denmark (1) and Turkey. This is, to our knowledge, the first report of A. nobilis on S. officinalis in Italy. The presence and importance of this disease is, at present, limited. References: (1) P. Neergaard. Danish species of Alternaria and Stemphylium. Oxford University Press, 1945. (2) E. G. Simmons. Mycotaxon 82:7, 2002. (3) E. G. Simmons. Alternaria: An Identification Manual. CBS Biodiversity Series 6, Utrecht, The Netherlands, 2007. (4) T. J. White et al. In: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Web Blight on Oregano (Origanum vulgare L.) Caused by Rhizoctonia solani AG-1-IB in Italy.

Origanum vulgare L., common name oregano, family Labiatae, is grown for its aromatic and medicinal properties and as ornamental. In the fall of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 6% of 30,000 50-day-old plants, grown in trays in a peat/perlite mix. Semicircular, water soaked lesions appeared on leaves and stems, starting from the basal ones. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. Eventually, infected plants died. Leaf and stem fragments taken from the margin of the diseased tissues belonging to 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani was consistently recovered. Three isolates of R. solani obtained from affected plants were successfully anastomosed with R. solani isolate AG 1 (ATCC 58946). Three pairings were made for each tester strain. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed. Results were consistent with other reports on anastomosis reactions (2). Isolates from oregano were paired with R. solani isolates AG 2, 3, 4, 6, 7, or 11 and examined microscopically. Anastomosis was not observed in any of the pairings. Tests were conducted twice. Mycelium of 10-day-old isolates from oregano appeared reddish brown, coarse, and radiate. Numerous dark brown sclerotia, 0.3 to 1.0 mm diameter (average 0.7) developed within 10 days after transfer of mycelia to PDA in 90 mm diameter petri dishes at 21 to 24°C. The descriptions of mycelium and sclerotia were typical for subgroup IB Type 1 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the 538 bp showed a 99% homology with the sequence of R. solani FJ746937, confirming the morphological identification of the species. The nucleotide sequence has been assigned the GenBank Accession KC493638. For pathogenicity tests, one of the isolates assigned to the anastomosis group AG-1-IB was tested by placing 9 mm diameter mycelial disks removed from PDA 10-day-old cultures of the fungus on leaves of 90-day-old oregano plants (n = 35). Thirty-five plants inoculated with non-inoculated PDA disks served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C with 12 h light/dark. The first symptoms, similar to those observed in the farm, developed 3 days after inoculation. Nine days after the artificial inoculation, 50% of plants were dead. About 10 colonies of R. solani were reisolated from infected leaves of inoculated plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on O. vulgare in Greece (3). This is, to our knowledge, the first report of blight of O. vulgare caused by R. solani in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res., 25:3389, 1997. (2) 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. (3) C. D. Holevas et al. Benaki Phytopathol. Inst., Kiphissia, Athens, 19:1-96, 2000. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

First Report of Web Blight on Rosemary (Rosmarinus officinalis) Caused by Rhizoctonia solani AG-1-IA in Italy.

Rosmarinus officinalis L., family Labiatae, is an evergreen shrub used in gardens as an aromatic or ground cover plant. In the summer of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 20% of 150,000 70-day-old plants, grown in trays. Water soaked lesions appeared on leaves and stems. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. A light mycelium spread on the substrate. Disease progressed from infected plants to healthy ones and, eventually, infected plants died. Leaf and stem fragments taken from the margin of the diseased tissues belonging to 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani was consistently and readily recovered. Three isolates of R. solani obtained from affected plants were successfully paired with R. solani tester strains AG 1, 2, 3, 4, 6, 7, or 11 and examined microscopically. Three pairings were made for each recovered isolate. The isolates of R. solani from rosemary anastomosed only with tester strain AG 1 (ATCC 58946). Results were consistent with other reports on anastomosis reactions (2). Tests were repeated once. Mycelium of 10-day-old isolates from rosemary appeared light brown, compact, and radiate. Numerous dark brown sclerotia, 0.7 to 2.0 mm diameter (average 1.3), developed within 10 days at 20 to 26°C. The descriptions of mycelium and sclerotia were typical for subgroup IA Type 2 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced (GenBank Accession No. KC005724). BLASTn analysis (1) of the 657-bp showed a 99% similarity with the sequence of R. solani GU596491. For pathogenicity tests, inoculum of R. solani was prepared by growing the pathogen on wheat kernels autoclaved in 1-liter glass flasks for 8 days. One of the isolates assigned to the anastomosis group AG 1 IA was tested. Fifteen 90-day-old rosemary plants were grown in 15-liter pots in a steam disinfested peat:pomice:pine bark:clay mix (50:20:20:10) infested with 3 g/liter of infested wheat kernels, placed at the base of the stem. Fifteen plants inoculated with non-infested wheat kernels served as control treatments. Plants were covered with plastic bags and arranged in a growth chamber at 20 to 24°C with 12 h light/dark for 15 days. The first symptoms, similar to those observed in the farm, developed 10 days after inoculation. About 10 colonies of R. solani were reisolated from infected leaves and stems of each inoculated plant. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on R. officinalis in United States (3), India, and Brazil. This is, to our knowledge, the first report of blight of R. officinalis caused by R. solani in Italy. This disease could cause serious economic losses, because rosemary is one of the most cultivated aromatic plants in the Mediterranean region. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) 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, 1996. (3) G. E. Holcomb. Plant Dis. 76:859, 1992. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

A Leaf Spot Caused by Phoma novae-verbascicola on Black Mullein (Verbascum nigrum L.) in Italy.

Verbascum nigrum L., common name black mullein, family Scrophulariaceae, is a rustic perennial plant belonging to the native flora in Italy. The plant, which produces bright yellow flowers densely grouped on the tall stem, is used in low-maintenance gardens. During fall 2012, plants grown in mixed planting borders in a garden located in Biella Province (northern Italy) showed extensive foliar disease. Approximately 100 plants were affected by the disease. Early symptoms were small, light brown, necrotic spots on leaves, later reaching 10 mm diameter, with an irregular shape, showing a chlorotic halo. Necrotic areas often coalesced surrounded by yellowing. In some cases, the internal part of the necrotic areas dried with the appearance of holes. The disease progressed from the base to the apex of plants. In some cases, most of leaves turned completely necrotic and plants were severely damaged. Symptomatic tissues were immersed in a solution containing 1% sodium hypochlorite for 2 to 3 s and rinsed with sterile distilled water. Small fragments were excised from the margin of lesions and plated on potato dextrose agar (PDA) medium. Petri dishes were incubated at temperatures ranging between 20 and 25°C under alternating daylight and darkness (12 h light, 12 h dark). A single fungus was consistently isolated and subcultured on malt extract agar (MEA). On MEA, colonies were felty, white cream, and produced dark globose or subglobose pycnidia measuring 68 to 185 × 62 to 177 (average 122 × 113) µm, containing hyaline (light grey in mass), ellipsoid, non-septate conidia measuring 3.1 to 5.7 × 1.5 to 2.7 (average 4.0 × 2.0) µm after 15 days. The internal transcribed spacer (ITS) and D1/D2 regions of rDNA were amplified using the primers ITS1/ITS4 and NL1/NL4, respectively, and then sequenced (GenBank Accession Nos. KC411473 and KF041823). BLAST analysis of both fragments showed 99% homology with the sequences GU237753 and JQ768403 of Phoma novae-verbascicola Aveskamp, Gruyter & Verkley (Basionym: Phyllosticta verbascicola Ellis & Kellerm.). Morphological characteristics of the fungus also were consistent with the descriptions of P. poolensis var. verbascicola (Ellis & Kellerm.) Aa & Boerema (2) (Syn.: P. novae-verbascicola). Pathogenicity tests were performed by spraying a conidial suspension (4 × 104 CFU/ml) obtained from 15-day-old PDA cultures of the fungus onto leaves of three healthy 3-month-old V. nigrum. Three plants inoculated with sterile water served as controls. Plants were maintained in a growth chamber for 5 days at 25 ± 1°C under 70 to 90% relative humidity. The first foliar lesions developed on leaves 2 days after inoculation and after 5 days, 80% of leaves were severely infected. Control plants remained healthy. The organism reisolated on PDA from leaf lesions was identical in morphology to the isolate used for inoculation. The pathogenicity test was carried out twice. Phoma spp. has been reported on Verbascum spp. P. novae-verbascicola has been very recently described (1). To our knowledge, this is the first report of the presence of P. novae-verbascicola on V. nigrum in Italy. At present, the economic importance of this disease is limited, but may become a more significant problem if the cultivation of this species increases. References: (1) M. M. Aveskamp et al. Studies in Mycology, 65: 1, 2010. (2) J. de Gruyter et al. Persoonia 15 (3): 369, 1993.

First Report of Sclerotinia Blight Caused by Sclerotinia sclerotiorum on Spearmint in Northern Italy.

Spearmint (Mentha spicata L.) is an aromatic plant belonging to the family Lamiaceae, grown as well as an ornamental potted plant. During the beginning of 2013, extensive wilting was observed on 4-month-old potted plants of M. spicata 'Moroccan' grown in a commercial, unheated, plastic house located near Albenga (Savona, northern Italy). Initial symptoms included stem necrosis and darkening and withering of leaves. Wilting of the plant occurred 2 to 4 days after the appearance of the initial symptoms. Infected plants were characterized by the presence of cottony soft rot. In the presence of high relative humidity, lesions became covered with a whitish mycelium and irregular, dark gray sclerotia (2.0 to 9.0 × 1.8 to 4.0, average 4.0 × 2.6 mm) were produced on the mycelium. Diseased tissue was surface sterilized for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 100 mg/l streptomycin sulfate. White colonies developed from infected stem pieces and produced sclerotia, mainly at the peripheries of the plates, measuring 2.0 to 8.0 × 2.0 to 6.0 (average 4.4 × 3.1) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1F/ITS4 and sequenced. BLAST analysis (1) of the 514-bp segment showed a 99% homology with the sequence of Sclerotinia sclerotiorum (JN012605). The nucleotide sequence has been assigned the GenBank Accession KC848769. The morphological and molecular identification permitted to identify as S. sclerotiorum (Lib.) de Bary (2) the causal agent of the disease observed on M. spicata. Pathogenicity of one isolate obtained from infected plants was confirmed by inoculating three 7-month-old plants cv. Moroccan transplanted in 1 liter pots in a glasshouse in a sphagnum peat/pomix/pine bark/clay (50:20:20:10) mix. Each plant was inoculated by placing 4 g of sterile wheat kernels infested with mycelium and sclerotia in the soil and around the collar. Three non-inoculated plants served as controls. Plants were maintained in a growth chamber at 24 ± 1°C and relative humidity >90%. The inoculation trial was carried out twice. All inoculated plants developed the symptoms, consisting of stem necrosis, 5 days after soil infestation, followed by leaf yellowing. White cottony mycelium and dark sclerotia developed on stems and at the base of all inoculated plants. Eventually, infected plants wilted. Control plants remained symptomless. S. sclerotiorum was reisolated from the stems of inoculated plants. To our knowledge, this is the first report of S. sclerotiorum on M. spicata in Italy as well as worldwide. The disease has been previously reported on M. piperita in the United States (4) and on M. arvensis in India (3). The economic importance of this disease in Italy is at present limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) N. F. Buchwald. Kongl. Veterisk Landb. Aarssk. 75, 1949. (3) K. Perveen et al. Indian Phytopathol. 62:310, 2009. (4) C. B. Skotland and J. D. Menzies. Plant Dis. Rep. 41:493, 1957.

First Report of Verticillium Wilt caused by Verticillium dahliae Kleb. on New Zealand Spinach (Tetragonia tetragonioides) in Italy.

Tetragonia tetragonioides (New Zealand spinach, Aizoaceae) is an Australasian annual species that occurs naturally in Italy, where it is cultivated for the edible young shoots and succulent leaves. In September 2011, a previously unknown wilt was observed in 10 private gardens, each 0.1 to 0.5 ha, near Castellaro, Northern Italy, on 7-month-old New Zealand spinach plants. Leaves wilted, starting from the collar and moving up the plant, and vascular tissues showed brown streaks in the roots, crowns, and stems. Diseased plants were stunted with small, chlorotic leaves. Infected stems and leaves then wilted, and plants often died. Of about 500 plants, 30% were affected. Stems of 10 diseased plants were disinfected with 1% NaOCl for 1 min. Sections of symptomatic vascular tissue were plated on potato dextrose agar. After 3 days at 23 ± 1°C, colonies developed that were white and turned a grey to dark green color. Irregular, black microsclerotia (32.0) 63.1 ± 16.8 µm (106.1) × (18.7) 39.1 ± 12.3 µm (65.8) developed in hyaline hyphae after 8 days. Hyaline, elliptical, single-celled conidia (2.7) 3.8 ± 0.6 µm (4.8) × (1.9) 2.6 ± 0.5 µm (3.5) developed on verticillate conidiophores with three phialides at each node. Based on these morphological characteristics, the fungus was identified as Verticillium dahliae (1). The internal transcribed spacer (ITS) region of rDNA was amplified for one isolate using the primers ITS1/ITS4 (3) and sequenced (GenBank Accession No. JX308315). BLASTn analysis of the 479-bp segment showed 100% homology with the ITS sequence of a V. dahliae isolate (AB551206). Pathogenicity tests were performed twice using 60-day-old plants of T. tetragonioides. Unwounded roots of eight plants were dipped for 1 min in a conidial suspension (5 × 107 conidia/ml) of one isolate of V. dahliae obtained from the original infected New Zealand spinach plants, and grown in potato dextrose broth. The inoculated plants were transplanted into 2-liter pots (1 plant/pot) containing steamed potting mix (sphagnum peat-perlite-pine bark-clay; 50:20:20:10) and maintained in a growth chamber at 20 to 24°C and 50 to 80% RH. Eight plants immersed in sterile water served as a control treatment. Wilt symptoms were observed 30 days after inoculation, with vascular discoloration in the roots, crowns and stems. V. dahliae was reisolated consistently from infected tissues, but not from the control plants that remained healthy. Pathogenicity was also tested using the same method on plants of four cultivars (five plants/cultivar) of Spinacia oleracea (Matador, Asti, Merlo Nero, and America). Wilt symptoms developed on all cultivars and V. dahliae was reisolated from each inoculated plant. No fungal colonies were reisolated from control plants, which remained healthy. To our knowledge, this is the first report of Verticillium wilt caused by V. dahliae on T. tetragonioides in Italy, as well in Europe. V. dahliae was reported on T. tetragonioides in Canada (2). At this time, the economic impact of Verticillium wilt on New Zealand Spinach in Italy is limited, although the use of this vegetable in Italy is increasing. References: (1) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002. (2) M. J. Richardson. Page 387 in: An Annotated List of Seed-Borne Diseases, Fourth Edition. International Seed Testing Association, Zurich, Switzerland, 1990. (3) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Postharvest Fruit Rot in Avocado (Persea americana) Caused by Lasiodiplodia theobromae in Italy.

Avocado (Persea americana Mill.) is grown in some areas of southern Italy. In spring 2011, a previously unknown rot was observed on fruit that was marketed in Torino (northern Italy). The decayed area started from the stalk, appeared irregular and soft, and was surrounded by a dark brown margin. The internal decayed area appeared rotten, brown, and surrounded by bleached tissue. Fragments (approximately 3 mm) were taken from the margin of the internal diseased tissues, cultured on potato dextrose agar (PDA), and incubated at temperatures between 21 and 25°C under alternating conditions of light and dark. Colonies of the fungus initially appeared whitish, later turning mouse gray to black. Mature mycelium was septate and produced a dark pigment. The fungus, grown on oat agar (2) and incubated at temperatures between 21 and 25°C under alternating light and darkness, produced grayish colonies with a fluffy aerial mycelium that became dark with age and produced black pigments. After 18 days of incubation, such colonies produced pycnidia aggregated into stromatic masses, emerging from decayed tissues, and up to 3 to 4 mm in diameter. Conidia produced in the pycnidia were initially unicellular, hyaline, granulose, ovoid to ellipsoidal, and measured 20.8 to 26.9 × 12.5 to 16.1 (average 24.4 × 13.5) µm. After 7 days, mature conidia became darker, uniseptate, and longitudinally striate. Paraphyses produced within the tissues of pycnidia were hyaline, cylindrical, nonseptate, and up to 63 µm long. Morphological characteristics of mycelia, pycnidia, and conidia observed with a light microscope permitted identify of the fungus as Lasiodiplodia theobromae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 488-bp segment showed a 100% similarity with the corresponding sequence (GenBank Accession No. GQ502453) of L. theobromae Pat. Griffon & Maubl. The nucleotide sequence of the strain used for pathogenicity tests was submitted to GenBank (Accession No. JN849098). Pathogenicity tests were performed by inoculating 10 avocado fruits after surface disinfesting in 1% sodium hypochlorite and then wounding. Mycelial disks (8 mm in diameter) obtained from PDA cultures of one strain were placed on wounds. Ten control fruits were inoculated with plain PDA. Fruits were incubated at 15 ± 1°C. The first symptoms developed 4 days after the artificial inoculation. After 7 days, the rot was evident and L. theobromae was consistently reisolated. Noninoculated fruit remained healthy. The pathogenicity test was performed twice. To our knowledge, this is the first report of the presence of L. theobromae causing postharvest fruit rot on avocado in Italy, as well as in Europe. The occurrence of postharvest fruit rot on avocado caused by L. theobromae was described in many avocado-producing areas such as the United States (4), South Africa, and Israel. In Italy, the economic importance of avocado cultivation is currently limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2). P. Narayanasamy. Microbial Plant Pathogens. Detection and Disease Diagnosis: Fungal Pathogens. Springer, Dordrecht, 2011. (3) E. Punithalingam. Sheet 519. CMI Description of Fungi and bacteria, 1976. (4) H. E. Stevens and R. B. Piper. Circular No. 582, USDA, 1941.

Golovinomyces biocellatus on Oregano (Origanum vulgare 'Compactum') in Italy.

Origanum vulgare L., common name oregano, also known as pot marjoram, Lamiaceae family, is grown for its aromatic and medicinal properties and as an ornamental. In particular, O. vulgare 'Compactum' is becoming popular as a potted plant. During January 2011, 3-month-old plants grown on a commercial farm located near Albenga (northern Italy) showed signs and symptoms of an unknown powdery mildew. Ninety percent of the plants were affected. The adaxial leaf surfaces were covered with white mycelia and conidia, while the abaxial surfaces were less infected. As the disease progressed, infected leaves turned yellow, wilted, and eventually fell off. Mycelia were also observed on stems. Conidia were hyaline, elliptical, borne single or in short chains (three to four conidia per chain), and measured 37.9 × 19.6 (31.2 to 45.1 × 14.9 to 26.2) µm. Conidiophores were erect with a cylindrical foot cell measuring 81.1 × 9.7 (54.2 to 112.4 × 7.9 to 11.6) µm followed by two to three shorter cells measuring 26.8 × 11.8 (16.6 to 38.1 × 8.5 to 15.3) µm. Fibrosin bodies were absent. Chasmothecia were not observed in the collected samples. The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1F/ITS4 and sequenced (3) (GenBank Accession No. JN594608). The 560-bp amplicon had 99% homology with the sequence of Golovinomyces biocellatus (GenBank Accession No. AB307675). Pathogenicity was confirmed through inoculation by spraying a conidial suspension (6 × 104 CFU/ml) prepared from diseased leaves onto leaves of healthy O. vulgare 'Compactum' plants. Four plants were inoculated while the same number of noninoculated plants served as a control. Plants were maintained in a glasshouse at temperatures ranging from 23 to 28°C. Ten days after inoculation, typical symptoms of powdery mildew developed on inoculated plants. The fungus observed on inoculated plants was morphologically identical to that originally observed. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. G. biocellatus on O. vulgare has been reported in Switzerland (2) and Argentina (4) and it is present on other plant genera in Italy. In Italy, on the same host, attacks of Erysiphe galeopsis have been previously reported (1). The economic importance of this disease is currently limited due to limited planting of this species. However, in the last years, potted aromatic plants represent a steady increasing crop in Italy. Voucher specimens are available at the Agroinnova Collection, University of Torino. References: (1) K. Amano. Host Range and Geographical Distribution of the Powdery Mildew Fungi. Japan Science Society Press, Tokyo, 1986. (2) A. Bolay. Cryptog. Helv. 20:1, 2005. (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. (4) S. M. Wolcan. J. Plant Patho. 91:501, 2009.

First Report of Root Rot Caused by Phytophthora cinnamomi on Mountain Laurel (Kalmia latifolia) in Italy.

Kalmia latifolia L., common name Mountain Laurel, is an evergreen shrub that is becoming increasingly popular in gardens. It is also grown as a potted plant for its round flowers that range from light pink to white and occur in clusters in late spring. During July 2011, 3-year-old plants of K. latifolia 'Olympic Fire' showed extensive chlorosis and root rot on several commercial nurseries close to Maggiore Lake (Verbano-Cusio-Ossola Province) in northwestern Italy. Twigs wilted and died and leaves dropped, although in some cases, wilted leaves persisted on stems. The whole root system was affected with vascular tissues on the lower stem exhibiting brown discoloration, followed by plant death. The disease was severe and widespread, affecting 5% of approximately 3,500 plants. Tissue fragments of 1 mm2 were excised from the margins of the lesions and plated. A Phytophthora-like organism was consistently isolated on an oomycete-selective medium (BNPRA + HMI at 25 µg/ml) (4) after disinfesting root pieces for 1 min in a 1% NaOCl solution. The pathogen was identified based on morphological and physiological features as Phytophthora cinnamomi (2). Oogonia didn't form in single culture. On V8 agar, the microorganism was characterized by coenocytic coralloid hyphae, with spherical lateral and terminal swellings 23 to 46 (average 34) µm in diameter (n = 50), single or in clusters, and produced spherical, terminal chlamydospores 35 to 47 (average 40) µm in diameter (n = 50). No sporangia were produced after growing pure cultures in sterilized soil extracts nor were they produced on V8 agar. The internal transcribed spacer (ITS) region of the rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 898-bp segment showed a 99% homology with the sequence of P. cinnamomi (GU799638). The nucleotide sequence has been assigned the GenBank Accession No. JQ951607. Pathogenicity of one isolate obtained from infected plants was confirmed by inoculating 18-month-old plants of K. latifolia 'Olympic Fire'. The isolate was grown for 50 days in a mixture of 70:30 wheat/hemp kernels and then mixed into a substrate containing sphagnum peat moss/pumice/pine bark/clay (50:20:20:10 v/v) at a rate of 3 g/l. One plant per 2-l pot was transplanted into the substrate and constituted the experimental unit. Five plants were inoculated. Noninoculated plants represented the control treatment and the trial was repeated once. All plants were kept in a greenhouse at 24 to 27°C. Two of five plants inoculated developed symptoms of chlorosis, wilting, and root rot after 70 days and remaining plants after about 80 days. P. cinnamomi was reisolated consistently from inoculated plants but not from controls that remained symptomless. To our knowledge, this is the first report of P. cinnamomi on K. latifolia in Italy and in Europe. The disease has been reported in the United States (3). The economic importance of the disease is uncertain because of the limited number of nurseries that grow this crop in Italy, although its importance could increase as the popularity of K. latifolia increases. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. American Phytopathological Society, St Paul, MN, 1996. (3) L. F. Grand. North Carolina Agric. Res. Serv. Techn. Bull. 240, 1985. (4) H. Masago et al. Phytopathology 67:425, 1977.

Podosphaera sp. on Euphorbia susannae and E. inermis in Italy.

Euphorbia susannae (common name Suzane's spurge) and E. inermis (Euphorbiaceae family) are grown in pots and commercialized in northern Italy. In March 2012, plants of these species grown on the same commercial farm showed signs of powdery mildew. On E. susannae, apexes of stems were covered with white mycelia and conidia, with the ultimate development of necrosis on symptomatic tissues more interested by the disease. Of 5,000 plants, 5% were affected. Conidia were hyaline, elliptical, borne in short chains (two to three conidia per chain), and 27.7 (24.4 to 30.6) µm long and 16.1 (13.6 to 19.1) µm wide. Conidiophores were erect, with a foot cell straight or slightly flexuous, 82.8 (52.7 to 117.1) µm long and 10.0 (9.2 to 11.2) µm wide, followed by two to three shorter cells 19.8 (14.7 to 28.9) µm long and 10.7 (8.7 to 13.0) µm wide. On E. inermis, a white mycelium covered the stems starting from the apexes, causing yellowing and necrosis on leaves finally killing infected tissues. Of 500 plants, 80% were affected. Conidia were 33.2 (25.7 to 42.0) µm long and 17.3 (12.6 to 21.6) µm wide. Conidiophores were erect, with a foot cell straight or slightly flexuous, 96.7 (67.0 to 138.6) µm long and 9.5 (7.7 to 11.7) µm wide, followed by two to three shorter cells 26.3 (17.5 to 42.2) µm long and 11.8 (8.3 to 16.2) µm wide. Fibrosin bodies were present on both hosts, while chasmothecia were not observed in samples from either host. The internal transcribed spacer (ITS) region of rDNA of samples of mycelium and conidia collected from the two hosts was amplified using the primers ITS1-ITS4 (3) and directly sequenced (GenBank Accession Nos. JX179221 and JX179219 for E. susannae and E. inermis, respectively). By performing BLAST analysis, the 692-bp amplicon from E. susannae and the 541-bp amplicon from E. inermis showed 100% homology with several sequences of Podosphaera spp. On the basis of morphological characteristics of the imperfect state and the ITS analysis, the causal agent of powdery mildew on E. susannae and E. inermis was identified as Podosphaera sp. Pathogenicity was confirmed by gently brushing healthy 20-month-old potted plants of E. susannae and E. inermis with mycelia and conidia from diseased plants. Four plants/treatment/species were used. Non-inoculated plants belonging to the two species served as control. Plants were maintained in a glasshouse at temperatures ranging from 18 to 25°C and relative humidity from 65 to 80%. About 20 days after inoculation, typical symptoms of powdery mildew developed on inoculated plants. The fungus observed on inoculated plants was morphologically identical to the original isolate. Non-inoculated plants did not exhibit symptoms. The pathogenicity test was performed in duplicate. Several agents of powdery mildew have been reported on Euphorbia spp. Among others, Podosphaera euphorbiae was reported on numerous Euphorbiaceae (1), P. euphorbiae-hirtae was observed on E. hirta (1) and P. euphorbiae-helioscopiae on E. pekinensis (2). To our knowledge, this is the first report of Podosphaera sp. on E. susannae and E. inermis in Italy. This disease is not presently of economic importance. Specimens are available at the Agroinnova Collection. References: (1) U. Braun. A Monograph of the Erysiphales (Powdery Mildews). J. Cramer, Berlin-Stuttgard, German Democratic Republic, 1987. (2) S. Y. Liu. Plant Dis. 95:1314, 2011. (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 Fusarium oxysporum Causing Wilt on Iceland Poppy (Papaver nudicaule) in Italy.

During fall 2011, symptoms of a wilt disease were observed in a commercial nursery near Ventimiglia as well as in the Research Center of Floriculture of Sanremo (northern Italy) on plants of Papaver nudicaule (Iceland poppy) of a local unnamed cultivar. In the commercial nursery, 15 to 20% of plants were affected, while about 3% of plants were affected at the Research Center. Symptoms consisted of chlorosis, premature leaf drop, and foliar wilting, followed by the stem wilting, bending, and eventually rotting from the base. Brown discoloration was observed in the stem vascular tissue. Using Komada's Fusarium-selective agar medium (2), a fungus was consistently and readily isolated from symptomatic vascular tissue of plants collected from both sites. The isolates were purified and subcultured on potato dextrose agar (PDA), on which medium both isolates produced pale violet, abundant, aerial mycelium, felted in old cultures, with pale purple pigments in the agar medium. The isolate generated short monophialides with unicellular, ovoid-elliptical microconidia of 3.9 to 6.7 × 1.4 to 3.0 (average 5.4 × 2.3) µm. On carnation leaf agar (CLA) (1), isolates produced pale orange sporodochia with macroconidia that were 3-septate, slightly falcate with a foot-shaped basal cell and a short apical cell, and 26.0 to 43.5 × 3.1 to 4.4 (average 35.3 × 3.7) µm. Chlamydospores were abundant, terminal, and intercalary, rough walled, mostly singles but sometime in short chains or clusters, and 5.2 to 10.1 µm in diameter. Such characteristics are typical of Fusarium oxysporum (3). The internal transcribed spacer (ITS) region of rDNA was amplified from the isolates using the primers ITS1/ITS4 (4), and sequenced. BLASTn analysis of the 507-bp ITS sequence of one isolate from P. nudicaule collected from the commercial nursery (GenBank Accession No. JX103564) showed an E-value of 0.0 and 100% identity with the ITS sequence of F. oxysporum (HQ649820). To confirm pathogenicity of one of the Iceland poppy isolates, tests were conducted on 2-month-old plants of the same cultivar on which symptoms were first observed. Plants (n = 14) were inoculated by dipping roots in a 1 × 107 CFU/ml conidial suspension of the isolate of F. oxysporum prepared from 10-day-old cultures grown in potato dextrose broth (PDB) on a shaker (90 rpm) for 10 days at 22 ± 1°C (12-h fluorescent light, 12-h dark). Non-inoculated control plants (n = 14) were dipped in sterilized water. All the plants were transplanted into pots filled with steamed potting mix (sphagnum peat/perlite/pine bark/clay at 50:20:20:10), and maintained in a glasshouse at 24 to 28°C. Inoculated plants showed typical symptoms of Fusarium wilt after 10 days. The stems then wilted and plants died. Non-inoculated plants remained healthy. F. oxysporum was reisolated from inoculated plants but not from control plants. The pathogenicity test was conducted twice with the same results. Since Fusarium wilt has not previously been described on Iceland poppy at any location, this is first report of F. oxysporum on P. nudicaule in Italy and anywhere in the world. References: (1) N. L. Fisher et al. Phytopathology 72:151, 1982. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) J. F. Leslie and B.A. Summerell. The Fusarium Laboratory Manual, Blackwell Professional, IA, 2006. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Leaf Spot of Garden Lupin (Lupinus polyphyllus) Caused by Pleiochaeta setosa in Italy.

Lupinus polyphyllus Lindl., common name garden lupin, is used in commercial, private, and public landscapes and sold as a cut flower. During summer 2011, extensive brown necrotic areas were observed on young and old leaves of plants grown in a private garden near Biella (northern Italy). The disease affected about 50 of 80 2-year-old plants. Early symptoms included circular to irregular-shaped brown lesions of alternating pale and dark brown concentric bands. Lesions coalesced and often were surrounded by chlorotic halos at an advanced development stage. Lesion expansion was not limited by leaf veins. When lesions covered much of the leaf area, the leaf curled and remained attached. However, expansion of stem lesions often resulted in plant death. A fungus was consistently isolated from 15 infected leaves on potato dextrose agar (PDA). Cultures were grown at 21 to 25°C under 16 h of light and 8 h of darkness. Mature colonies were dark olive-green and produced orangeochre pigments in the medium. Ten isolates were obtained and three strains were used in the morphological study. The mycelium had olivaceous, septate hyphae that produced abundant dark, intercalary chlamydospores. The conidia were cylindrical to elliptical, slightly curved, with a truncated base, five to seven transverse septa and three hyaline appendages. Apical and basal cells were subhyaline, whereas the intermediate cells were olivebrown. The conidia measured 76 to 94 × 14 to 19 (average 85 × 16) µm. Appendages were up to 84 µm long. On the basis of its morphological characteristics the pathogen was identified as Pleiochaeta setosa Kirchn. DNA was extracted using Terra PCR Direct Polymerase Mix (Clontech). The internal transcribed spacer region of rDNA was amplified using primers ITS 1 and 4 (4) and sequenced. BLAST analysis (1) of the 570 bp fragment showed a 100% homology with a P. setosa isolate submitted to GenBank (accession no. EU167563). The nucleotide sequence was submitted to GenBank (JQ358708). Pathogenicity was verified on healthy 5-month-old garden lupin plants by placing 8-mm mycelial disks from 15-day-old cultures on 10 unwounded leaves per plant with five plants per treatment. Ten leaves of five plants were inoculated with PDA disks to serve as a negative control. Plants were covered with plastic bags for 4 days after inoculation and maintained in a growth chamber at 20 ± 1°C. Lesions developed on 80% of leaves 3 days after inoculation, whereas control plants remained healthy. P. setosa was consistently isolated from these lesions. The pathogenicity test was carried out twice. The presence of P. setosa on L. polyphyllus was reported in Australia, United States (2), and Poland (3). This is, to our knowledge, the first report of P. setosa in Italy. The impact of this disease is currently limited. References: (1) S. F. Altschul et al. Nucleic Acids Res., 25:3389, 1997. (2) A. M. French. California Plant Disease Host Index. Calif. Dept. Food Agric. Sacramento, 1989. (3) W. Mulenko et al. A Preliminary Checklist of Mycromycetes in Poland Polish Academy of Sciences, 1982. (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.

First Report of Fruit Rot in Pear Caused by Botryosphaeria dothidea in Italy.

Pear (Pyrus communis L.) is widely grown in Italy, the leading producer in Europe. In summer 2011, a previously unknown rot was observed on fruit of an old cultivar, Spadoncina, in a garden in Torino Province (northern Italy). The decayed area of the fruit was soft, dark brown, slightly sunken, circular, and surrounded by an irregular margin. The internal decayed area appeared rotten and brown and rotted fruit eventually fell. To isolate the causal agent, fruits were soaked in 1% NaOCl for 30 s and fragments (approximately 2 mm) were taken from the margin of the internal diseased tissues, cultured on potato dextrose agar (PDA), and incubated at temperatures between 20 and 28°C under alternating light and darkness. Colonies of the fungus initially appeared whitish, then turned dark gray. After about 30 days of growth, unicellular elliptical hyaline conidia were produced in pycnidia. Conidia measured 16 to 24 × 5 to 7 (average 20.1 × 5.7) µm (n = 50). The morphological characteristics are similar to those of the fungus Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not. (4). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 473-bp segment showed a 100% similarity with the sequence of the epitype of B. dothidea AY236949. The nucleotide sequence has been assigned the GenBank Accession No. JQ418493. Pathogenicity tests were performed by inoculating six pear fruits of the same cultivar (Spadoncina) after surface disinfesting in 1% sodium hypochlorite and wounding. Mycelial disks (8 mm diameter), obtained from 10-day-old PDA cultures of one strain, were placed on wounds. Six control fruits were inoculated with plain PDA. Fruits were incubated at 25 ± 1°C in plastic boxes. The first symptoms developed 3 days after inoculation. After 5 days, the rot was very evident and B. dothidea was consistently reisolated. Noninoculated fruits remained healthy. The pathogenicity test was performed twice. B. dothidea was identified on decayed pears in the United States (2), South Africa, New Zealand, Japan, and Taiwan (3). To our knowledge, this is the first report of the presence of B. dothidea on pear in Italy, as well as in Europe. In Italy, the economic importance of the disease on pear fruit is at present limited, although the pathogen could represent a risk for this crop. References: (1) S. F. Altschul et al. Nucleic Acids Res., 25:3389, 1997. (2) L. F. Grand. Agr. Res. Serv. Techn. Bull. 240:1, 1985. (3) Y. Ko et al. Plant Prot. Bull. (Taiwan) 35:211, 1993. (4) B. Slippers et al. Mycologia 96:83, 2004.