First Report of Phytophthora taxon niederhauserii Causing Root and Stem Rot of Mimosa in Italy.

Mimosa [Acacia dealbata Link, syn. Acacia decurrens (Wendl. F.) Wild. var. dealbata (Link) F. Muell., Fabaceae] is an evergreen shrub native to southeastern Australia that is cultivated as an ornamental plant in warm temperate regions of the world. In spring 2010, in a commercial nursery in Liguria (northern Italy), 6- to 10-month-old potted plants of A. dealbata showed symptoms of sudden collapse, defoliation, and wilt associated with root and basal stem rot. An abundant gum exudate oozed from the basal stem. A Phytophthora species was consistently isolated from roots and stem on BNPRAH selective medium (4). On V8 agar (V8A), axenic cultures obtained by single hyphal transfers formed stellate to radiate colonies with aerial mycelium whereas on potato dextrose agar (PDA) the colonies grew more slowly than on V8A and showed stoloniform mycelium and irregular margins. Minimum and maximum growth temperatures on PDA were 10 and 35°C, with the optimum at 30°C. In water, all isolates produced catenulate or single fusiform hyphal swellings and ellipsoid, nonpapillate, persistent sporangia. Dimensions of sporangia were 46.1 to 65.4 × 23.1 to 30.8 µm (mean l/b ratio 2.1). All isolates were A1 mating type and produced spherical oogonia with amphyginous antheridia when paired with A2 mating type of P. drechsleri Tucker on V8A plus ß-sytosterol (4). Internal transcribed spacer (ITS) regions of rDNA of the representative Phytophthora isolate IMI 500394 from A. dealbata were amplified and sequenced in both directions with primers ITS6/ITS4. The consensus sequence (GenBank Accession No. JF900371) was 99% similar to sequences of several isolates identified as Phytophthora taxon niederhauserii Z.G. Abad and J.A. Abad (e.g., GQ848201 and EU244850). Pathogenicity tests were performed on 1-year-old potted plants of A. dealbata with isolate IMI 500394. Twenty plants were transplanted into pots (12-cm-diameter) filled with soil infested (4% v/v) with the inoculum of IMI500394 produced on kernel seeds. Plants were kept in a greenhouse with natural light at 25 ± 2°C and watered to field capacity weekly. All inoculated plants showed symptoms of wilt, leaf chlorosis, and basal stem rot within 3 to 4 weeks. Twenty control plants transplanted in autoclaved soil mix remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants, thus fulfilling Koch's postulates. Since 2003, this pathogen has been found on bottlebrush and rock rose grown in a nursery in Sicily (southern Italy), as well as on Banksia in a nursery in Liguria (2,3). To our knowledge, this is the first report of P. taxon niederhauserii on A. dealbata. P. taxon niederhauserii, recently described as P. niederhauserii sp. nov. (1), is a polyphagous pathogen that was originally reported on arborvitae and ivy in North Carolina in 2001. References: (1) Z. G. Abad et al. Mycologia (in press), 2013. (2) S. O. Cacciola et al. Plant Dis. 93:1075, 2009. (3) S. O. Cacciola et al. Plant Dis. 93:1216, 2009. (4) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

First Report of Phytophthora × pelgrandis Causing Root Rot and Lower Stem Necrosis of Common Box, Lavender and Port-Orford-Cedar in Hungary.

In 2008 and 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown common box (Buxus sempervirens L.), lavender (Lavandula angustifolia Mill. 'Hidcote'), and Port-Orford-cedar (Chamaecyparis lawsoniana (A. Murray) Parl. 'Columnaris') in three ornamental nurseries in western Hungary. Number of affected plants ranged from approximately 100 (Port-Orford-cedar) to 250 (lavender). Isolations from necrotic root collars of each host plant species yielded four Phytophthora isolates developing uniform colonies on carrot agar with a maximum growth temperature of 35 to 36°C. The isolates were homothallic with smooth-walled oogonia (32.2 ± 2.3 to 35.9 ± 3.5 µm), aplerotic oospores (27.5 ± 1.8 to 32.1 ± 3.1 µm) and both amphigynous and paragynous antheridia, and produced chlamydospores (25.8 ± 3.9 to 29.1 ± 5.2 µm) and papillate sporangia (35.2 ± 2.5 to 43.5 ± 5.6 µm long and 27.6 ± 2.2 to 32.0 ± 3.8 µm wide), mostly obpyriform to nearly spherical or rarely distorted with two or three apices. In spring water, sporangia were both caducous with short pedicel and non-caducous. Multiplex ITS-PCR assay of DNA from all isolates, using primers specific for P. nicotianae (NICF1 and NICR2.1) and P. cactorum (CACTF1 and CACTR1) (1), amplified DNA fragments of the expected size for each Phytophthora species. In addition, isoenzyme analysis revealed a characteristic banding pattern of one heterodimer and two homodimer bands at both loci of the dimeric enzyme malate dehydrogenase. These bands comigrated with those of P. × pelgrandis (Gerlach et al.) (CBS 123385) and isolate PD 93/1339 (courtesy of W. A. Man in 't Veld), two natural hybrid strains of P. nicotianae and P. cactorum (2,3), proving that our four isolates can be referred to as this interspecific hybrid. Pathogenicity was tested on 1- or 3-year-old plants of the original host species and cultivars (for common box, cv. Faulkner was used). Cultures were grown for 4 to 6 weeks at 20°C on autoclaved millet grains moistened with V8 broth. Infested and uninfested grains were mixed with autoclaved soil in a ratio of 6% (w/v), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth chamber (20°C, 70% RH, 12-h photoperiod). Pots were flooded for 24 h on the 1st and 21st day after transplanting. All plants in infested potting mix showed symptoms of wilt associated with basal stem and root necrosis, similar to those observed on the plants from the field, within 2 and 3 months on lavender and both common box or Port-Orford-cedar, respectively. Additionally, a reduction of root weight ranging from 35 to 68% compared to the control was recorded. Growth reduction was significant at P ≤ 0.019 according to Mann Whitney test. Control plants remained healthy. The same Phytophthora hybrid was reisolated solely from inoculated plants. In Europe, hybrid isolates of P. nicotianae × P. cactorum have been reported on several ornamental plants, including lavender, in the Netherlands and Germany (2,3). However, to our knowledge, this is the first report of this hybrid in Hungary and as a pathogen of common box and Port-Orford-cedar in the world. References: (1) P. J. M. Bonants et al. Phytopathology 90:867, 2000. (2) W. A. Man in 't Veld et al. Phytopathology 88:922, 1998. (3) H. I. Nirenberg et al. Mycologia 101:220, 2009.

First Report of Canker Disease Caused by Diplodia olivarum on Carob Tree in Italy.

The evergreen carob tree (Ceratonia siliqua L., Fabaceae), also called locust, is widespread in the Mediterranean Region. Carob pods have been traditionally consumed as animal and human food and seeds are mainly used in the pharmaceutical and cosmetic industries. In July 2009, symptoms of canker, branch dieback, and foliage reddening were observed on carob trees in several natural areas in the province of Ragusa, Italy. Disease incidence ranged from 5 to 80% across different sites and for most areas it was nearly 15%. All affected trees showed dark necrotic tissue in the bark, cambium, and sapwood of the trunk and branches. Cankers often girdled the stem or branch, causing wilting and death of the portions beyond the canker. Black, subepidermal pycnidia developed in and erupted through the dead bark. Fragments of discolored wood were collected from 36 symptomatic carob trees (12 trees for each area), transferred onto potato dextrose agar (PDA), and incubated for 5 days at 21°C in the dark. Fungal colonies were consistently obtained from these diseased tissues. They initially were pale, becoming gray-green and finally black. After 30 days of incubation at room temperature in the natural light, colonies produced pycnidia identical to those observed in nature. A total of 500 conidia on 10 isolates were examined with a compound microscope. Conidia were initially hyaline, smooth, oblong to ovoid, both ends rounded, and aseptate; at maturity they were pale brown, one-septate, and measured 24 to 28 × 10 to 13.5 µm (means ± S.D. = 24.3 ± 1.4 × 12.1 ± 1 µm, L/W = 2.0 ± 0.18). The nucleotide sequences of the ß-tubulin (GenBank Accession No. HQ660080) and TE-1α (No. HQ660078) genes and ITS-rDNA region (No. HM028640) for a representative isolate (IMI 390972) from carob showed 100, 100, and 98% similarity, respectively, when compared with the sequences HQ660079, EU392279, and EU392302, respectively, of the ex-type isolate of Diplodia olivarum (strain CBS 121887). On the basis of morphological and molecular characters, the fungus was identified as D. olivarum A.J.L. Phillips, Frisullo & Lazzizera; teleomorph unknown (1). Two-year-old trees were wounded with a scalpel through the full thickness of the bark along 1-cm longitudinal direction and inoculated by applying a 5-mm-diameter plug of mycelial (isolate IMI 390972) on PDA to the wound site. Three control seedlings were similarly wounded and plugs of sterile PDA applied. Plugs were held in place by Parafilm. The inoculated seedlings were maintained at 20 to 22°C and a 12-h light/dark cycle. Sixty days after inoculation, all inoculated trees showed leaf chlorosis, sunken, necrotic bark at the inoculation sites and finally pycnidia of D. olivarum. All treated seedlings were killed within 6 months from the inoculation. No symptoms were observed in the control plants. The pathogen was consistently reisolated from all the inoculated trees, but not from the control plants. D. olivarum has been found on rotting olive drupes in Apulia (southern Italy) and was first described as a new species in 2008 (1). This fungal species could be phenotypically misidentified as the closely related species D. mutila, which differs by having larger mean dimensions of conidia. To our knowledge, this is the first report of D. olivarum inducing canker and dieback on carob tree. Reference: (1) C. Lazzizera et al. Fungal Divers. 31:63, 2008.

Bud and Root Rot of Windmill Palm (Trachycarpus fortunei) Caused by Simultaneous Infections of Phytophthora palmivora and P. nicotianae in Sicily.

In June 2009 in a commercial nursery in eastern Sicily (Italy), 3-year-old potted windmill palms (Trachycarpus fortunei (Hooker) H. Wendl.) showed a decline in growth, wilt, droop, and basal rot of the youngest leaves. The rot progressed inward and killed the bud. Initially, older leaves remained green but eventually the entire plant collapsed. Root rot was consistently associated with aboveground symptoms. Two Phytophthora species were consistently isolated from the petiole base, heart, roots, and rhizosphere soil of symptomatic plants on a selective medium (2) and occasionally recovered from roots and rhizosphere soil of asymptomatic plants. Pure cultures were obtained by single-hypha transfers and the two species were identified on the basis of morphological and molecular characters as Phytophthora palmivora and P. nicotianae. Both species were recovered from all symptomatic plants. From multiple tissue samples per plant, we recovered either or both species. On potato dextrose agar (PDA), P. palmivora isolates grew between 10 and 35°C, with the optimum at 27°C. On V8 juice agar, they produced elliptical to ovoid, papillate, caducous sporangia (32 to 78 × 23 to 39 µm) with a mean length/breadth (l/b) ratio of 1.8:1 and a short pedicel (mean pedicel length = 5 µm). Isolates of P. nicotianae produced arachnoid colonies on PDA, grew at 37°C but did not grow at 40°C. Sporangia (29 to 55 × 23 to 45 µm) were spherical to ovoid (l/b ratio 1.3:1), papillate and often bipapillate, and noncaducous. Isolates of both species produced amphigynous antheridia and oogonia only when paired with reference isolates of P. nicotianae of the A2 mating type. The internal transcribed spacer (ITS) region of rDNA of two isolates of P. palmivora (IMI 398987 and IMI 398988) and an isolate of P. nicotianae (IMI 398989) from T. fortunei was amplified with primers ITS6/ITS4 and sequenced (1). Blast analysis of the sequences of isolates IMI 398987 and IMI 398988 (GenBank Accession Nos. HQ596556 and HQ596558) showed 99% homology with the sequence of two reference isolates of P. palmivora (GQ398157.1 and GU258862), while the sequence of isolate IMI 398989 (HQ596557) showed 99% homology with a reference isolate of P. nicotianae (EU331089.1). Pathogenicity of isolates IMI 398987 and IMI 398989 was proved by inoculating separately each isolate on 1-year-old potted plants of T. fortunei (10 plants per isolate). A zoospores suspension (2 × 104 zoospores/ml) was pipetted onto the petiole base of the three central leaves (200 µl per leaf) of each plant. Sterile water was used for control plants. All plants were incubated at 25 ± 2°C with 100% humidity for 48 h and then maintained in a greenhouse at 24 to 28°C. Within 3 weeks, all inoculated plants showed symptoms of bud rot. Control plants remained healthy. P. palmivora and P. nicotianae were reisolated only from inoculated plants. Bud rot of palms caused by P. palmivora was reported previously in Italy (3). However, to our knowledge, this is the first report of simultaneous infections of P. palmivora and P. nicotianae as causal agents of this disease. Outbreak of bud rot may have been favored by overhead sprinkler irrigation. The recovery of P. palmivora and P. nicotianae from rhizosphere soil and roots of asymptomatic plants suggests infested soil was the primary inoculum source. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) A. Pane et al. Plant Dis. 91:1059, 2007.

First Report of Root and Basal Stem Rot Caused by Phytophthora nicotianae on Tree Aeonium (Aeonium arboreum) in Italy.

The genus Aeonium, family Crassulaceae, comprises approximately 35 species that are native to northern Africa and the Canary Islands. Tree aeonium (Aeonium arboreum (L.) Webb & Berthel.) is a bushy, perennial succulent with rosettes of tender, waxy leaves at the apex of few-branched or occasionally single, naked stems. Mature rosettes bear yellowish inflorescences. Aeoniums are cultivated as ornamentals in gardens and containers. During the summer of 2009, in a garden in eastern Sicily (southern Italy), 3-year-old potted plants of tree aeonium showed stunting, shrivelling, and chlorosis of leaves and drop of external leaves associated with root and basal stem rot. Drops of an amber exudate oozed from the basal stem. Tissues of the basal stem were soft, but no external necrosis was visible. A species of Phytophthora was consistently isolated from symptomatic roots and basal stem tissues on a medium selective for Oomycetes (2). Axenic cultures were obtained by single-hypha transfers. The pathogen was identified by morphological criteria as Phytophthora nicotianae B. de Haan; it formed stoloniferous colonies on potato dextrose agar and grew between 8 and 38°C, with the optimum at 30°C. On V8 juice agar it produced spherical, intercalary chlamydospores (mean diameter of 26 µm) and persistent, mono- and bipapillate, spherical to ovoid, ellipsoid, obpyriform sporangia that measured 29 to 56 × 22 to 45 µm with a mean length/breadth ratio of 1.3:1. All isolates were A2 mating type and formed spherical oogonia (mean diameter 28 ± 2 µm) with smooth walls and amphigynous antheridia in dual cultures with a reference isolate of the A1 mating type of P. nicotianae. BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of a representative isolate from aeonium (IMI 398812, GenBank Accession No. HQ433333) amplified by PCR using the ITS6/ITS4 universal primers (1), revealed 99% similarity with the sequences of a reference isolate of P. nicotianae available in GenBank (Accession No. EU331089.1). Pathogenicity of isolate IMI 398812 was demonstrated by transplanting cuttings of A. arboreum into pots filled with a mixture of steam-sterilized sandy loam soil and inoculum (4% vol/vol) produced by growing the isolate for 20 days on wheat kernels. Ten plants were transplanted into 3-liter pots (two plants per pot) while 10 plants, transplanted into pots filled with a mixture of steam-sterilized soil and noninoculated kernels, were used as controls. Plants were kept in a greenhouse at 25 to 28°C and watered daily to field capacity. Thirty to forty days after the transplanting into infested soil, cuttings developed the same symptoms observed on plants with natural infections. Control plants remained symptomless. P. nicotianae was reisolated from symptomatic plants, thereby completing Koch's postulates. To our knowledge, this is the first report of P. nicotianae on an Aeonium species worldwide. The economic relevance of this disease is minor because aeoniums are not cultivated on a large scale. Moreover, the disease may be easily prevented by avoiding excess irrigation water since aeoniums need a well-drained soil or potting mix and do not tolerate soil waterlogging. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977.

Root and Basal Stem Rot of Mandevillas Caused by Phytophthora spp. in Eastern Sicily.

Approximately 150,000 potted mandevillas (Apocynaceae) are produced each year in the Etna District of eastern Sicily. Since 2004, leaf chlorosis, wilt, and sudden collapse of the entire plant associated with root and basal stem rot of 6- to 12-month-old potted mandevillas, including Mandevilla × amabilis 'Alice du Pont', M. splendens, and M. sanderi 'Alba', 'My Fair Lady', and 'Scarlet Pimpernel', have been observed in six nurseries. Incidence of affected plants varied from 5 to 40%. Four Phytophthora species were consistently isolated from rotted roots and stems on a selective medium (2). Pure cultures of the first species produced colonies with a camellia pattern on potato dextrose agar and grew between 10 and 37°C with an optimum of 27°C. On V8 juice agar they produced ellipsoid to obpyriform (length/breadth [l/b] 1.45:1), nonpapillate sporangia with internal proliferation, coralloid, spherical hyphal swellings and both terminal and intercalary chlamydospores. In dual cultures with A1 and A2 isolates of P. nicotianae, all isolates produced oogonia with amphyginous antheridia only with A2 isolates. Isolates of the second species formed petaloid colonies, had an optimum growth temperature of 25°C, and produced mono- and bipapillate, ovoid to limoniform sporangia (l/b 1.40:1); they did not produce gametangia. Isolates of the third species formed colonies with a slight petaloid pattern and grew between 2 and 30°C with an optimum of 25°C. Sporangia were obpyriform (l/b 1.48:1), nonpapillate, and proliferous. All isolates were A2 mating type. The isolates of the fourth species formed arachnoid colonies, grew between 8 and 38°C with an optimum of 30°C, and produced mono- and bipapillate, ellipsoid, and obpyriform (l/b 1.3:1) sporangia and apical chlamydospores. All isolates were A2 mating type. DNA was extracted from mycelium and amplified by PCR using the ITS 4/ITS 6 primers (1). Blast search of the rDNA-ITS sequence of isolate IMI 397618 (GenBank Accession No. GQ388261) of the first species showed 100% identity with the ITS sequence of an isolate of P. cinnamomi var. parvispora (EU748548). The sequences (GQ463703 and GQ463704) of isolates IMI 397471 and IMI 397472 of the second species showed 99% similarity with the sequences of a P. citrophthora isolate (EU0000631). The sequence of isolate IMI 397473 (GQ463702) of the third species showed 99% similarity with the sequence of a P. cryptogea isolate (AY659443.1), while the sequence of isolate IMI 397474 (GU723474) of the fourth species showed 99% similarity with the sequence of a P. nicotianae isolate (EU331089). The pathogenicity of individual isolates IMI 397618, IMI 397471, IMI 397472, IMI 397473, and IMI 397474 was tested on 3-month-old potted plants (10 plants per isolate) of mandevilla 'Alice du Pont' by applying 10 ml of a suspension (2 × 104 zoospores/ml) to the root crown. Plants were maintained at 25°C and 95 to 100% relative humidity. All inoculated plants wilted after 4 weeks, while noninoculated control plants remained healthy. The four Phytophthora spp. were subsequently reisolated only from symptomatic plants. To our knowledge, this is the first report of P. cinnamomi var. parvispora in Italy and on mandevilla worldwide. In recent years, Phytophthora root and stem rot has become the most serious disease of potted mandevillas in Sicily. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977.

First Report of a Decline and Wilt of Young Olive Trees Caused by Simultaneous Infections of Verticillium dahliae and Phytophthora palmivora in Sicily.

In summer 2008, leaf chlorosis, defoliation, exceptional fruit set, twig dieback, and wilt were observed on 4-year-old olive (Olea europea L.) trees cv. Tonda Iblea in a drip-irrigated orchard in eastern Sicily. Rot of fine roots was associated with these symptoms and on ~15% of symptomatic trees rot extended to the crown and basal stem. Trees declined slowly or collapsed suddenly with withered leaves still attached. Incidence of affected trees was ~10%. A fungus identified as Verticillium dahliae Kleb. was isolated from the xylem of main roots and basal stem. An oomycete identified as Phytophthora palmivora (Butler) Butler was isolated from roots and basal trunk bark. Both pathogens were recovered from symptomatic trees with mean frequency of positive isolations per tree of 80 and 30% for V. dahliae and P. palmivora, respectively. To isolate V. dahliae, wood chips were surface disinfested in 0.5% NaOCl for 1 min and plated onto potato dextrose agar (PDA). The fungus was identified on the basis of microsclerotia, verticillate arrangement of phialides on conidiophores, and hyaline single-celled conidia. Ten monoconidial isolates were characterized by PCR using primer pairs INTND2f/INTND2r and DB19/espdef01 (3). Only 824-bp amplicons, diagnostic of the virulent, nondefoliating V. dahliae pathotype, were obtained. P. palmivora was isolated on selective medium (2) and pure cultures were obtained by single-hypha transfers. Colonies grew on PDA between 10 and 35°C (optimum at 27°C). Chlamydospores and elliptical to ovoid, papillate, caducous (mean pedicel length = 5 µm) sporangia (length/breadth ratio of 1.8) were produced on V8 juice agar. All isolates were paired with reference isolates of P. nicotianae and produced gametangia only with isolates of the A2 mating type. PCR amplicons of a representative isolate generated using primers ITS 6 and ITS 4 (1) were sequenced and found to be identical to those of a reference isolate of P. palmivora (GenBank No. AY208126). Pathogenicity of V. dahliae (IMI 397476) and P. palmivora (IMI 397475) was tested on 6-month-old rooted cuttings of olive cv. Tonda Iblea. Ten cuttings were transplanted into pots with steam-sterilized soil and inoculum of P. palmivora (4% vol/vol) produced on wheat kernels. Ten olive cuttings were inoculated with V. dahliae by injecting the stem with 150 µl of a conidial suspension (107 conidia ml-1) and 10 cuttings were stem inoculated with V. dahliae and transplanted into soil infested with P. palmivora. Controls were 10 noninoculated cuttings transplanted into steam-sterilized soil. Pots were kept in a greenhouse (25 ± 3°C) for 4 months. No aerial symptoms were observed on cuttings transplanted into soil infested with P. palmivora. However, root dry weight was reduced by 40% in comparison with the controls. Cuttings inoculated solely with V. dahliae had a 15% reduction in height compared with the controls but only four cuttings wilted. All cuttings inoculated with P. palmivora and V. dahliae wilted, indicating a synergism between the two pathogens. Controls remained healthy. Each pathogen was reisolated solely from inoculated cuttings and both pathogens were reisolated from cuttings with double inoculations. A similar syndrome 'seca' (drying) was reported in Spain (4). References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) J. Mercado-Blanco et al. Plant Dis. 87:1487, 2003. (4) M. E. Sánchez-Hernández et al. Eur. J. Plant Pathol. 104:34, 1998.

Callistemon citrinus and Cistus salvifolius, Two New Hosts of Phytophthora taxon niederhauserii in Italy.

Bottlebrush (Callistemon citrinus (Curtis.) Skeels., Myrtaceae) and rock rose (Cistus salvifolius L., Cistaceae) are evergreen shrubs native to Australia and the Mediterranean Region, respectively. In the spring of 2003, approximately 2% of a nursery stock of 12-month-old potted plants of C. citrinus and 8% of a nursery stock of 12-month-old potted plants of Cistus salvifolius grown in the same nursery in Sicily, showed symptoms of leaf chlorosis, defoliation, and wilt associated with root and collar rot. A Phytophthora species was consistently isolated from roots and basal stems on BNPRAH selective medium (2). One isolate from rock rose (IMI 391708) and one from bottlebrush (IMI 391712) were characterized. On potato dextrose agar (PDA), the colonies showed stoloniform mycelium and irregular margins; on V8 juice agar (V8A), colonies were stellate to radiate. Minimum and maximum temperatures on PDA were 10 and 35°C, respectively, with the optimum at 30°C. Mean radial growth rate of isolates on this substrate was 9.9 and 11.3 mm/day, respectively. In saline solution (1), both isolates produced catenulate hyphal swellings and ellipsoid, nonpapillate, persistent sporangia with internal proliferations and dimensions of 52 to 70 × 30 to 42 µm and 51 to 85 × 39 to 45 µm. Mean l/b ratio of sporangia for both isolates was 1.8 ± 1. On V8A plus ß-sytosterol, both isolates produced amphyginous antheridia and spherical oogonia in dual cultures with an A2 tester of P. drechsleri Tucker. Conversely, they did not produce gametangia with an A1 tester of P. cryptogea Pethybr., indicating they were A1 mating type. The internal transcribed spacer (ITS)-rDNA sequences of rock rose and bottlebrush isolates showed 100% similarity with those of two reference isolates of P. taxon niederhauserii from GenBank (Accession Nos. FJ648808 and FJ648809). On the basis of the analysis of the DNA, the species isolated from bottlebrush and rock rose were identified as Phytophthora taxon niederhauserii. Pathogenicity tests were carried out on 6-month-old potted plants of C. salvifolius and C. citrinus (10 plants of each plant species for each isolate) transplanted into pots (12 cm in diameter) containing a mixture of 1:1 steam-sterilized, sandy loam soil (vol/vol) with 4% inoculum produced on autoclaved kernel seeds. Plants were maintained at 25 to 28°C and watered to soil saturation once a week. After 2 to 3 weeks, all inoculated plants developed symptoms identical to those observed on plants with natural infections. Ten control plants transplanted into pots containing noninfested soil remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants. To our knowledge, this is the first report of P. taxon niederhauserii on C. citrinus and C. salvifolius in Italy. This Phytophthora taxon has been reported recently on rock rose in Spain (3). References: (1) D. W. Chen and G. A. Zentmyer. Mycologia 62:397, 1970. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) E. Moralejo et al. Plant Pathol. 58:100, 2009.

Effect of rituximab on clinical and laboratory features of antiphospholipid syndrome: a case report and a review of literature.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by a hypercoagulable state related to persistently elevated levels of antiphospholipid antibodies (aPL). Current treatment for APS is only partially effective and new therapies are strongly needed. We report on a case of a 50 years old man with APS who suffered from recurrent thromboembolic episodes despite conventional anticoagulant treatment. Eight years after the first thrombotic manifestation he was diagnosed with a large B cell non-Hodgkin lymphoma. Treatment with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) plus rituximab was started with partial clinical remission of lymphoma and normalization of aPL levels with a three years follow-up period free of thrombotic episodes.A review of the literature revealed that only 12 case reports on the use of rituximab in patients with primary, secondary and catastrophic APS have been published. Current knowledge clearly suggests the need for clinical trials to evaluate the effect of rituximab in the treatment of resistant APS.

Iloprost therapy acutely decreases oxidative stress in patients affected by systemic sclerosis.

BACKGROUND: Oxidative stress has been considered a leading factor in the pathogenesis of systemic sclerosis (SSc). Consistently with this hypothesis the determination of urinary isoprostanes, a reliable method for evaluation of oxidative stress, has recently showed increased levels of isoprostanes in SSc patients. Data about the effect on oxidative stress of accepted therapies for SSc such as iloprost therapy are lacking. OBJECTIVE: The aim of this prospective study was to verify whether iloprost therapy in patients with SSc acutely reduces oxidative stress assessed by determination of 8-Iso PGF2alpha urinary levels. METHODS: urine samples were obtained before and after a five-day cycle of iloprost infusion and urinary 8-Iso PGF2alpha levels were determined using a commercially available enzyme immunoassay. RESULTS: Consistent with previous reports, we found an increased level of oxidative stress in SSc patients with respect to healthy controls. Basal urinary 8-iso PGF2alpha levels in SSc patients were significantly higher than those in healthy controls [2002(1122-3575) pg/mg creatinine vs. 334(225.7-441) pg/mg creatinine, p<0.001]. Moreover, as expected, urinary 8-iso PGF2alpha levels after iloprost therapy were significantly lower than basal levels [1277.5 pg/mg creatinine (742.7-2017.3) vs. 2002 pg/mg creatinine (1122-3575), p=0.001] but persisted significantly elevated respect to the levels of healthy controls (p<0.001). The effect of iloprost on oxidative stress appeared significant in patients with early and limited form of disease. CONCLUSIONS: This prospective open-label explorative study suggests that standard course of iloprost therapy may acutely reduce oxidative stress in SSc patients. This effect appears to be more consistent in the early phases and in the limited subset of disease. Further larger trials are needed to confirm our results and to explain the pathway of such reduction, its clinical significance and potential therapeutic implications.

Simvastatin maintains steady patterns of GFR and improves AER and expression of slit diaphragm proteins in type II diabetes.

The factors determining the course of glomerular filtration rate (GFR) and albumin excretion rate (AER) and the expression of mRNA of slit diaphragm (SD) and podocyte proteins in microalbuminuric, hypertensive type II diabetic patients are not fully understood. GFR, AER, and SD protein mRNA were studied in 86 microalbuminuric, hypertensive, type II diabetics at baseline and after 4-year random double-blind treatment either with 40 mg simvastatin (Group 1) or with 30 g cholestyramine (Group 2) per day. Both groups had at baseline a GFR decay per year in the previous 2-4 years of 3 ml/min/1.73 m(2). Both Groups 1 and 2 showed a significant decrease of low-density lipoprotein cholesterol levels after simvastatin and cholestyramine treatment (P<0.01). No change from base line values was observed as for hs-C-reactive protein and interleukin-6. A significant decrease of 8-hydroxydeoxyguanosine urinary excretion was observed after simvastatin treatment. GFR did not change from baseline with simvstatin, whereas a decrease was observed with cholestyramine treatment (simvastatin vs cholestyramine: -0.21 vs -2.75 ml/min/1.73 m(2), P<0.01). AER decreased in Group 1 (P<0.01), but not in Group 2 patients. Real-time polymerase chain reaction measurement of mRNA SD proteins (CD2AP, FAT, Actn 4, NPHS1, and NPHS2) significantly increased in kidney biopsy specimens after simvastatin, but not cholestyramine treatment. Simvastatin, but not cholestyramine, 4-year treatment maintains steady patterns of GFR, and improves AER and expression of SD proteins in type II diabetes, despite similar hypocholesterolemic effects in circulation.

[Diagnostic and prognostic value of antibodies to cyclic citrullinated peptide (Anti-CCP) in rheumatoid arthritis]. / Il significato diagnostico e prognostico degli anticorpi anti-peptide citrullinato ciclico (Anti-CCP) nell'artrite reumatoide.

There is strong evidence that the determination of autoantibodies against filaggrine is a very useful tool for the diagnosis of rheumatoid arthritis (RA). Anti-cyclic citrullinated peptide antibodies (Anti-CCP)-ELISA appear to be the most efficient test among those available for the detection of antifilaggrine autoantibodies, as it has the best diagnostic accuracy for the diagnosis of RA. Furthermore, the anti-CCP-ELISA determination in early arthritis is a good predictor of disease persistence and radiographic joint damage. The positivity of Anti-CCP some years before the onset of the RA and the high concentration of autoantibodies in synovial fluid suggest a possible pathogenetic role of citrullination. However, at present, it is unclear whether anti-CCP antibodies have a better diagnostic performance than rheumatoid factor in recent onset synovitis and if they confer any additional value to the prognostic evaluation obtained with validated predictors of outcome (FR, joint count, duration of disease).

Identification of a new mutation in the alpha4(IV) collagen gene in a family with autosomal dominant Alport syndrome and hypercholesterolaemia.

BACKGROUND: Alport syndrome (AS) is a hereditary disease of the glomerular basement membrane in the kidney characterized by progressive renal failure, sensorineural deafness, and/or ocular abnormalities. In contrast to the well-known X-linked phenotype, very little is known about the autosomal dominant form. Rare autosomal forms of AS have been described with mutations in COL4A3 and COL4A4 at chromosome region 2q35-q37, but there have been no descriptions of dominant forms due to a mutation in COL4A4. METHODS: We describe a Sardinian family with a classical AS-phenotype plus hypercholesterolaemia, a clinical feature also present in Fechtner syndrome (FS), a disease that segregates as an autosomal dominant trait. RESULTS: A suggestive linkage (LOD=2.7) between AS and the COL4A3/A4 locus at 2q35-q37 was identified. Other candidate collagen genes encoding basement membrane collagen (COL4A1/A2 and COL4A5/A6) were excluded by linkage analysis (13q33-q34 and Xq22), or by sequence (COL4A3). DNA sequence analysis of the COL4A4 gene revealed that the Lys325Asn mutation was present in all affected family members, but was absent in all unaffected members and in a random sample of the Sardinian population. A clear indication of a gene-dosage effect was seen in the most severely affected family member, since she carried the mutation in the homozygous form. CONCLUSIONS: These data confirm the importance of collagen 4A4 as a component in the structural integrity of the glomerular basement membrane and confirm the phenotypic and genetic heterogeneity of collagen disorders.

Isolation and identification of Mycobacterium neoaurum from a patient with urinary infection.

Mycobacterium neoaurum is a novel species of Mycobacteria, until now only isolated from catheters in immunosuppressed patients. This report describes the isolation and identification of M. neoaurum from urine obtained from a hospitalized patient.

Urinary glycosaminoglycans in patients with systemic lupus erythematosus.

OBJECTIVE: Several investigations indicate that glycosaminoglycans (GAG) are important components of the glomerular basement membrane (GBM) and that they play a remarkable role in the control of charge-selectivity in the glomerular capillary wall. In order to evaluate the possible use of GAG as a marker of glomerular disease, we evaluated urinary GAG excretion in 37 patients with systemic lupus erythematosus (SLE) grouped by disease activity and kidney involvement and in 17 healthy controls. METHODS: GAG were isolated from urine by using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: Total GAG levels were significantly increased only in active extra-renal SLE patients. Qualitative analysis of urinary GAG revealed the presence of a low sulphated chondroitin sulphate-protein complex (LSC-PG), whose frequency was higher in patients compared to controls. Moreover, inactive SLE was characterized by an alteration of the chondroitin sulphate/heparan sulphate ratio. CONCLUSION: These variations suggest the presence of an abnormal permeability of the renal filter in patients without other appreciable signs of kidney alteration. Therefore, qualitative-quantitative urinary GAG analysis could represent an additional diagnostic approach.

Relationship between cerebrospinal fluid pressure and plasmatic ADH.

In a healthy human being, the extracellular volume is kept constant by homeostatic systems. One of these is represented by the antidiuretic hormone (ADH). ADH release is modulated by osmoreceptors and baroreceptors which respond to an increase in osmolality of extracellular fluid and a decrease in blood volume, respectively. In previous studies we investigated the existence of additional structures sensitive to plasma volume modifications. We found evidence of the presence of such receptors in the inner ear, with nervous connections to supraoptic and paraventricular nuclei. However, the possibility that the cerebral ventricle wall contained stretch sensors could not be excluded. To test our hypothesis, we studied 19 rats divided into three groups: Group 1 (n =7), Group 2 (n =7) and Group 3 (control group n =5). In each rat, under total anaesthesia, a femoral cannula was inserted into the left artery and a 22 gauge stainless steel cannula was implanted into the left cerebral ventricle. In the first group an isotonic fluid, similar to the animal's cerebrospinal fluid (CSF), was infused intracerebroventricularly (ICV) at a rate of 0.6 microl min-1 continuously for 6 h. In the second group, under the same conditions, CSF was aspirated; the third group was used as the control. In all animals, plasma modifications of ADH (pADH), osmolality (pOSM), Na+(pNa+) and K+(pK+) were evaluated before and after the experimental procedures. Mean arterial pressure (MAP) and heart rate (HR) were recorded throughout the experiment. At the end of the experiment no significant changes in pNa+, pK+, MAP and HR were observed. Plasma osmolality was significantly lower in Group 2 before and during the experimental procedure, since we deliberately expanded the volume in animals of Group 2 to partially suppress ADH, in order to evaluate its modifications. Plasma ADH fell in the first experimental group (-37.4%+/-6.3 sem) after the ventricular pressure had been increased, and rose in the second (+47.3%+/-14.7 sem) after ventricular decompression. These changes were statistically significant in comparison with those occurring in control subjects (-0.9+/-18.9 sem;P =0.07 and P =0.03, respectively). These results suggest the presence of additional volume receptors probably located in the cerebral ventricles, capable of controlling ADH. The importance of these receptors in physiological situations of plasma volume contraction or expansion remains to be established.

Inner ear pressure changes modify ADH secretion in freely moving guinea pig.

The present study was designed to elucidate the relationship between endolymphatic pressure and plasma ADH levels in conscious guinea pigs. Plasma ADH (pADH) was measured in basal conditions and after having applied positive or negative pressure of 20 cmH2O to the inner ear. The experimental protocol was designed to avoid any interference on ADH release caused by anesthesia and surgical stress. There was no change in blood pressure, heart rate, plasma Na (pNa) and osmolality (pOsm) after inner ear pressure (IEP) modifications. However, pADH was inversely related with IEP: pADH averaged 31.4 +/- 7.0 pg/ml (mean +/- S.D.) in basal conditions, rising to 48.8 +/- 19.3 when IEP was lowered and falling to 16.6 +/- 10.3 when IEP was raised. These results confirm that structures in the inner ear help control of ADH release.

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.

Outbreak of a Leaf Disease Caused by Pseudocercospora ceratoniae on Carob in Sicily.

Carob (Ceratonia siliqua L.), an evergreen tree typical of the Mediterranean flora, has been grown in Sicily (Italy) from time immemorial for its fruits, used mainly as food for cattle and horses as well as for industrial production of alcohol. Although the economic importance of carob as a commercial crop has declined over the last decades, carob trees still are a characteristic aspect of the landscape in southeastern Sicily. In early April 1998, a severe outbreak of a foliar disease was noted on carob trees in the Ragusa province. Symptoms initially consisted of small (2 to 3 mm wide) dark brown, vein-limited spots, visible on both sides of the leaf and, later in the season, surrounded by a pale halo. In a humid atmosphere, spots scattered over the leaf blade but usually were most numerous along the midrib, enlarged, and coalesced, forming large blotches. Severely affected leaflets dropped, leaving the petiole attached to the tree. As a result, the trees appeared defoliated. Severely defoliated trees did not produce fruits. The causal agent of this disease was identified as Pseudocercospora ceratoniae (Pat. & Trab.) Deighton (1), an hyphomycetous fungus reported previously as a pathogen of carob under the name Cercospora ceratoniae Pat. & Trab. (3). On carob leaflets, P. ceratoniae formed grayish caespituli, confined to the lower surface of the necrotic spots. Caespituli consisted of dense fascicles of conidiophores (up to more than 50 conidiophores per fascicle) emerging through the stomata. Conidiophores were simple, slightly ampulliform, and geniculate at the conidial scars, which were conspicuous and unthickened. Old scars often were situated laterally on a short denticle. Conidia, borne singly as terminal blastospores and varying considerably in length, were pluriseptate, filiform, substraight or slightly curved, frequently slightly obclavate, with an obtuse apex and a short constriction at the base toward the truncate, unthickened hilum. Conidia from pure cultures of the fungus grown on water agar under black light were suspended in water and sprayed onto pot-grown carob plants. Inoculated plants were kept in a moist chamber for 48 h and subsequently transferred to the greenhouse. After 12 to 14 days leaf spots similar to those observed on naturally infected trees developed on inoculated plants and the pathogen was reisolated. Control plants sprayed with distilled water remained symptomless. C. ceratoniae had been recorded previously on carob in various Mediterranean countries, including Italy (2), but since has attracted little attention, being regarded as a sporadically occurring pathogen. Both mild temperatures during the winter and exceptionally frequent and persistent rain during the spring may have favored the epidemic outbreak of the disease caused by this fungus. References: (1) F. C. Deighton. 1976. Mycol. Pap. No. 140. Commonw. Mycol. Inst., Kew, England. (2) R. Parisi. Boll. Orto Bot. R. Univ. Napoli 10:155, 1932. (3) L. Roger. 1953. Phytopathologie des Pays Chauds. Vol. 2. P. Lechevalier, Paris.