Tracking the invasion: dispersal of Hymenoscyphus fraxineus airborne inoculum at different scales.

Ash dieback is caused by an invasive pathogen Hymenoscyphus fraxineus, which emerged in Europe in the 1990s and jeopardizes the management of ash stands. Although the biological cycle of the pathogen is well understood, its dispersal patterns via airborne spores remain poorly described. We investigated the seasonal and spatial patterns of dispersal in France using both a passive spore-trapping method coupled with a real-time PCR assay and reports of ash dieback based on symptom observations. Spores detection varies from year to year with a detection ability of 30-47% depending on meteorological conditions, which affect both production of inoculum and efficiency of the trapping. Nevertheless, our results are consistent and we showed that the sporulation peak occurred from June to August and that spores were detected up to 50-100 km ahead of the disease front, proving the presence of the pathogen before any observation of symptoms. The spore dispersal gradient was steep, most of inoculum remaining within 50 m of infected ashes. Two dispersal kernels were fitted using Bayesian methods to estimate the mean dispersal distance of H. fraxineus from inoculum sources. The estimated mean distances of dispersal, either local or regional scale, were 1.4 km and 2.6 km, respectively, the best fitting kernel being the inverse power-law. This information may help to design disease management strategies.

One-Step Detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a Multiplex Real-Time PCR Assay.

Brown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.

Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex.

Alder decline has been a problem along European watercourses since the early 1990s. Hybridization was identified as the main cause of this emerging disease. Indeed, the causal agent, a soil-borne pathogen named Phytophthora alni subsp. alni (Paa) is the result of interspecific hybridization between two taxa, Phytophthora alni subsp. multiformis (Pam) and Phytophthora alni subsp. uniformis (Pau), initially identified as subspecies of Paa. The aim of this work was to characterize the ploidy level within the P. alni complex that is presently poorly understood. For that, we used two complementary approaches for a set of 31 isolates of Paa, Pam and Pau: (i) quantification of allele copy number of three single-copy nuclear genes using allele-specific real-time PCR and (ii) comparison of the genome size estimated by flow cytometry. Relative quantification of alleles of the three single-copy genes showed that the copy number of a given allele in Paa was systematically half that of its parents Pau or Pam. Moreover, DNA content estimated by flow cytometry in Paa was equal to half the sum of those in Pam and Pau. Our results therefore suggest that the hybrid Paa is an allotriploid species, containing half of the genome of each of its parents Pam and Pau, which in turn are considered to be allotetraploid and diploid, respectively. Paa thus results from a homoploid speciation process. Based on published data and on results from this study, a new formal taxonomic name is proposed for the three taxa Paa, Pam and Pau which are raised to species status and renamed P. ×alni, P. ×multiformis and P. uniformis, respectively.

First Report of Pineapple Black Rot Caused by Ceratocystis paradoxa on Ananas comosus in French Guiana.

Ceratocystis paradoxa (Dade) C. Moreau is a polyphagous wound parasite causing black rot post-harvest disease in pineapple. This fungus is responsible for high losses of fruit destined for the fresh market and is a common problem in many countries (2). C. paradoxa is officially listed as a quarantine pathogen for French Guiana. In November 2013, the Plant Protection Service of French Guiana observed damage in crops of Ananas comosus var. perolera located in Corossony (4°19'10.8″ N, 52°10'17.1″ W) and Wayabo (5°01'02.3″ N, 52°36'18.7″ W) (eastern and middle part of the country). The three plants collected at each location showed a soft base rot of the stem and of young leaves, and developed a foul smell. Plant tissues were collected from the edge of the lesions, chopped in small pieces, and plated on malt extract agar supplemented with 100 ppm chloramphenicol. The plates were incubated at 25°C with a 12-h photoperiod. After 5 days, a fungal colony, first white and downy, then becoming black and velvety after 10 days, was transferred on potato dextrose agar (PDA) and incubated in the same conditions. After 7 days, the colonies produced phialides releasing cylindrical or doliform conidia that were unicellular, colorless to pale brown, in long chains (3.09 to 20.17 × 3.1 to 5.57 µm, n = 20) and oval, pyriform, brown chlamydospores (8.02 to 21.32 × 4.20 to 9.76 µm, n = 20), occurring in long chains or singly with a vertical slit, usually not very visible. Furthermore, the colonies emitted a fruity odor. On the basis of these morphological characteristics, the fungus was identified as the anamorph of C. paradoxa (Thielaviopsis paradoxa (De Seynes) Höhn.) (1). The species designation was confirmed by sequencing the ITS region of the rDNA followed by comparison with reference sequences available in GenBank. Fungal material was collected from PDA culture by scraping the mycelium with a sterile needle and transferring into 2-ml microtubes. Fungal total DNA was then extracted and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair. Nucleotide sequence was determined and deposited in GenBank (KJ667047). BLAST analysis showed 100% identity with C. paradoxa. The pathogenicity of the fungus was confirmed by inoculating two pineapples with mycelium from the C. paradoxa isolate grown on PDA. The peel of fruits and the base of the crowns were wounded with a sterile scalpel blade, each at five locations. Mycelial plugs (avg. 4 mm diameter) were placed on the wounds. Inoculation sites were wrapped with Parafilm to prevent dehydration and to hold the mycelial plugs in position. Negative controls received five sterile PDA plugs. The samples were incubated at 25°C in a moist chamber with a 12-h photoperiod. Eight days after inoculation, negative controls remained symptomless, whereas characteristic soft, watery, and black rot lesions developed on the base of all the crowns that were inoculated with C. paradoxa. The pathogen was successfully re-isolated from symptomatic tissues, fulfilling Koch's postulate. To our knowledge, this is the first report of C. paradoxa on A. comosus in French Guiana, and quarantine measures have been enforced to prevent the spread of this pathogen that might also cause severe losses on other susceptible plant species that are important for the local market (e.g., banana, coconut, sugar cane). Pineapple has become a major crop in French Guiana, and is now subjected to a more intensive monitoring, which may explains why this disease was discovered recently. References: (1) T. R. Nag Raj and W. B. Kendrick. A Monograph of Chalara and Allied Genera. Wilfrid Laurier University Press, Waterloo, Ontario, 1975. (2) R. C. Ploetz et al., eds. Compendium of Tropical Fruit Diseases. American Phytopathological Society, St. Paul, MN, 1994.

First Report of Dothistroma pini, a Recent Agent of the Dothistroma Needle Blight, on Pinus radiata in France.

Dothistroma needle blight (DNB), also known as red band needle blight, is an important fungal disease of Pinus spp. that occurs worldwide. On the basis of molecular and morphological studies of the anamorphic stage, Barnes et al. (1) showed that two closely related species were involved in DNB: Dothistroma septosporum (Dorog.) Morelet and D. pini Hulbary. D. septosporum (teleomorph: Mycosphaerella pini Rostr.) has a worldwide distribution and is reported as the species that caused past epidemics of DNB. This species is reported on more than 80 different pine species, and Pinus radiata D. Don (Monterey pine) is classified as a highly or moderately susceptible species, depending on the published sources (4). D. pini (telemorph: unknown) was initially found on needles of P. nigra J. F. Arnold collected from 1964 to 2001 in the north central United States (Minnesota, Nebraska, and Michigan). It was subsequently found in Ukraine and southwestern Russia, where it has been associated with the emergence of DNB on P. nigra subsp. pallasiana (Lamb.) Holmboe, in Hungary on P. nigra, and in Russia on P. mugo Turra (1). In France, D. pini was reported for the first time on P. nigra, and was sometimes found in association with D. septosporum on the same needles (3). Later on, a more intensive survey of DNB was launched in France and 216 stands of Pinus sp. were studied. D. septosporum and D. pini were detected in 133 and 123 stands, respectively. Both species co-occurred in 40 stands but D. pini was only found on P. nigra (subsp. laricio and austriaca) (2). Up to now, D. pini was therefore only reported on European pine species, mainly on the different allopatric subspecies belonging to the black pine complex and on one occasion on P. mugo, which belongs to the same section and subsection as P. nigra. In March 2011, typical symptoms of DNB (needles with orangey-red brown distal ends, dark red bands, and green bases; small and black fruit bodies within the bands) were observed in a 50- to 60-year old P. radiata stand of ~3 ha located in Pyrénées Atlantiques close to the Spanish border (1°36'08″ W, 43°19'51″ N). The density of pine was relatively low and patches of natural regeneration were present. Although nearly all of the trees showed DNB symptoms, less than 50% of their needles were affected by the disease. In this stand, needles showing typical DNB symptoms were randomly taken from four pines and mixed together to form a single sample for analysis. Total DNA was extracted from symptomatic needle pieces. The presence of D. pini was confirmed by a specific multiplex real-time PCR analysis using the D. pini-specific primers/probe combination DPtef-F1-/R1/-P1 (3), and by sequencing a D. pini-specific amplicon generated by another conventional PCR (3) using DPtef-F/DPtef-R primers (GenBank Accession KC853059) (3). D. septosporum was not detected in the sample. To our knowledge, this is the first report worldwide of D. pini on P. radiata, a pine species largely planted in Spain and in the Southern Hemisphere. This is also the first report of this pathogen on a non-European pine species. The original native range and the host range of D. pini remain unknown and there is currently no data about host preferences or aggressiveness on different pine species. References: (1) I. Barnes et al. For. Pathol. 41:361, 2011. (2) B. Fabre et al. Phytopathology 102:47, 2012. (3) R. Ioos et al. Phytopathology 100:105, 2010. (4) M. Watt et al. For. Ecol. Manage. 257:1505, 2009.

First Report of Begonia Elatior Wilt Disease Caused by Fusarium foetens in France.

Fusarium foetens is a destructive vascular pathogen on Begonia, mainly on cultivars of Begonia elatior hybrids (Begonia × hiemalis), which has recently been identified in Europe and Northern America (1,3). This Fusarium species has been responsible for severe damage in the begonia flower industry (1) and is listed as an EPPO A2 quarantine pathogen since 2007. In May 2007, wilted potted plants of B. elatior showing chlorotic leaves and basal stem rot were observed in a nursery located in the west of France (La Flèche, Sarthe). Symptomatic foliar and basal stem pieces were plated on a Fusarium semi selective medium, dichloran chloramphenicol peptone agar (DCPA), and on malt agar medium supplemented with 100 ppm chloramphenicol. Homogeneous mycelium of a Fusarium species developed from both types of tissue and on both media, and was transferred to potato dextrose agar (PDA) and to spezieller nährstoffarmer agar (SNA) media for morphological examination. Microscope slides were then prepared by pressing gently a clear self-adhesive tape onto the surface covered by mycelium and sporodochia, which was further stained with lactic acid/methylene blue. Typical multiseptate (often three septa), hyaline, slightly curved Fusarium macroconidia 29.2 to 41.8 (32.5) × 3.6 to 4.5 (4.3) µm were collected in sporodochia. In the aerial mycelium, long and short conidiophores with mono- or polyphialidic cells bearing false heads of ellipsoidal microconidia were observed. In addition, a pungent distinctive odor was produced by the mycelium grown on PDA. These features were consistent with F. foetens (2). To support the diagnosis, total DNA was further extracted from the pure culture and a partial region of the translation elongation 1 (tef1) gene was amplified by PCR using EF1-EF2 primer pair (4). Nucleotide sequence was determined and deposited on GenBank (Accession No. JX298790). Analysis of the sequence by BLAST showed that it was 100% identical with all the available F. foetens sequences, which confirmed our morphological diagnosis. To our knowledge, this is the first official report of F. foetens in France. Since this first detection, F. foetens was again identified in 2010 in another nursery located in the Pays de la Loire on collapsed B. elatior. Approximately 15 to 20% of the Begonia plants showed typical Fusarium wilt symptoms and the infected lots were systematically destroyed. The origin of these infections could not be traced back since the mother plants tested negative. The disease is considered as eradicated in France but causes major economic losses to Begonia growers and marketers in regions where the disease is established (2). References: (1) H. Huvenne et al. Eur. J. Plant Pathol. 131:705, 2011. (2) H. J. Schroers et al. Mycologia 96:393, 2004. (3) X. L. Tian et al. Plant Dis. 94:1261, 2010. (4) D. Geiser. Eur. J. Plant Pathol. 110:473, 2004.

Is the emergence of Dothistroma needle blight of pine in France caused by the cryptic species Dothistroma pini?

Dothistroma needle blight (DNB) emerged in France in the past 15 years. This disease is induced by two closely related species: Dothistroma septosporum and D. pini. Although both species are currently present in France, only D. septosporum was reported in the past. We investigated whether a recent arrival of D. pini in France could be a cause of the DNB emergence. We analyzed herbarium specimens of pine needles with DNB symptoms using polymerase chain reaction techniques to study the past frequency of D. pini in France. We also determined the present distribution within the country of D. septosporum and D. pini and compared it with the spatial pattern of DNB reported in the Département de la Santé des Forêts (DSF; French forest health monitoring agency) database. Although D. pini was detected on herbarium specimens from 1907 and 1965, it was not frequent in France in the past. Today, it is frequent, although not present throughout the country, being absent from the north and the east. There is no relationship between the D. pini distribution in France and the spatial pattern of DNB reported in the DSF database. Thus, the emergence of DNB in France cannot be explained by a recent arrival of D. pini.

First Report of Powdery Mildew Caused by Podosphaera pannosa on Prunus cerasus in France.

Podosphaera pannosa (Wallr.:Fr.) de Bary (anamorph Oïdium leucoconium Desm.) is described as the most frequent species causing powdery mildew of members of the Rosaceae family, especially on Rosa spp. and Prunus spp. P. pannosa is reported as cosmopolitan, but its occurrence on Prunus cerasus (cherry) is limited to Hungary (3). During spring 2011, typical symptoms of powdery mildew were observed in a Prunus cerasus orchard located in La Roche de Glun (southeastern France). On average, 25% of the shoots per individual tree were affected by this disease. Although several different cultivars were grown in the orchard, cultivar Bigalise alone displayed powdery mildew symptoms. The lower surface of the leaves was covered with superficial, white, dense mycelium, whereas the upper side showed discoloration, necrosing patches, and blisters. Microscopic slides were prepared from fresh material by gently pressing a clear adhesive tape onto the lower surface covered by mycelium, which was further stained with lactic acid/methyl blue. The presence of powdery mildew was confirmed by the observation of typical microscopic features of the anamorphic stage of the fungus (2). Conidiophores were erect. Conidia (oïdia) were hyaline and keg-shaped, and developed basipetally in chains of six to eight conidia. Conidial dimensions were 17 to 29 (23) × 9 to 17 (14) µm. No cleistothecia (teleomorphic state of the fungus) were observed. Species identity was determined by sequencing the ITS region of the rDNA followed by comparison with reference sequences available on GenBank (1). Fungal material was collected from infected leaves by scraping the mycelium with a sterile needle, and was transferred into 2-ml microtubes. Fungal total DNA was then extracted using a commercial plant DNA extraction kit and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair (4). Nucleotide sequence was determined and deposited in GenBank (Accession No. JN654341). Analysis of the sequence by BLAST showed 100% identity with Podosphaera pannosa. To our knowledge, this is the first report of Podosphaera pannosa on Prunus cerasus in France. This species was hitherto scarcely reported on cherry trees, and may deserve more attention in the future. References: (1) M. Gàbor et al. Eur. J. Plant Pathol. 131:135, 2011. (2) G. Grove et al. Page 12 in: Compendium of Stone Fruit Diseases. American Phytopathological Society, St Paul, MN, 1995. (3) L. Vajna et al. New Dis. Rep. 12:15, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, 1990.

First Report of Blight Disease on Buxus Caused by Cylindrocladium buxicola in France.

Cylindrocladium buxicola Henricot causes twig blight on Buxus spp., severe defoliation, and eventually death of plants, especially in young seedlings (1). The disease was first observed in the United Kingdom and New Zealand in the 1990s and recently, the fungus was detected in other European countries (1). In November 2006, box blight symptoms were observed in a Buxus sempervirens nursery located in the South West of France (La Reole). Since then, more diseased samples from other French sites, including forestry areas and ornamental garden nurseries, have been received, indicating that this disease is spreading. Symptomatic twig samples were sent for lab analysis and dark brown spots were observed on the leaves, sometimes coalescing to cover the entire leaf surface, with black streaks on the stems. Fungal fruiting structures were observed directly on the leaf surface and were examined with a stereomicroscope. Microscopic slides were then prepared by gently pressing a clear adhesive tape onto the surface covered by mycelium and spores, which was further stained with lactic acid and methyl blue. Cylindrical, straight, biseptate, hyaline conidia, 53.8 to 75.3 (64.4) × 4.4 to 5.2 (4.6) µm, were observed, sterile hyphae with terminal vesicles ended with a pointed apex, and conidiophores were penicilliate; all of those characters were consistent with C. buxicola (2). To support the diagnosis, fruiting structures were plated on malt agar media supplemented with 100 ppm of chloramphenicol. The pure culture obtained showed a whitish mycelium with a tan brown center that was in line with the original description of C. buxicola (2). DNA was extracted from the pure culture and the internal transcribed spacer (ITS) region was amplified by PCR using the ITS1-ITS4 primer pair (4). Nucleotide sequence was determined and deposited on GenBank (Accession No. JQ743502). BLAST analysis of the sequence showed 100% identity with all currently available C. buxicola ITS sequences, which confirmed our morphological diagnosis. To our knowledge, this is the first report of C. buxicola (teleomorph Calonectria pseudonaviculata) in France. The occurrence of this disease in France worries the nursery industry since losses can sometimes be dramatic as seen in United Kingdom, where the disease is widespread (3). References: (1) B. Henricot. The Plantsman 9:153, 2006. (2) B. Henricot and A. Culham. Mycologia 94:980, 2002. (3) B. Henricot et al. Plant Pathol. 49:805, 2000. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, 1990.

First Report of Black Sigatoka Disease in Banana Caused by Mycosphaerella fijiensis on Martinique Island.

Mycosphaerella fijiensis Morelet (anamorph Pseudocercospora fijiensis Morelet), the causal agent of black Sigatoka disease of banana, is considered to be the greatest economical threat for export-banana cultivation throughout the world because most cultivars are highly susceptible. The disease has a worldwide distribution throughout the humid tropical regions, but was still absent in some Caribbean islands hitherto. In Martinique Island, an intensive survey has been conducted by the plant protection service and the Fédération Régionale de Défense Contre les Organismes Nuisibles (FREDON) since April 2008 to detect as early as possible any outbreak of infection by M. fijiensis. In September 2010, typical symptoms of black Sigatoka were observed in a plantain crop located in Ducos Municipality (14°35.702'N, 60°58.221'W) in the west-central area of the island. Typical early symptoms were 1- to 4-mm long brown streaks on the abaxial leaf surface. The presence of the disease was further confirmed by the in situ observation of microscopic features of the anamorphic form of the pathogen (3). Typical pale brown, straight or slightly geniculate conidiophores were observed occurring singly or in little groups without any stroma, with a thickened wall at the conidial scars. Conidia were hyaline to pale olive, straight or slightly curved, with one to eight septa, and a conspicuous scar at the basal cell. The diagnosis was confirmed by real-time PCR targeting M. fijiensis-specific regions within the ß-tubulin gene (1). Positive results were consistently obtained with DNA extracted from infected banana tissue samples, and the identity of the amplicon was confirmed by sequencing (Accession No. HQ412771) and comparison with reference sequences deposited on GenBank. After this first finding, the survey was intensified and black Sigatoka symptoms were also observed in several other locations on the island, affecting a large range of susceptible cultivars (Grande Naine, French, and Figue Sucrée), and in plantations, backyards, and private gardens. The presence of the fungus in the samples was confirmed by PCR analysis of DNA extracted from symptomatic leaves with a M. fijiensis-specific ITS-based primer pair (2). The pathogen may have been introduced into Martinique by ascospores, from islands where black Sigatoka is present, that were blown by continuous southerly winds over a 2-week period in August 2010 that was immediately followed by heavy rains that favor disease development. To our knowledge, this is the first report of M. fijiensis on Martinique Island, showing that the disease is still spreading northward in this region of the Caribbean. References: (1) M. Arzanlou et al. Phytopathology 97:1112, 2007. (2) J. Henderson et al. Page 59 in: Mycosphaerella Leaf Spot Disease of Bananas: Present Status and Outlook. L. Jacome et al., eds. International Network for the Improvement of Banana and Plantain (INIBAP), Montpellier, France. 2003. (3) M. F. Zapater et al. Fruits 63:389, 2008.