First Report of Phytophthora palmivora on Grevillea spp. in Italy.

The genus Grevillea (family Proteaceae) comprises over 300 species and is a popular and widely cultivated group of Australian plants. In the last 3 years, numerous potted grevilleas with symptoms of decline associated with a rot of feeder roots were found in ornamental nurseries in Sicily. Aboveground symptoms were reduced growth, yellowing of foliage, wilt, dieback, and death of the entire plant. The disease was observed on many commercial cultivars and was especially severe on G. alpina (mountain grevillea), G. juniperina (juniper-leaf grevillea), G. lavandulacea (lavender grevillea), and G. rosmarinifolia (rosemary grevillea) as well as the hybrid cultivars Clearview David (G. lavandulacea × rosmarinifolia) and Poorinda Rondeau (G. baueri × lavandulacea), while G. lanigera (woolly grevillea) cv. Mount Tamboritha and G. thelemanniana subsp. obtusifolia appeared resistant. A species of Phytophthora was consistently isolated from rotted roots of symptomatic plants using a selective medium (4), and pure cultures were obtained by single-hypha transfers. The species was identified as P. palmivora (E.I. Butler) E.I. Butler on the basis of morphological and cultural characters. On solid media, all isolates produced elliptical to ovoid, papillate sporangia with a mean length/width ratio of 1.8. Sporangia were caducous with a short pedicel (5 µm) and a conspicuous basal plug. All isolates were heterothallic (mating type A1) and produced oogonia and oospores only when paired with A2 mating type reference isolates of P. nicotianae and P. palmivora. Antheridia were amphyginous. Identification was confirmed by electrophoresis of mycelial proteins in polyacrylamide slab gels (1). The electrophoretic patterns of total soluble proteins and six isozymes (alkaline phosphatase, esterase, fumarase, NAD-glucose dehydrogenase, malate dehydrogenase, and superoxide dismutase) of isolates from grevillea were identical to those of a reference isolate of P. palmivora from Coronilla valentina subsp. glauca (2) but distinct from those of reference strains of eight other papillate species of Phytophthora included in Waterhouse's taxonomic group VI. Koch's postulates were fulfilled using 6-month-old rosemary grevillea plants that were transplanted into pots filled with soil that was artificially infested with chlamydospores (50 per gram of soil) produced in submerged cultures (3) by grevillea isolate IMI 390579. Plants were maintained in a glasshouse at 20 to 28°C and watered to field capacity once a week. One month after transplanting, infected plants showed decline symptoms similar to those of naturally infected plants. Control plants grown in pots containing noninfested soil remained healthy. P. palmivora was reisolated from roots of symptomatic plants. It appears that P. palmivora has become a widespread root pathogen in commercial ornamental nurseries in Italy (2). References: (1) S. O. Cacciola et al. EPPO Bull. 20:47, 1990.D. (2) S. O. Cacciola et al. Plant Dis. 86:327, 2002. (3) J. Y. Kadooka and W. H. Ko. Phytopathology 63:559, 1973. (4) H. Masago et al. Phytopathology 67:425, 1977.

First Report of Septoria Spot on Bergamot.

More than 95% of bergamot (Citrus bergamia Risso & Poit) essence production utilized in the international perfume industry comes from the coastal growing area in the Province of Reggio di Calabria in the Calabria Region of southern Italy. Fruit and leaf spots were observed on bergamot cv. Fantastico in some orchards during February and March 2001. Symptoms affected ≈20% of the fruits and resembled those caused by Septoria citri Pass. on lemon in the nearby Sicily Region (2). Symptoms on fruits were characterized by reddish-brown pits, 1 to 2 mm in diameter, often in proximity, and extending no deeper than the flavedo; or characterized by larger confluent brown spots, sunken and extending into the albedo. Leaf symptoms occurred on both sides of the blade and were characterized by irregular brown spots surrounded by a yellow halo. Spherical, dark-walled pycnidia were observed on brown spots on fruits. Pycnidia contained hyaline, nonseptate, or 1 to 3 septate, cylindrical conidia, rounded at the apex, measuring 8 to 18 × 1.5 to 2.0 µm (8 to 29 × 1.5 to 2.0 µm in pure culture), differing from the tapered conidia of S. citri. The fungus was subsequently identified as S. limonum Pass., a species first described in the 19th century on lemon in unheated glasshouses in northern Italy and later reported from other countries (3). It is still an open question whether S. limonum is distinct from S. citri. Previously, in fact, different species of Septoria from citrus have been considered synonyms of S. citri on the basis of isozyme electrophoretic phenotype (1). The fungus was isolated on artificial media from infected leaf and fruit tissues (pits and larger spots). Brown spot symptoms were reproduced by artificial inoculation of detached bergamot fruits. A spore suspension (1 × 106 spores per ml) of the fungus was sprayed on fruit wounded by a needle (1 mm in diameter) to a depth of 2 mm and washed in sterile water. After inoculation, the fruits were incubated 10 days at 22°C and 100% relative humidity. The fungus was reisolated from inoculated tissues. The damage caused by this disease appears to be more important on bergamot than on other citrus fruits since it affects oil-bearing tissue and consequently the production of essential oil. References: (1) M. R. Bonde et al. Phytopathology 81:517, 1991. (2) S. Grasso and R. La Rosa. Riv. Patol. Veg. 19:15, 1983. (3) L. J. Klotz. Color Handbook of Citrus Diseases, 4th ed. University of California, Division of Agricultural Sciences, Berkeley, 1973.

Collar and Root Rot of Olive Trees Caused by Phytophthora megasperma in Sicily.

Olive (Olea europea L.) is grown on about 154,000 ha in Sicily (southern Italy). In the summer of 1999, a few 3-year-old olive trees with decline symptoms were observed in a recently planted commercial orchard in the Enna province (Sicily). The trees were propagated on wild olive (O. europea L. var. sylvestris Brot.) rootstock. Aerial symptoms, consisting of leaf chlorosis, wilting, defoliation, and twig dieback followed in most cases by plant death, were associated with root rot and basal stem cankers. A Phytophthora sp. was consistently isolated from rotted rootlets and trunk cankers using the BNPRAH (benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol) selective medium. Pure cultures were obtained by single-hypha transfers. The species isolated from symptomatic olive trees was identified as P. megasperma Drechsler on the basis of morphological and cultural characteristics. All isolates were homothallic, with paragynous antheridia. The diameter of oospores varied from 28 to 42 µm (mean ± SE = 36.3 ± 0.4) when they were produced on potato-dextrose agar (PDA) and from 30 to 43 µm (mean ± SE = 37.8 ± 0.4) when they were produced in saline solution. Sporangia were non-papillate. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was about 6 mm per day. The identification was confirmed by the electrophoresis of mycelial proteins on a polyacrylamide slab gel. The electrophoretic banding patterns of total soluble proteins and three isozymes (esterase, fumarase, and malate dehydrogenase) of the isolate from olive were identical to those of two isolates of P. megasperma obtained from cherry and from carrot in Italy and characterized previously (1). Conversely, they were clearly distinct from the electrophoretic patterns of four isolates of P. megasperma var. sojae Hildebr. from soybean (= P. sojae Kauf. & Ger.), from those of three isolates from asparagus tentatively identified as P. megasperma sensu lato (1) and from those of reference isolates of various species producing non-papillate sporangia, including P. cambivora (Petri) Buisman, P. cinnamomi Rands, P. cryptogea Pethybr. & Laff., P. drechsleri Tucker, and P. erythroseptica Pethybr. Pathogenicity of the isolate from olive was tested in the greenhouse at 18 to 25°C using 18-month-old rooted cuttings of olive cv. Biancolilla. Cuttings were inoculated on the lower stem by inserting a 3-mm plug taken from actively growing colonies on PDA into an incision made with a sterile scalpel. The wound was sealed with waterproof tape. Agar plugs with no mycelium were placed into the stem of cuttings used as a control. The bark was stripped and lesion areas were traced and measured 60 days after inoculation. The isolate from olive produced a brown necrotic lesion (mean size = 500 mm2) around the inoculation wound and was reisolated from the lesion. Conversely, the wound healed up on control plants. P. megasperma has previously been recognized as a pathogen of olive in Greece and Spain (3). However, this is the first report of P. megasperma causing root and collar rot of olive in Italy. References: (1) S. O. Cacciola et al. Inf. Fitopatol. 46:33, 1996. (2) D. C. Erwin and O. K. Ribeiro, 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. (3) M. E. Sánchez-Hernádez et al. Plant Dis. 81:1216, 1997.

First Report of Phytophthora palmivora as a Pathogen of Olive in Italy.

Olive (Olea europea L.) is an economically important crop in Italy and is planted on about 1 million ha. The Apulia, Calabria, and Sicily regions of Southern Italy account for about 70% of the production. Many new plantations have been established during the last 10 years. In summer 1999, 1- to 2-year-old olive trees (cv. Carolea) with decline symptoms were observed in new plantations in Catanzaro Province (Calabria). The symptoms associated with the root rot were leaf chlorosis, defoliation, wilting, twig dieback, and eventual plant collapse. In some cases, more than 40% of the trees were affected. A Phytophthora sp. was isolated consistently from rotted rootlets of diseased trees using a selective medium (2). Single-zoospore isolates were obtained from the colonies. The species isolated from olive roots was identified as P. palmivora (E. Butler) E. Butler on the basis of morphological and cultural characters according to Erwin and Ribeiro (1). All isolates produced papillate sporangia, which were elliptical to ovoid, caducous (mean pedicel length = 5 µm), with a length-breadth ratio of 1.8. In addition, some isolates produced subglobose, non-papillate sporangia with a length-breadth ratio ranging from 1.2 to 1.5. Electrophoresis of mycelial proteins on polyacrylamide gels confirmed that all isolates were pure cultures and that they all belonged to the same species. The electrophoretic banding patterns of total soluble mycelial proteins and eight isozymes (alkaline phosphatase, esterase, fumarase, NAD-glucose dehydrogenase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, and superoxide dismutase) of all olive isolates were identical to those of two strains of P. palmivora from parlor palm and from pittosporum. Conversely, they were clearly distinct from the electrophoretic banding patterns of reference isolates of P. cactorum (Lebert & Cohn) Schroter, P. capsici Leonian, P. citrophthora (R. E. Sm. & E. H. Sm.) Leonian, and P. nicotianae van Breda de Haan. All isolates of P. palmivora from olive were of the A1 mating type. The pathogenicity of four P. palmivora isolates from olive, two producing only typical and two producing both types of sporangia, was tested in the greenhouse at 18 to 25°C, using 20 1-year-old rooted cuttings of olive cv. Carolea for each isolate. Twenty noninoculated cuttings were used as a control. The inoculum for pathogenicity tests was produced on autoclaved rice grains moistened with V-8 juice. Cuttings were transplanted to pots filled with soil infested with inoculum at 2% (wt/vol). Control plants were grown in pots containing a mixture of soil and 2% autoclaved rice. After transplanting, all pots were flooded once a week for 24 h by plugging the drain hole of the pot. One month after planting, all the plants in infested soil had died and no difference in virulence was observed among the isolates. Control plants remained healthy. P. palmivora was reisolated from the roots of symptomatic plants. Pathogenicity tests were repeated twice with similar results. In a survey of nurseries in Southern Italy, P. palmivora was recorded frequently from roots of young olive trees suggesting that infections originated from nurseries. This is the first report from Italy of P. palmivora on olive. This species has been described recently as a pathogen of olive in Spain (3). References: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society. St. Paul, MN. (2) H. Masago et al. Phytopathology 67:25, 1977. (3) M. E. Sanchez Hernandez et al. Eur. J. Plant Pathol. 104:347, 1998.

Insensitivity to Metalaxyl Among Isolates of Phytophthora capsici Causing Root and Crown Rot of Pepper in Southern Italy.

Pepper (Capsicum annuum L.) has become an economically important crop in the coastal provinces of Catanzaro and Vibo Valentia, in Calabria (southern Italy). An old local selection Riggitano, very susceptible to root and crown rot caused by Phytophthora capsici Leonian, is the prevalent cultivar in this area. Although repeated applications of metalaxyl are used as a soil drench, severe outbreaks occur each year on greenhouse crops. To examine metalaxyl resistance in P. capsici, 60 single-hypha isolates of P. capsici were tested in vitro for their level of sensitivity to metalaxyl. The isolates were collected from 1992 to 1997, during epidemic outbreaks of root and crown rot, from two commercial greenhouse pepper crops, near Vibo Valentia and Lametia Terme (Catanzaro). Fungicide sensitivity was determined by plating mycelial plugs onto potato dextrose agar (PDA) amended with metalaxyl. The fungicide was added to PDA after autoclaving, at final concentrations of 0.1, 1, 5, 10, 50, 100, and 200 µg/ml a.i. The percentage of inhibition of radial growth on metalaxyl-amended medium compared with the growth on unamended medium was determined after 6 days of incubation in the dark at 25°C. Three replicate petri dishes were used per treatment and each test was performed twice. The isolates were paired in culture on V8 agar with isolates of P. capsici of known mating type and all proved to be A2 mating type. Significant variation was observed among the isolates tested in responce to metalaxyl. The ED50 values for in vitro inhibition of mycelial growth by metalaxyl ranged from 1 to 11 µg/ml, whereas an ED 50 value of 0.1 µg/ml had been reported for a wild-type isolate of P. capsici obtained from pepper in northern Italy (3). The variation observed among the isolates from Calabria appeared unrelated to both the geographical origin and the year of isolation. The isolates from Calabria were inhibited by between 1 and 12% at 0.1 µg/ml and by between 7 and 27% at 1 µg/ml, proving to be less sensitive to metalaxyl than isolates from Capsicum spp. originating from Central America, tested by other authors (1). According to the criterion used in a recent screening for sensitivity to metalaxyl (2), 19% of the isolates from Calabria should be considered sensitive, as they were inhibited by more than 60% at 5 µg/ml, while all the others were intermediate, as they were inhibited less than 60% at 5 µg/ml but more than 60% at 100 µg/ml. On the basis of this preliminary screening, we report the presence of insensitivity to metalaxyl in field isolates of P. capsici in southern Italy. Although no isolate tested appeared highly resistant to metalaxyl, the presence of a high proportion of isolates with an intermediate level of resistance should be a reason for the growers to use metalaxyl more cautiously to control root and collar rot. References: (1) M. D. Coffey and L. A. Bower. Phytopathology 74:502, 1984. (2) G. Parra and J. Ristaino. Plant Dis. 82:711, 1998. (3) M. L. Romano and A. Garibaldi. La difesa delle piante 3:153, 1984.