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 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.

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.

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 Web Blight on Winter Savory (Satureja Montana "Repandens") Caused by Rhizoctonia solani AG-1-IA in Italy.

Satureja montana L. (winter savory "Repandens") is an evergreen shrub. In late summer 2010, blight was observed on a farm near Albenga (northern Italy) on 3% of 500 potted 2-month-old plants. Semicircular, water-soaked lesions appeared first on stems then on leaves. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage within 5 to 6 days. Stem fragments taken from the margin of the diseased tissues of 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 100 µg/liter streptomycin sulfate. A fungus with morphological characters of Rhizoctonia solani was consistently isolated. 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 tested strain. 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 winter savory 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 repeated once. Mycelium of 10-day-old isolates from winter savory appeared light brown, compact, and radiate. Numerous, dark brown sclerotia, 1 to 4 mm in diameter (average 1.7), developed within 20 days after transfer of mycelia to PDA in 90-mm-diameter petri dishes and incubated (11-h daylight, 13-h dark) at 21 to 24°C. Descriptions of mycelium and sclerotia were typical for subgroup IA Type 2 (3). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the 696 bp showed a 99% homology with the sequence of R. solani. The nucleotide sequence has been assigned GenBank No. JQ313811. For pathogenicity tests, inoculum of R. solani was prepared by growing the pathogen on wheat kernels autoclaved in 1-liter glass flasks (30 min at 121°C and 1 atm) for 15 days. One of the isolates assigned to the anastomosis group AG 1 IA was tested. Five 90-day-old plants of S. montana were inoculated. Each plant grown in 2-liter pots in a steam disinfested peat/pumice/pine bark/clay mix (50:20:20:20:10) was inoculated with 10 g of infested wheat kernels placed at the base of the stem. Five plants inoculated with noninfested wheat kernels served as the control. Plants covered with plastic bags were arranged randomly in a growth chamber at 20 ± 1°C with 12-h light/dark for 5 days. Symptoms, similar to those observed in the farm, developed 4 days after inoculation. Ten 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. Symptoms caused by R. solani have been recently observed on S. hortensis in Poland (4). This is, to our knowledge, the first report of blight of S. montana caused by R. solani in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. Carling. Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, The Netherlands, 1996. (3) R. T. Sherwood. Phytopathology 59:1924, 1969. (4) B. Zimowska. Herba Polonica 56:29, 2010.

First Report of Fusarium oxysporum Causing Wilt on Jade Plant (Crassula ovata) in Italy.

During summer 2010, symptoms of a wilt disease were observed in a commercial farm in northern Italy on Crassula ovata (jade plant). First symptoms consisted of chlorosis and premature drop of still turgid leaves. As the disease progressed, leaves turned yellow and wilted before dropping off and the stem wilted, bent, and eventually rotted starting from the base. In some cases, the stem broke or the basal portion of the leaf rotted. Brown discolorations were observed in the vascular system. Of 10,000 plants, 65% (cv. Mini) and 5% of 600 plants (cv. Magical Tree) were affected. Premature dropping of leaves was more frequent on cv. Magical Tree. Using the Komada's Fusarium-selective medium, a fungus was consistently and readily isolated from symptomatic vascular tissues of plants belonging to both cultivars. Isolates obtained from both cultivars were purified, subcultured on potato dextrose agar (PDA), and single-spore cultures were obtained. On PDA, both isolates produced pale violet, abundant, aerial mycelium, felted in old cultures, with purple pigments in the agar. The isolates were grown on Spezieller Nährstoffarmer agar for characterization of macroconidia and microconidia (1). Both isolates produced sparse, 3 to 5 septate, nearly straight macroconidia measuring 30 to 56 × 3 to 5 (average 40 × 4) µm with a short apical cell and a foot-shaped basal cell. Sporodochia were not observed. Unicellular, oval-elliptical microconidia measuring 5 to 13 × 3 to 4 (average 8 × 3) µm were produced on short monophialides. Chlamydospores were abundant, single and sometime in pairs, terminal and intercalary, rough walled, and measured 6 to 9 µm. Such characteristics are typical of Fusarium oxysporum (3). The ITS region (internal transcribed spacer) of rDNA was amplified with primers ITS1/ITS4 (4) and sequenced. BLASTn analysis of an isolate from C. ovata cv. Mini (515 bp, Accession No. HQ682196) and C. ovata cv. Magical Tree (509 bp, Accession No. HQ682197) showed an E-value of 0.0 with F. oxysporum. For these sequences, pairwise alignment of EMBOSS (E.B.I. - The European Bioinformatics Institute) revealed identity and similarity of 99.0%. To confirm pathogenicity, tests were conducted on 5-month-old plants of cvs. Mini and Magical Tree. Plants (three per treatment) were inoculated by dipping roots in a 1 × 106 CFU/ml conidial suspension of the two isolates of F. oxysporum prepared from 10-day-old cultures grown on casein liquid medium (2), shaken (90 rpm) for 10 days at 24°C ± 1 (12-h fluorescent light, 12-h dark). Inoculated plants were transplanted into pots filled with steamed mix (sphagnum peat/perlite/pine bark/clay; 50:20:20:10) and maintained in a plant growth chamber at 25 ± 1°C under a regimen of 12 h per day of fluorescent light. Inoculated plants belonging to both cultivars showed typical first symptoms of Fusarium wilt after 13 days. In the following days, leaves dropped, stems wilted, and plants died. Noninoculated plants remained healthy. F. oxysporum was reisolated from inoculated plants. The pathogenicity test was conducted twice. This is, to our knowledge, the first report of F. oxysporum on C. ovata in Italy or worldwide. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Professional, Ames, IA, 2006. (2) A. Minuto et al. Phytoparasitica 36:294, 2008. (3) B. A. Summerell et al. Plant Dis. 87:117, 2003. (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 Basal Stem Rot of Apple Cactus (Cereus peruvianus monstruosus) Caused by Fusarium oxysporum in Italy.

During the summer of 2010, 20% of 7,000 4-month-old plants of apple cactus (Cereus peruvianus monstruosus) showed symptoms of a basal stem rot in a commercial nursery located in Liguria (northern Italy). Affected plants showed yellow orange-to-pale brown color from the crown level to the stem apex and a water-soaked rot was observed on the stem starting from the base. Brown discoloration was observed in the vascular system. Eventually stems bent, plants collapsed and died, and affected tissues dried out. A Fusarium sp. was consistently and readily isolated from symptomatic tissue on Komada selective medium. Isolates were purified and subcultured on potato dextrose agar (PDA). Single-spore cultures on PDA, Spezieller Nährstoffarmer agar (SNA) (3), and carnation leaf-piece agar (CLA) (2) were incubated at 26 ± 1°C (12-h fluorescent light, 12-h dark). On PDA, cultures produced a thick growth of white-to-pink mycelium and pale pink pigments in the agar. On SNA, cultures produced short monophialides with unicellular, ovoid-elliptical microconidia measuring 4.3 to 8.2 × 2.3 to 3.8 (average 6.0 × 2.8) µm. Chlamydospores were abundant, single or paired, terminal and intercalary, rough walled, and 6 to 8 µm in diameter. On CLA, cultures produced orange sporodochia with macroconidia that were 3 to 4 septate, nearly straight with a foot-shaped basal cell and a short apical cell, and measured 31.1 to 51.5 × 4.4 to 3.5 (average 43.2 × 3.8) µm. Such characteristics are typical of Fusarium oxysporum (3). Amplification of the ITS (internal transcribed spacer) of the rDNA using primers ITS1/ITS4 (4) yielded a 498-bp band. Sequencing and BLASTn analysis of this band showed an E-value of 0.0 with F. oxysporum. The nucleotide sequence has been assigned GenBank Accession No. JF422071. To confirm pathogenicity, five 6-month-old healthy plants of C. peruvianus monstruosus were inoculated by dipping roots in a conidial suspension (2.4 × 106 CFU/ml) of F. oxysporum isolated from affected plants. Inoculum was obtained from pure cultures of three single-spore isolates grown for 10 days on casein hydrolysate liquid medium. Roots were not wounded before the inoculation. Plants were transplanted into pots filled with steam-sterilized substrate (sphagnum peat/perlite/pine bark/clay 50:20:20:10). Five noninoculated plants served as a control. Plants were placed in a climatic chamber at 25 ± 1°C (12-h fluorescent light, 12 h-dark). Basal stem rot and vascular discoloration in the crown and stem developed within 30 days on each inoculated plant. Noninoculated plants remained healthy. F. oxysporum was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. F. oxysporum has been reported on Cereus spp. in the United States (1). To our knowledge, this is the first report of F. oxysporum on C. peruvianus monstruosus in Italy as well as in Europe. Currently, this disease is present in a few nurseries in Liguria. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (2) N. L. Fisher et al. Phytopathology 72:151, 1982. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, 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, 1990.

Powdery Mildew Caused by Golovinomyces biocellatus on Spearmint (Mentha spicata) in Italy.

Spearmint (Mentha spicata) is grown for its aromatic and carminative oil and as an ornamental. During the fall of 2009, 4-month-old plants grown on a commercial farm located near Albenga (northern Italy) showed signs and symptoms of an unknown powdery mildew. 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 and wilted. Mycelia were also observed on stems. Conidia were hyaline, elliptical to doliform, borne in short chains (two to three conidia per chain), and measured 35 × 21 µm (30 to 43 × 18 to 26 µm). Conidiophores measured 86 × 11 µm (76 to 97 × 9 to 13 µm) followed by one to three shorter cells, measuring, respectively, 22 × 11 µm (13 to 28 × 9 to 15 µm). Fibrosin bodies were absent. Chasmothecia were not observed in the collected samples. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced (1) (GenBank Accession No. HM053470). The 567-bp amplicon had 100% homology with the sequence of Golovinomyces biocellatus (GenBank Accession No. AB307675). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of healthy M. spicata plants. Three plants were inoculated, while the same number of noninoculated plants served as a control. Plants were maintained at temperatures from 18 to 25°C. Twelve 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 has been reported on Mentha spp. in Australia (3) and the United States (4) and the pathogen is present on other plant genera in Italy. A similar powdery mildew of M. spicata was attributed to Erysiphe orontii in the United States (2). The economic importance of this disease is currently limited in Italy because of the limited planting of this host, but potted aromatic plants represent a steadily increasing crop in Italy. Voucher specimens are available at the Agroinnova Collection, University of Torino. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) S. T. Koike and G. S. Saenz. Plant Dis. 83:399, 1999. (3) J. R. Liberato and J. H. Cunnington. Australas. Plant Dis. Notes 2:83, 2007. (4) D. B. Marcum et al. Plant Dis. 94:276, 2010.

First Report of Botrytis Blight Caused by Botrytis cinerea on Lavandula stoechas in Italy.

Lavandula stoechas or French lavender (Labiatae) is a perennial shrub that produces pinkish purple flowers and is endemic to the Mediterranean Region. In the Albenga area (northern Italy), this species is grown as a potted plant. In October 2008, symptoms of a previously unknown blight were observed in a commercial glasshouse in the Savona Province (northern Italy) on 10% of 3-month-old 'Sugarberry Ruffles' potted plants. Glasshouse temperatures ranged between 11 and 32°C (average of 21°C) and plants were overhead irrigated. Initially, leaves and stems appeared chlorotic. Subsequently, necrotic lesions developed on infected tissues. After 10 days, fluffy, gray mycelium became apparent on symptomatic tissue, especially on the basal parts of the plants. Severely infected plants eventually died. Tissues were excised from diseased leaves, immersed in an aqueous solution of 1% sodium hypochlorite for 10 s, and then cultured on potato dextrose agar (PDA). A fungus developed abundant mycelium when incubated under constant fluorescent light at 22 ± 1°C. Numerous small sclerotia developed on PDA plates incubated for 20 days at 8 ± 1°C. Sclerotia were dark and irregular and measured 2 to 5 × 1 to 2 mm. Conidia were smooth, gray, unicellular, ovoid, measured 9.4 to 13.6 × 6.2 to 7.9 (average 11.4 × 7.2) µm, and similar to those described for Botrytis cinerea (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 573-bp segment was 100% similar to the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). The nucleotide sequence has been assigned GenBank Accession No. GQ375747. Pathogenicity tests were performed by spraying leaves of 9-month-old healthy potted L. stoechas 'Blue Star', 'Madrid Blue', 'Madrid Purple', and 'Madrid White' plants with a 7.5 × 104 conidia/ml spore suspension obtained from 7-day-old PDA cultures. Each plant received 5 ml of inoculum. Plants sprayed with water only served as controls. Four plants per treatment and per cultivar were used. Plants were covered with plastic bags for 4 days after inoculation and maintained in a growth chamber at 20 ± 1°C. The first lesions developed on flowers 5 days after inoculation, and 2 days later, lesions developed on leaves and stems. Lesions were similar to those observed in the commercial glasshouse. Control plants remained healthy. B. cinerea was consistently reisolated from these lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of B. cinerea on L. stoechas in Italy as well as worldwide. Botrytis blight previously has been described on L. angustifolia in Japan (4) and Poland (3). In Italy, the economic importance of the disease is currently still limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England, 1971. (3) L. B. Orlikowski and A. Valjiuskaite. Acta Mycol, 42:193, 2007. (4) J. Takeuch and H. Horie. Annu. Rep. Kanto-Tosan Plant Prot. Soc. 53:87, 2006.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Figmarigold in Italy.

Lampranthus spp. N.B. Brown (figmarigold) belongs to the Aizoaceae family and is grown as a ground cover in gardens or as a potted plant. In January 2009, severe outbreaks of a previously unknown wilt were observed at a commercial farm in Liguria (northern Italy) where 7-month-old potted plants were grown outdoors in a mix of peat/clay/pumice at pH 6.5. In cultivars with pink flowers, 12% of plants were affected, while only a few cultivars with red flowers were diseased. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. Subsequently, infected tissues wilted and stopped growing, stems and leaves appeared desiccated, and infected plants died. Stems of 10 pink-flowered plants were severed with a knife, cut ends sealed with wax, and surfaces disinfected with 1% sodium hypochlorite. Cross-sections (1 mm long) through symptomatic vascular tissue were plated onto potato dextrose agar. After 10 days at 22°C, 90% of the stems tested positive for Verticillium. Irregular, dark microsclerotia, 22 to 128 × 13 to 66 (average 51 × 29) µm, developed in hyaline hyphae after 10 days of growth at 22 ± 1°C (12-h photoperiod). Hyaline, elliptical, single-celled conidia, 2.9 to 4.8 × 1.3 to 2.4 (average 4.1 × 1.7) µm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as V. dahliae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 (2) and sequenced. BLASTn analysis (1) of the 476-bp segment showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. GQ 149479. Pathogenicity tests were performed twice using five 40-day-old plants of a pink-flower cultivar of a Lampranthus sp. grown in 1-liter pots containing a 50:20:20:10 mix of peat moss/pumice/pine bark cortex/clay. The substrate was infested with a conidial suspension (1.0 × 107/ml) of one isolate of V. dahliae recovered from infected plants. Inoculum (50 ml) were added to each pot, drenching the top of the soil. Noninoculated plants served as controls. Plants (five per treatment) were maintained in a glasshouse at daily average temperatures between 20 and 26°C and at 50 to 70% relative humidity. The first wilt symptoms and a vascular discoloration in the roots, crown, and veins developed 30 days after inoculation. V. dahliae was consistently reisolated. Noninoculated plants remained healthy. In a second test, the susceptibility of purple-, white-, yellow-, red-, and orange-flowered cultivars was tested. Ten rooted cuttings of each cultivar were inoculated as described above. The severity of Verticillium wilt was evaluated and each cultivar was classified as resistant, partially resistant, average susceptible, susceptible, or highly susceptible. All tested cultivars were susceptible or highly susceptible to Verticillium. Only the purple cultivar showed an average susceptibility. To our knowledge, this is the first report of Verticillium wilt on Lampranthus spp. in Italy as well as worldwide. Today, the economic importance of Verticillium wilt on figmarigold in Italy is still limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. A. Innis et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

First Report of Sclerotinia Blight Caused by Sclerotinia sclerotiorum on Petunia × hybrida in Italy.

Petunia × hybrida (Solanaceae) includes several hybrids that are grown as ornamental plants and are very much appreciated for their long-lasting flowering period. During the spring of 2009, extensive wilting was observed on 2-month-old potted plants of Petunia × hybrida cv. Sanguna Lilac Vein grown in a commercial glasshouse near Albenga (northern Italy). First symptoms included stem necrosis and darkening and withering of leaves. Wilt occurred a few days after the appearance of the first symptoms. Infected plants were characterized by the presence of soft, watery tissues. In the presence of high relative humidity, lesions became covered with a whitish mycelium and irregular, dark gray sclerotia (3.0 to 6.5 × 2.0 to 5.0 mm, average 4.8 × 3.3 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 of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary (3) was consistently recovered from infected stem pieces. Sclerotia produced on PDA measured 2.0 to 6.0 × 1.5 to 5.0 mm (average 3.9 × 3.1) mm. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 548-bp segment showed a 100% homology with the sequence of S. sclerotiorum. The nucleotide sequence has been assigned GenBank Accession No. GQ375746. Pathogenicity of one isolate obtained from sclerotia of infected plants was confirmed by inoculating three 90-day-old plants belonging to cv. Sanguna Lilac Vein transplanted in 22-cm-diameter pots in a glasshouse in a sphagnum peat/pomix/pine bark/clay (50:20:20:10) mix. Inoculum that consisted of 2 g/liter of substrate of wheat kernels infested with mycelium and sclerotia was placed in the soil and around the base of each plant. Three noninoculated plants served as controls. Plants were maintained in greenhouse at temperatures ranging between 22 and 26°C and relative humidity >90%. The inoculation trial was carried out twice. All inoculated plants developed leaf yellowing by 20 days after soil infestation. 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 Petunia × hybrida in Italy. The disease has been previously reported on Petunia × hybrida in Bermuda (2) and the United States (4). References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) R. M. Brien. N.Z. J. Sci. Tech., A, 24, 62, 1942. (3) N. F. Buchwald. Kongl. Veterisk Landb. Aarssk. 75, 1949. (4) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.

First Report of Gray Mold Caused by Botrytis cinerea on Stevia rebaudiana in Italy.

Stevia rebaudiana (sweetleaf) is a perennial shrub belonging to the Asteraceae family and is widely grown for its sweet leaves. With its extracts having as much as 300 times the sweetness of sugar, this species is used in many countries for the production of sugar substitutes. However, in Italy, as well as in other countries, this species cannot be grown for the use of its leaf extracts. This plant is grown in a few nurseries in the Albenga Region (northern Italy) as potted plants. In February of 2008, 3-month-old plants grown in plastic pots (14-cm diameter) under glasshouse on heated benches started showing symptoms of a previously unknown blight. The temperature in the glasshouse ranged between 16 and 20°C and plants were watered by sprinkle irrigation. Leaves, starting from the basal ones, showed small, brown spots that spread across the entire leaf surface. Subsequently, the crown and stem were infected, and the pathogen developed abundant, soft, gray mycelium on leaves and stems and in the middle of the heads of S. rebaudiana. Flowers were not present when the symptoms appeared. Severely infected leaves dried out and became necrotic. The disease was observed in one nursery in which 5% of the plants were affected. The margins of the lesions were excised from leaves, immersed in a solution containing 1% sodium hypochlorite, and then cultured on potato dextrose agar (PDA) medium. A fungus produced abundant mycelium when incubated under constant fluorescent light at 22 ± 1°C after 10 days. The conidia were smooth, hyaline, ovoid, measuring 15.5 to 8.3 × 11.1 to 7.3 (average 11.6 × 8.6) µm, and were similar to those described for Botrytis cinerea. Conidiophores were slender and branched with enlarged apical cells bearing conidia on short sterigmata. The identity of the fungus was also confirmed by the production of numerous, small, black sclerotia on PDA plates incubated for 20 days at 8 ± 1°C. Sclerotia were dark and irregular with a diameter ranging from 1 to 2 mm. These morphological characters identified the fungus as B. cinerea (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 780-bp segment showed a 100% homology with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). The nucleotide sequence has been assigned GenBank Accession No. FJ486270. Pathogenicity tests were performed by spraying leaves of six healthy 6-month-old potted S. rebaudiana plants with a 105 conidia/ml suspension. Six plants sprayed with water only served as controls. Plants were covered with plastic bags for 3 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 20 ± 1°C. The first foliar lesions developed on leaves 4 days after inoculation, whereas control plants remained healthy. B. cinerea was consistently reisolated from these lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of B. cinerea on S. rebaudiana in Italy. The disease has been reported in Ukraine (3) and more recently in Japan (4). The economic importance of this disease is at the moment limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. Burgess Publishing Company, Minneapolis, MN, 1972. (3) J. Takeuch and H. Horie. Annu. Rep. Kanto-Tosan Plant Prot. Soc. 53:87, 2006. (4) V. F. Zubenko et al. Zash. Rast. 18, 1991.

First Report of Botrytis Blight Caused by Botrytis cinerea on Gaura lindheimeri in Italy.

Gaura lindheimeri (wand flower) is a perennial plant belonging to the Onagraceae family that is used for perennial borders in xeric and mesic landscapes. It produces flowers floating above the plant like small, dancing butterflies. This plant is becoming popular in the Albenga Region (northern Italy) where white and rose varieties are grown as potted plants. In January of 2008, 5-month-old 'Whirling Butterflies' plants grown in plastic pots (14 cm in diameter) in the open field started showing symptoms of a previously unknown blight. When the disease developed, temperatures ranged between 3 and 17°C (average 9°C) and average relative humidity was 64%. Small, brown spots appeared on the basal portion of leaves first, eventually spreading to cover entire leaves. Subsequently, the pathogen developed abundant, soft gray mycelium on affected leaf tissue. Severely infected leaves eventually became completely rotten and desiccated. Sixty percent of plants were affected by the disease. Tissues were excised from diseased leaves, immersed in a solution containing 1% sodium hypochlorite for 10 s, and then cultured on potato dextrose agar (PDA) medium. The fungus produced abundant mycelium on PDA medium when incubated under constant fluorescent light at 22 ± 1°C. The conidia were smooth, hyaline, globoid, measuring 11.8 to 9.4 × 8.3 to 6.6 (average 10.7 × 7.4) µm, and are similar to those described for Botrytis cinerea. The identity of the pathogen was also confirmed by the production of numerous sclerotia on PDA plates incubated for 20 days at 8 ± 1°C. Sclerotia were dark, irregular, and measured 3 to 4 × 2 to 3 mm. The fungus was identified as B. cinerea on the basis of these characters (1). Pathogenicity tests were performed by spraying leaves of healthy, potted 8-month-old G. lindheimeri 'Whirling Butterflies' plants with a 105 conidia/ml suspension. Plants sprayed with water only served as controls. Five plants per treatment were used. Plants were covered with plastic bags for 6 days after inoculation and maintained in a growth chamber at 20 ± 1°C. The first foliar lesions developed on leaves 5 days after inoculation, whereas control plants remained healthy. B. cinerea was consistently reisolated from these lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of B. cinerea on G. lindheimeri in Italy. The economic importance of this disease will increase with the increased cultivation of this species. Reference: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. Burgess Publishing Company, Minneapolis, MN, 1972.

First Report of Collar and Stem Rot Caused by Pythium aphanidermatum on Figmarigold (Lampranthus sp.) in Italy.

Lampranthus sp. N.B. Brown (figmarigold) of the Aizoaceae family is used as groundcover in gardens. In October of 2008, severe outbreaks of a previously unknown rot were observed in a nursery located in Liguria, near Savona (northern Italy), on 35-day-old rooted cuttings grown in a peat substrate. Approximately 50% of rooted cuttings of red-flowered cultivars were affected. Lesions on collars and young stems were brown, water soaked, and soft. Plants eventually collapsed as roots rotted. Thin, aerial hyphae were visible on the surface of the stems and substrate. Later, a thick, light yellow, mycelial mat surrounded infected plants. Tissue fragments were excised from the margins of the lesions, dipped in a solution containing 1% sodium hypochlorite, and plated on potato dextrose agar and a medium selective for Oomycetes (4). Plates were incubated under constant fluorescent light at 23 ± 1°C for 4 to 5 days. Hyphae of five isolates grown on V8 medium were aseptate and 4.2 to 7.9 (average 6.2) µm wide. Sporangia consisted of complexes of swollen hyphal branches. Oogonia were globose, smooth, and 23.5 to 28.0 (average 25.9) µm in diameter. Antheridia were barrel shaped, intercalary, and diclinous. Oospores were globose and 19.4 to 23.6 (average 21.4) µm in diameter. The internal transcribed spacer (ITS) region of rDNA of a single isolate (DB24112008) was amplified with primers ITS4/ITS6 and sequenced. A BLAST analysis (1) in GenBank of the 1,074-bp segment showed a 100% homology with the sequence of Pythium aphanidermatum (Accession No. EU245039). The nucleotide sequence has been assigned the GenBank Accession No. FJ492745. Pathogenicity tests were performed twice on a red-flower cultivar of a Lampranthus sp. grown in 1-liter pots containing a peat moss substrate infested with wheat and hemp kernels colonized with one isolate of P. aphanidermatum at a rate of 20 g/liter. Ten plants were grown in infested media and 10 plants were grown in noninfested media. Greenhouse temperatures were 18 to 24°C. The first symptoms of stem and root rot developed 15 days later, while control plants remained healthy. P. aphanidermatum was consistently reisolated from the lesions. To our knowledge, this is the first report of P. aphanidermatum on a Lampranthus sp. in Italy. The disease has been reported in Japan (3) in 2008, while in the United States, a Pythium sp. was reported on L. aureus and L. glomeratus (2). Currently, the economic importance of Pythium rot on figmarigold in Italy is still limited. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (3) H. Kawarazachi et al. J. Gen. Plant Pathol. 74:94, 2008. (4) H. Masago et al. Phytopathology, 67, 425, 1977.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Borago officinalis in Italy.

Borago officinalis L. (common borage) is a flowering plant belonging to the Boraginaceae with several culinary and medicinal uses and grown in soil or as potted plants in Liguria (northern Italy). At the end of winter in 2008, extensive chlorosis was observed on potted plants of B. officinalis grown in containers on a farm near Albenga (northern Italy). Initial symptoms included stem necrosis at the soil level and darkening of leaves. Disease was characterized by the presence of soft, watery tissues, particularly on affected leaves. As stem and foliar necrosis progressed, infected plants wilted and died. Wilt occurred within a few days on young plants. Symptomatic plants were found on 10 farms: average disease incidence in some nurseries reached 20%. Necrotic tissues became covered with a whitish mycelium that produced dark sclerotia. The diseased stem tissue was surface disinfected for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 100 ppm of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary (2) was consistently recovered from infected stem pieces. Sclerotia observed on infected plants at the soil level measured 2 to 9 × 1.5 to 7 (average 6 × 3) mm. Sclerotia produced on PDA measured 3 to 7 × 2 to 4 (average 4.4 × 3.1) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 (3) and then sequenced. BLAST analysis of the 521-bp segment showed a 100% homology with the sequence of S. sclerotiorum. The nucleotide sequence has been assigned GenBank Accession No. EU627005. Pathogenicity of four isolates obtained from infected plants and used in mixture was confirmed by inoculating 10 30-day-old plants grown in 14-cm-diameter pots that were maintained in the greenhouse. Inoculum, consisting of 1-cm2 mycelial plugs excised from a 10-day-old PDA culture of each isolate, was placed on the soil surface around the base of each plant. Ten noninoculated plants served as a control. Plants were covered with a plastic bag for 4 days after inoculation. The inoculation trial was repeated once. All plants were kept at an average temperature of 20°C and watered as needed. All inoculated plants developed symptoms of leaf yellowing within 4 days, followed by the appearance of white mycelium and sclerotia and eventual wilt. Control plants remained symptomless. S. sclerotiorum was reisolated from the stems of inoculated plants. To our knowledge, this is the first report of white mold on B. officinalis in Italy as well as in Europe. The disease was recently reported in North Dakota (1). The economic importance of this disease is currently low; however, the request of borage for culinary uses is increasing. References: (1) C. A. Bradley et al. Plant Dis. 89:208, 2005. (2) N. F. Buchwald. Den. Kgl. Veterin.er-og Landbohojskoles Aarsskrift 5:1949. (3) D. E. I. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997.