Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy.

Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

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.

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.

Phytophthora taxon niederhauserii, a New Root and Crown Rot Pathogen of Banksia spp. in Italy.

In the last 10 years, various species of Banksia (family Proteaceae) endemic to Australia have been introduced into Italy where cultivation as flower plants is expanding. In the spring of 2003, a decline associated with root and basal stem rot of 2- to 3-year-old plants of Banksia speciosa R. Br., B. baxteri R. Br., and B. prionotes Lindl. grown in the ground was observed in a commercial nursery in Liguria (northern Italy). Aboveground symptoms included leaf chlorosis and wilt. Plants collapsed within 1 to 2 weeks after the appearance of leaf symptoms. A Phytophthora species was consistently isolated from roots and basal stem on BNPRAH selective medium (3). On V8 juice agar (V8A), axenic cultures obtained by single hyphal transfers formed stellate to radiate colonies with aerial mycelium; on potato dextrose agar (PDA). the colonies showed stoloniform mycelium. Minimum and maximum growth temperatures on PDA and V8A were between 5 and 10°C and 38 and 40°C, respectively, with the optimum at 30°C on PDA (mean radial growth rate of 10 isolates ranged between 9.3 and 10.2 mm per day) and 25 to 30°C on V8A (14 mm per day). In saline solution and soil extract, all isolates produced catenulate hyphal swellings and ellipsoid, nonpapillate, persistent sporangia. Sporangia in saline solution varied from 47 to 70 × 30 to 44 µm (mean l/b ratio of 1.5). All isolates were A1 mating type and produced oogonia with amphyginous antheridia when paired with A2 mating type of P. drechsleri Tucker on V8A plus ß-sytosterol (3). The electrophoretic patterns of total mycelial proteins and two isozymes (esterase and malate dehydrogenase) (2) of all isolates from Banksia plants were identical, but distinct from the patterns of isolates of other Phytophthora species, including P. drechsleri, P. megasperma sensu stricto, and P. sojae. Internal transcribed spacer (ITS) regions of rDNA were amplified with primers ITS4/ITS6 and sequences of two isolates, IMI 393960 from B. speciosa and 466/03 from B. baxteri (GenBank Nos. FJ648808 and FJ648809), were 100% identical to sequences of isolates identified as Phytophthora taxon niederhauserii Z. G. Abad and J. A. Abad (GenBank Nos. AY550916, AM942765, and EU244850). Pathogenicity tests were performed on 1-year-old potted plants of B. speciosa with isolates IMI 393960 and 466/03. Twenty plants per each isolate were transplanted into 12-cm-diameter pots containing infested soil prepared by mixing steam-sterilized sandy loam soil with 1% of inoculum produced on autoclaved wheat kernels. Twenty control plants were grown in autoclaved soil mix. Plants were kept in the greenhouse with natural light at 25 ± 2°C and watered to field capacity weekly. All Banksia plants transplanted in infested soil showed symptoms of wilt, leaf chlorosis, and basal stem rot within 2 to 3 weeks. Noninoculated plants remained healthy. P. taxon niederhauserii was reisolated solely from inoculated plants. P. taxon niederhauserii has been reported recently from Banksia spp. in Australia (1), but to our knowledge this is the first report from Italy. P. taxon niederhauserii may represent a threat to the cultivation of many ornamentals including Cystus spp., English ivy, and laurel (4). References: (1) T. I. Burgess et al. Plant Dis. 93:215, 2009. (2) S. O. Cacciola et al. EPPO Bull. 20:47, 1990. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) E. Moralejo et al. Plant Pathol, 58:100, 2009.

First Report of Phytophthora tentaculata Causing Root and Stem Rot of Oregano in Italy.

Oregano (Origanum vulgare L.; Lamiaceae) is cultivated for culinary and medicinal purposes and as an ornamental. In October of 2007, 1- to 2-year-old potted plants of oregano showed symptoms of decline associated with root and basal stem rot in a nursery in Liguria (northern Italy) that produces 1 million to 1.5 million potted aromatic plants per year. Aboveground symptoms included leaf russeting and chlorosis, wilt, defoliation and dieback of twigs, browning of the basal stem, and subsequent collapse of the entire plant. Approximately 80% of the plants died within 30 days after the appearance of the first symptoms on the canopy. Approximately 20% of a stock of 30,000 oregano plants was affected. Stocks of other aromatic species, such as mint, lavender, rosemary, and sage, appeared healthy. A Phytophthora species was consistently isolated from symptomatic stems and roots of oregano plants on BNPRAH selective medium (2). Ten pure cultures were obtained by single-hypha transfers, and the species was identified as Phytophthora tentaculata Kröber & Marwitz by morphological criteria and sequencing of the internal transcribed spacer (ITS) region of rDNA using the ITS 4 and ITS 6 universal primers for DNA amplification. Isolates from oregano formed stoloniferous colonies with arachnoid mycelium on potato dextrose agar and had a growth rate of 2 to 3 mm per day at 24°C with optimum, minimum, and maximum temperatures of 24, 8, and 34°C, respectively. Sporangia formed in soil extract solution and were papillate and spherical or ovoid to obpyriform with a length/breadth ratio of 1.3:1. Few sporangia were caducous and all had a short pedicel (<5 µm). Hyphal swellings and chlamydospores were produced in sterile distilled water and corn meal agar, respectively. All isolates were homothallic and produced globose terminal oogonia (mean diameter of 34 µm) with one or occasionally two paragynous, monoclinous, or diclinous antheridia. Amphigynous antheridia were also observed. The sequence of the ITS region of the rDNA (GenBank No. FJ872545) of an isolate from oregano (IMI 395782) showed 99% similarity with sequences of two reference isolates of P. tentaculata (Accession Nos. AF266775 and AY881001). To test for pathogenicity, the exposed root crowns of 10 6-month-old potted plants of oregano were drench inoculated with 10 ml of a suspension of 2 × 104 zoospores/ml of isolate IMI 395782. Sterile water was pipetted onto the roots of 10 control plants. All plants were maintained in 100% humidity at 22 to 24°C in a greenhouse under natural light and watered once a week. Within 3 weeks after inoculation, all inoculated plants developed symptoms identical to those observed in the nursery and died within 30 to 40 days after the appearance of the first symptoms. Control plants remained healthy. P. tentaculata was reisolated solely from symptomatic plants. P. tentaculata has been reported previously on several herbaceous ornamental plants (1,3). However, to our knowledge, this is the first report of this species on O. vulgare. Root and basal stem rot caused by P. tentaculata is the most serious soilborne disease of oregano reported in Italy so far. References: (1) G. Cristinzio et al. Inf. Fitopatol. 2:28, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) H. Kröber and R. Marwitz. Z. Pflanzenkr. Pflanzenschutz 100:250, 1993.

Four Phytophthora Species Causing Foot and Root Rot of Apricot in Italy.

In the summer of 2006, 1-year-old apricot (Prunus armeniaca L.) trees with leaf chlorosis, wilting, and defoliation associated with root and crown rot were observed in a nursery in Sicily (Italy). Of 3,000 plants, ~2% was affected. Four Phytophthora spp. (45, 25, 20, and 10% of the isolations of the first, second, third, and fourth species, respectively) were isolated from decayed roots and trunk bark on BNPRAH (3). Axenic cultures were obtained by single-hypha transfers. Isolates of the first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth temperature of 25°C. On V8 agar (VA), they produced persistent, papillate (often bipapillate), ovoid to limoniform sporangia (length/breadth ratio 1.4:1). They did not produce gametangia when paired with A1 and A2 isolates of Phytophthora nicotianae. The second species formed arachnoid colonies, had an optimum growth of 30°C, and produced uni- and bipapillate, ellipsoid, ovoid or pyriform sporangia (length/breadth ratio 1.3:1). All isolates were A2. The third species formed rosaceous colonies on PDA, had an optimum temperature of 28 to 30°C, and produced papillate (sometime bipapillate), ellipsoid or limoniform (length/breadth ratio 2:1), caducous sporangia with a tapered base and a long pedicel (as much as 150 µm). All isolates were A1 type. The fourth species formed petaloid-like colonies on PDA and had an optimum growth of 26 to 28°C. On VA, it produced papillate (sometimes bipapillate), ovoid (length/breadth ratio 1.3:1), and decidous sporangia with a short pedicel (<4 µm). The isolates were homothallic and produced oogonia (25 to 31 µm in diameter) with paragynous antheridia and aplerotic oospores. On the basis of morphological and cultural characters, the species were identified as P. citrophthora, P. nicotianae, P. tropicalis and P. cactorum. Identification was confirmed by the electrophoretic analysis of total mycelial proteins and four isozymes (acid and alkaline phosphatases, esterase, and malate dehydrogenase) on polyacrylamide gel (1). Analysis of internal transcribed spacer (ITS) regions of rDNA using the ITS 4 and ITS 6 primers for DNA amplification (2) revealed 99 to 100% similarity between apricot isolates of each species and reference isolates from GenBank (Nos. AF266785, AB367355, DQ118649, and AF266772). The ITS sequence of a P. citrophthora isolate from apricot (IMI 396200) was deposited in GenBank (No. FJ943417). In the summer of 2008, pathogenicity of apricot isolates IMI 396200 (P. citrophthora), IMI 396203 (P. nicotianae), IMI 396201 (P. tropicalis), and IMI 396202 (P. cactorum) was tested on 3-month-old apricot seedlings (10 plants for each isolate) that were transplanted into pots filled with soil prepared by mixing steam-sterilized sandy loam soil (4% vol/vol) with inoculum produced on autoclaved kernel seeds. Ten control seedlings were grown in autoclaved soil. Seedlings were maintained in a screenhouse and watered daily to field capacity. Within 40 days of the transplant, all inoculated seedlings showed leaf chlorosis, wilting, and root rot. Control seedlings remained healthy. All four Phytophthora spp. were reisolated solely from inoculated plants. To our knowledge, this is the first report of Phytophthora root and crown rot of apricot in Italy and of P. tropicalis on this host. References: (1) S. O. Cacciola et al. Plant Dis. 90:680, 2006. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977.

First Report of Phytophthora spp. as Pathogens of Pandorea jasminoides in Italy.

In the summer of 2005, approximately 5% of a nursery stock of 12-month-old potted plants of bower vine (Pandorea jasminoides (Lindl.) K. Schum.) in Sicily (Italy) showed wilt, leaf chlorosis, defoliation, root rot, and collapse of the entire plant. Three Phytophthora spp. (20, 50, and 30% of the isolations of the first, second, and third species, respectively) were isolated from rotted roots on BNPRAH selective medium (2). Single-hypha isolates of the first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth temperature of 25°C (9.3 mm/day); on V8 juice agar, they produced uni- and bipapillate, ovoid to limoniform sporangia with mean dimensions of 45 × 30 µm and a mean length/width (l/w) ratio of 1.4:1. They did not produce gametangia when paired with A1 and A2 isolates of Phytophthora nicotianae. The second species formed arachnoides colonies on PDA, had an optimum growth temperature of 30°C (6.9 mm/day) and produced sporangia that were uni- and bipapillate, ellipsoid, ovoid, or pyriform to spherical (dimensions 44 × 34 µm; l/w ratio 1.3:1). All isolates were A2 mating type and produced amphyginous antheridia and spherical oogonia with smooth walls. The third species formed rosaceous colonies on PDA, had an optimum growth temperature of 28 to 30°C (11.9 mm/day), and produced uni- and bipapillate, ellipsoid or limoniform, caducous sporangia (dimensions 52 × 26 µm; l/w ratio 2.1:1) with a tapered base and a long pedicel (as much as 150 µm). All isolates were A1 type and produced amphigynous antheridia and spherical oogonia with smooth walls. The three species were identified as P. citrophthora, P. nicotianae, and P. tropicalis, respectively. The electrophoretic analysis of the mycelial proteins and four isozymes (1) confirmed the identification. Blast analysis of the sequence of the internal transcribed spacer region of the rDNA of a P. tropicalis isolate from bower vine (GenBank Accession No. EU076731) showed 99% similarity with the sequence of a P. tropicalis isolate from Cuphea ignea (GenBank Accession No. DQ118649). The pathogenicity of three isolates from bower vine, IMI 395552 (P. citrophthora), IMI 395553 (P. nicotianae), and IMI 395346 (P. tropicalis), was tested on 3-month-old potted bower vine plants (10 plants for each isolate) by applying 10 ml of a suspension (2 × 104 zoospores/ml) to the root crown. The plants were maintained at 24°C and 95 to 100% relative humidity. All inoculated plants wilted after 4 weeks. Noninoculated control plants remained healthy. The three Phytophthora spp. were reisolated from symptomatic plants. To our knowledge, this is the first report of Phytophthora root rot of bower vine in Italy. References: (1) S. O. Cacciola et al. Plant Dis. 90:680, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

The unresponsiveness of the immune system of the rat to hypergravity.

The immune response in rats exposed to simulated hypergravity (2.1 G and 3.1 G) by chronic centrifugation was assessed. Rats were immunized with sheep red blood cells (SRBC), either on the day of initial exposure to hypergravity (hyper-G), or after being centrifuged for 28 d and remaining on the centrifuge thereafter. Pair-fed and ad libitum fed noncentrifuged controls were used. Although there were some alterations in leukocyte counts, hyper-G did not systematically affect the primary or secondary anti-SRBC response, hematocrits, or the sizes of the liver, spleen, kidneys, thymus, or adrenal glands. The immune system is thus remarkably homeostatic under hypergravity conditions which do affect other physiologic parameters.