Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

Dothistroma septosporum Identified in Greece on Pinus brutia and Pinus nigra Plantations.

Dothistroma needle blight (DNB) is caused by two ascomycete fungi, Dothistroma septosporum with a worldwide distribution and D. pini found in the United States, Russia, Ukraine, Hungary, and France (1). DNB has been known in Greece since the early 1980s (3) and the species responsible for the disease was reported as D. pini. In December 2011, needles were collected from three trees in Lagada, Thessaloniki regional unit of Central Macedonia (northern Greece), where the disease was first recorded. DNB infection seems to be limited to a valley in this area in Pinus nigra and P. brutia plantations established more than 50 years ago in an originally deciduous oak forest. Infections were observed over an area of 50 to 60 ha of pine plantations. Although the majority of pine trees were infected by the pathogen, the severity of the disease was relatively low and mortality of infected trees was not observed. Infections were limited to the lower branches in the 50-year-old trees, while on a limited number of younger trees of P. brutia (10 to 15 years old), infection was more severe, extending to the entire crowns of the trees. DNB does not appear to be very common in Greece. Infected needles had reddish-brown bands, usually with necrotic tips or entirely necrotic needles. Black sub-epidermal fruiting bodies (acervuli) were observed with the needle epidermis split and raised. Isolations were made from fruiting structures on needles of P. nigra and P. brutia (50-year-old trees) and P. brutia (10-year-old trees) after surface disinfection with 70% ethanol. Conidia from single fruiting bodies were transferred onto 3% malt extract agar (MEA) in petri dishes and incubated at 20°C. Colonies on MEA had a radial growth rate of 1.3 to 1.6 mm per week, were crustose, brown to grey-brown, and partly covered with slimy masses of conidia. The agar surrounding the colonies had a reddish color. Conidia from acervuli on the needles and the cultures were similar in shape and size, filiform, hyaline, 2 to 4 septate, and 1.8 to 2.5 (3.3) × 22 to 47 µm long. DNA was extracted from three cultures: one from P. nigra (CMW 37966) and two from P. brutia (CMW 37965, CMW 37967) using a standard phenol/chloroform method. The internal transcribed spacer (ITS) region was amplified and sequenced (1). Sequences were 100% identical to D. septosporum isolates in GenBank (e.g., AY808291). In addition, up to 12 randomly selected acervuli were excised directly from the needles of each of the three trees and DNA was extracted using PrepMan solution (Applied Biosystems). The mating types of these samples were determined using species-specific mating type primers for D. pini and D. septosporum (2). All acervuli were confirmed to be those of D. septosporum. Both mating types were found on needles from P. brutia and P. nigra. However, the teleomorph of D. septosporum was not detected on infected needles. References: (1) I. Barnes et al. For. Pathol. 41:361, 2011. (2) M. Groenewald et al. Phytopathology 97:825, 2007. (3) D. S. Kailidis and S. Markalas. Dasika Chronika 24:257, 1981 (in Greek).

First Record of Phytophthora nicotianae Causing Leaf Blight on Rhododendron in Greece.

Species of Rhododendron and resulting hybrids are very important hosts of the quarantine pathogen Phytophthora ramorum, since they play a major role in the spread of the pathogen in Europe. However, many other Phytophthora species infect these hosts, causing similar symptoms. Widmer (4) listed 17 Phytophthora species as foliar pathogens of rhododendron in the United States. A survey was conducted in Greece in October 2009, in which potted plants of Rhododendron spp. were inspected for symptoms of necrotic lesions on leaves and buds caused by P. ramorum. Symptomatic plants were observed in one of the nurseries inspected in the Triphylia Region in southwestern Peloponnese. Isolations from symptomatic leaves on PARBhy-V8 selective agar medium (1) yielded Phytophthora isolates. Colonies on V8 juice agar appeared white and cottony, with a radial growth of 4.2 to 4.6 mm per day at 28°C with a maximum growth temperature of 36°C. Sporangia were produced abundantly on the medium surface and in water; the sporangia were broadly ovoid and papillate and 35 to 50 × 25 to 35 µm. Chlamydospores, 25 to 40 µm in diameter, were observed in 2-week-old cultures, while no sexual structures were observed. Three of the isolates examined were identified as P. nicotianae B. de Haan on the basis of morphological and physiological features (3,4). Genomic DNA was extracted from pure cultures of an isolate and the internal transcribed spacer (ITS) region was amplified using the ITS4/5 primer pair. Sequence analyses by BLAST indicated that the isolates were most similar to P. nicotianae (GenBank Accession No. AJ 854295.1) with sequence identity values of 99%. One of the isolates was deposited in the culture collection of the University of Athens (ATHUM 6519). Detached wounded leaves of Rhododendron hybrid cv. Red Jack were inoculated with agar plugs. Necrotic lesions, similar to those observed in the nursery, appeared on the inoculated leaves after 7 days of incubation at 26°C, while no symptoms developed on control leaves inoculated with sterile agar plugs. The pathogen was consistently reisolated from infected leaves, but not from the controls. P. nicotianae, being a thermophilic species, is the most common Phytophthora species in Greece, reported on more than 30 plant species (2). This pathogen has been reported on Rhododendron spp. in the United States (3,4), but to our knowledge, there was no record of this pathogen on these hosts as yet in Europe and this is the first published report of the pathogen on Rhododendron in Greece. References: (1) A. Belisario et al. Plant Dis. 87:101, 2003. (2) K. Elena. Technical Bulletin No 13. Benaki Phytopathological Institute. Athens, Greece (in Greek), 1999. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) T. L. Widmer. Online publication. doi: 10.1094/PHP-2010-0317-01-RS, Plant Health Progress, 2010.

First Report of Phytophthora ramorum on Rhododendron in Greece.

In April 2010, during a survey conducted in Fthiotis Prefecture of central Greece, symptoms of stem necrosis and leaf lesions were observed on two container-grown plants of Rhododendron, hybrid 'Kate Waterer' in a nursery. From symptomatic leaves, a Phytophthora species was isolated on PARPH-V8 selective agar medium (2) with typical morphological characters of Phytophthora ramorum S. Werres & A.W.A.M. de Cock (4). The whole block of plants was under probation until molecular verification of the pathogen was completed. The nursery was reexamined 6 weeks after the first encounter, whereas spread of the pathogen was noticed to neighboring plants in the same block; five more Rhododendron plants with similar symptoms were recorded while one of the originally infected plants was dead. Isolates of Phytophthora with similar morphology were obtained from symptomatic leaves of three new plants as well as from the potting mix of a severely infected plant that was baited in a Rhododendron leaf assay (2). All Rhododendron plants in the block belonged to the same consignment imported from Belgium and covered by a phytosanitary plant passport. Colonies on 10% clarified V8 juice agar appeared with coralloid, coenocytic mycelium with radial growth at 1.7 mm per day at 20°C and maximum temperature 26 to 27°C. Propagules characteristic of P. ramorum, including semipapillate, caducous, sporangia measuring 35 to 55 × 15 to 30 µm (1.9 length/width ratio) and large chlamydospores (45 to 80 µm), were observed on V8 agar. One isolate was confirmed as P. ramorum by sequence analysis of the internal transcribed spacer region of rDNA and was deposited in the culture collection of the University of Athens (ATHUM 6522). Comparison of amplicon sequences (using ITS4/5 primer pair) of approximately 875 bp long was carried out using MEGABLAST search for highly similar sequences. Alignment data revealed the highest and most significant homology to P. ramorum (GenBank Accession No. AY594198.1) at 99%. Pathogenicity tests were carried out using detached leaves of Rhododendron hybrid 'Red Jack' and Arbutus unedo L., which were slightly wounded and inoculated with mycelium agar plugs (3). Necrotic lesions appeared on the inoculated leaves of both plant species 10 days after incubation at 20°C, while no symptoms developed on control leaves inoculated with sterile agar plugs. P. ramorum was consistently reisolated from artificially infected leaves of both plant species. Following confirmation of pathogen presence, eradication measures were applied in the nursery. The adverse weather conditions encountered in summer, with temperatures very often above 35°C, are expected to favor pathogen eradication. However, not all plants of the same consignment imported from Belgium were traced and it is possible that other infected plants have been sold in other areas of Greece. So far, P. ramorum had been reported in 21 other European countries; Serbia is the nearest country where the pathogen was detected (1). To our knowledge, this is the first report of P. ramorum in Greece. References: (1) A. Bulajic et al. Plant Dis. 94:703, 2010. (2) E. J. Fichtner et al. Phytopathology 97:1366, 2007. (3) R. G. Linderman et al Online publication. doi:10.1094/PHP-2007-0917-01-RS. Plant Health Progress, 2007. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.