D. Bertetti

Effect of leaf wetness duration and temperature on infection of downy mildew (Peronospora sp.) of basil

The effect of leaf wetness duration and temperature on the development of downy mildew of basil, incited by Peronospora sp., was studied under controlled conditions. In several trials carried out under growth chamber and or glasshouse conditions, downy mildew was particularly severe when plants were kept wet for a period of at least 6-12 h immediately after inoculation. The pathogen was mostly active at a temperature of 20†C. At temperatures of 12†C and of 27†C no disease development was observed. A leaf wetness of at least 24 h after symptom appearance is necessary for sporulation. The results obtained are critically discussed also in relation to the possibility of managing this disease through proper cultural practices.ZusammenfassungDer Einfluss von Blattnässedauer und Temperatur auf die Entwicklung des Falschen Mehltaus an Basilikum (Peronospora sp.) wurde unter kontrollierten Bedingungen untersucht. Ein hoher Befall trat in Klimakammern und im Gewächshaus besonders nach Nässeperioden von mindestens 6-12 h nach der Inokulation auf. Das Pathogen zeigte die höchste Aktivität bei einer Temperatur von 20†C, während bei 12†C und of 27†C kein Befall beobachtet wurde. Eine Blattnässedauer von mindestens 24 h ist für die Sporulation erforderlich. Die Versuchsergebnisse werden auch im Hinblick auf eine Kontrolle der Krankheit duch geeignete Kulturmaßnahmen kriitisch diskutiert.

Fusarium wilt of gerbera in soil and soilless crops in Italy.

In 2002, gerbera (Gerbera jamesonii cv. Kaiki) plants that were grown for cut flowers in a soilless cultivation system (rockwool substrate) at Albenga (Savona) in northern Italy were observed exhibiting symptoms of a wilt disease. During the summer of 2002, in a commercial gerbera farm in the province of Imperia (northern Italy), a similar wilt was also observed on cvs. Red Bull, Anedin, and Gud finger that were grown in soil. In both cases, the planting material originated from the Netherlands. During 2003, wilted plants (cvs. Red Bull, Basic, and Cirill) were repeatedly observed in other commercial greenhouses located in the same area. Affected plants were stunted and developed yellowed leaves with initially brown and eventually black streaks in the vascular system. The vascular streaks in the yellow leaves were continuous with a brown discoloration in the vascular system of the crown and upper taproot. In some cases, the leaves of affected plants turned red. From these plants, Fusarium spp. were consistently and readily isolated from symptomatic vascular tissue onto a Fusarium-selective medium (2). Colonies were identified as F. oxysporum after subculturing on potato dextrose agar. Healthy rooted 30-day old plants (cv. Dino) were inoculated by dipping roots into a conidial suspension (5 × 107 conidia per ml) in one of six test isolates of F. oxysporum. Plants were transplanted (1 plant per pot) into pots (3.5 l vol) containing rockwool-based substrate. Noninoculated plants served as control treatments. Plants (21 per treatment) were grown in a glasshouse with an average day temperature of 31°C and night temperature of 25°C (minimum of 20°C and maximum of 42°C). Wilt symptoms and vascular discoloration in the roots, crown, and veins developed within 30 days on each inoculated plant, while noninoculated plants remained healthy. F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was conducted twice. To our knowledge, this is the first report of the presence of F. oxysporum on gerbera in Italy. A wilt of gerbera was described in the Netherlands in 1952 (1) but its presence was not confirmed in further observations (3). Reference: (1) J. Arx and J. A. von Tijdschr. PlZiekt. 58:5, 1952 (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) G. Scholten. Neth. J. Plant Pathol. 76:212, 1970.

FIRST REPORT OF A LEAF SPOT OF SALVIA LEUCANTHA CAUSED BY ALTERNARIA sp. IN ITALY

During summer-autumn 2014, extensive necroses were observed on Salvia leucantha plants growing in a private garden near Biella (northern Italy, ca 45°39’N 8°00’E). A high proportion (70%) of about 100 plants was diseased. Pale-brown, small, irregular lesions were present on the leaves, which were followed by extensive chlorosis and, eventually, leaf drop. A fungus was consistently isolated on potato dextrose agar (PDA) which on potato carrot agar (PCA), produced a greenish mycelium, with brown, septate, ovoid or obclavate conidia with both transverse and longitudinal septa and a prominent tapered beak, measuring 14.3- 42.3×5.4-12.6 μm (average: 22.8×9.1 μm). These morphometric traits were consistent with those of Alternaria sp. (Simmons, 2007). DNA was extracted using the Nucleospin plant kit (Macherey Nagel, Germany) and PCR carried out using ITS1/ITS4 primers. A 483 bp PCR product was amplified and sequenced (GenBank accession No KP280314) and a BLASTn search (Altschul et al., 1997) confirmed that the sequence corresponded to Alternaria sp. In pathogenicity tests, leaves of three healthy plants of S. leucantha were inoculated by spraying with a spore and mycelium suspension (1×105 CFU/ml) of the fungus grown on PDA. Plants inoculated only with sterile PDA fragments suspended in water served as control. Inoculated plants were covered with plastic bags and maintained at 18 to 25°C. At about six days post inoculation lesions developed only on inoculated leaves and Alternaria sp. was consistently reisolated. This is the first report of Alternaria sp. on S. leucantha in Italy as well as in the world.

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.

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 Komadas 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 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 powdery mildew caused by Golovinomyces cichoracearum on orange coneflower (Rudbeckia fulgida) in Italy.

Rudbeckia fulgida (orange coneflower), a flowering plant belonging to the Asteraceae, is increasingly used as a border in parks and gardens. In September 2007, severe outbreaks of a previously unknown powdery mildew were observed on plants in a public park in Torino (northern Italy). More than 90% of the plants were affected by the disease. Both surfaces of leaves of affected plants were covered with white mycelia and conidia. As the disease progressed, infected leaves turned yellow and wilted. Mycelia and conidia also were observed on stems and flower calyxes. Conidia were hyaline, ellipsoid, borne in chains (as many as three to four conidia per chain) and measured 34 × 23 (30 to 39 × 21 to 25) μm. Conidiophores measured 129 × 12 (89 to 181 × 11 to 13) μm and showed a foot cell measuring 88 × 12 (48 to 129 × 11 to 13) μm followed by two shorter cells. 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. BLASTn analysis (1) of the 619 bp showed a 100% homology with the sequence of Golovinomyces cichoracearum (3). The nucleotide sequence has been assigned GenBank Accession No. EU 233820. Pathogenicity was confirmed through inoculations by gently pressing diseased leaves onto leaves of healthy R. fulgida plants. Twenty plants were inoculated. Fifteen noninoculated plants served as the control. Plants were maintained in a greenhouse at temperatures ranging from 18 to 22°C. Eight 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. To our knowledge, this is the first report of powdery mildew on R. fulgida in Italy. Powdery mildew on Rudbeckia spp. was previously reported in the United States (4), Poland, and more recently, India and Switzerland. Particularly, in Switzerland the disease has been observed on R. laciniata and R. nitida (2). The economic importance of this disease is currently limited. 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) A. Bolay. Cryptogam. Helv. 20:1, 2005. (3) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (4) D. F. Farr et al. Page 82 in: Fungi on Plants and Plants Products in the United States. The American Phytopathological Society, St Paul, MN, 1989.

A NEW FORMA SPECIALIS OF FUSARIUM OXYSPORUM ON CRASSULA OVATA

Symptoms of wilting were observed in Crassula ovata cvs Mini and Magical tree in northern Italy during summer 2010. Fourteen fungal isolates recovered from diseased tissues were analyzed by ITS sequences and identified as Fusarium oxysporum. For pathogenicity assays, roots of both cultivars plants were dipped into a conidial suspension. Inoculated plants showed typical symptoms of Fusarium wilt, confirming the virulence of all isolates. For phylogenetic analysis three genomic regions [endopolygalacturonase gene (pg5), exopolygalacturonase gene (pgx1) and Mat alpha (Mat 1-1)] were amplified by PCR, sequences were aligned with those of other formae speciales of Fusarium oxysporum from GenBank and used for constructing phylogenetic trees. Regardless of the sequenced region Crassula ovata isolates grouped together in a cluster clearly separated from other known formae speciales. Recently, a wilt disease of Cactaceae caused by F. oxysporum f. sp. opuntiarum has been reported from Italy, whose presence among the C. ovata isolates was checked by sequencing the elongation factor EF1a. The occurrence of several gaps in the sequence alignment excluded the presence of F. oxysporum f. sp. opuntiarum. Based on the results of biological and molecular investigations, it seems plausible to conclude that the F. oxysporum infecting C. ovata plants represents a novel forma specialis, for which the name F. oxysporum f. sp. crassulae, f. sp. nov. is proposed.

First Report of a Leaf Spot Caused by Alternaria compacta on Hydrangea anomala subsp. petiolaris in Italy

Hydrangea anomala subsp. petiolaris (synonym H. petiolaris and H. scandens), also known as climbing hydrangea, is cultivated as an ornamental for landscaping in parks and gardens. This species, belonging to the Hydrangeaceae and native to the woodlands of Japan and coastal China, is widely appreciated for its abundant, creamy white flowers with a sweet aroma, particularly in shade gardens. During the summer of 2006, extensive necroses were observed on leaves and young stems of 3-year-old plants grown outdoors in several gardens of Piedmont of northern Italy. In many cases, on the upper side of the leaves, necrotic spots (4 to 10 mm in diameter) turned progressively black. Lesions often coalesced, generating larger (2 to 6 cm in diameter) necrotic areas. Necroses initially developed mainly at leaf margins and near petioles, and severely affected plants were defoliated. Infected plants rarely died, but the presence of lesions reduced the aesthetic quality and subsequently the commercial value. The disease occurred on 50 of 100 plants. A fungus was consistently isolated from infected leaves on potato dextrose agar (PDA) and identified on the basis of its morphological characteristics as an Alternaria sp. Conidia were dark gray, multicellular, clavate to pear shaped, measuring 23 to 54 × 10 to 13 μm (average 38 × 12 μm), with five longitudinal crosswalls and a relatively short apical beak. DNA was extracted with a Nucleospin Plant Kit (Macherey Nagel, Brockville, ON, Canada) and PCR was carried out with ITS 6/ITS 4 primer (2). A 557-bp PCR product was sequenced, and a BLASTn search (1) confirmed that the sequence corresponded to Alternaria compacta (99% homology). The nucleotide sequence has been assigned GenBank Accession No. EU 128529. Pathogenicity tests were performed by spraying leaves of healthy 1-year-old potted H. anomala plants with an aqueous 105 CFU/ml spore suspension. The inoculum was obtained from cultures of the fungus grown on sterilized host leaves placed on PDA for 20 days in light/dark at 23 ± 1°C. Plants sprayed only with water served as controls. Five plants were used for each treatment. Plants were covered with plastic bags for 3 days after inoculation and maintained between 12 and 22°C. Lesions developed on leaves 8 days after inoculation with the spore suspension, whereas control plants remained healthy. A. compacta was consistently reisolated from these lesions. The pathogenicity test was repeated twice. The presence of an Alternaria sp. on H. macrophylla was reported in the United States (3), whereas A. hortensiae was observed in Spain on H. hortensis. Recently, A. alternata belonging to the alternata group was reported on H. macrophylla in Italy (4). This is, to our knowledge, the first report of A. compacta on H anomala subsp. petiolaris in Italy. References: (1) S. F. Altschud et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (3) M. L. Daughtrey et al. Page 9 in: Compendium of Flowering Potted Plant Diseases. American Phytopathological Society. St. Paul, MN, 1995. (4) A. Garibaldi et al. Plant Dis. 91:767, 2007.

First report of bacterial leaf spot caused by Pseudomonas cichorii on Phlox paniculata in Italy.

Phlox paniculata L. (fall phlox) is a perennial garden species belonging to the Polemoniaceae family. During the spring of 2003 and 2004, leaf spot symptoms were observed on fall phlox plants in some private gardens in the Biella area (northern Italy). Lesions were first observed on leaves at the collar level and later on the entire plant. Lesions started as water-soaked areas, which in few days developed on the upper side of the leaves into irregular, shrunken, reddish brown spots from 1 to 2 mm in diameter. Lesions on the lower surface sometimes had a translucent halo. In many cases, the leaves were completely withered. Disease was particularly severe during the spring and fall and its incidence ranged from 10 to 25%. No fungal structures were observed within the lesions. Small fragments of tissue from affected leaves were macerated in nutrient yeast dextrose broth (NYDA), and dilutions of the resulting suspension were streaked onto NYDA and potato dextrose agar (PDA). Isolations were made from at least 25 leaves. Plates were maintained at 22 ± 1°C for 48 h. No fungi were isolated from the spots on NYDA or PDA. Colonies typical of Pseudomonas species were consistently isolated on NYDA. Isolates were negative for levan, potato soft-rot (pectolytic activity), and arginine dehydrolase while positive for oxidase and hypersensitivity on tobacco leaves. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein analysis (1) indicated that the bacterium isolated was similar to Pseudomonas cichorii (Swingle) Stapp NCPPB 943 and 3283 strains. On Kings medium B, (2) a typical fluorescent pigment was produced. The pathogen was identified as Pseudomonas cichoriii. Pathogenicity of 10 colonies was tested by growing inoculum in nutrient-broth shake cultures for 48 h, suspending bacterial cultures in water, diluting to 106 CFU/ml, and spraying 10 1-year-old healthy plants of P. paniculata. Ten control plants were sprayed with sterile nutrient broth. Inoculated and control plants were kept covered with plastic bags for 72 h. After 8 days in a growth chamber at 20 ± 1°C, leaf spots identical to those observed in the field developed on leaves of inoculated plants. Control plants remained symptomless. The pathogenicity test was repeated once. Bacteria were reisolated from the spots and identified as P. cichoriii. To our knowledge, this is the first record of bacterial leaf spot of Phlox paniculata in Italy as well as in the world. References: (1) D. H. Bergey et al. Bergeys Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (2) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.