Needle blight of pine caused by two species of Dothistroma in Hungary

Dothistroma septosporum and D. pini cause the serious pine needle disease, Dothistroma needle blight (DNB). Of these, D. septosporum has a global distribution, while D. pini is known only from the USA, Russia, Ukraine and France. During a study of D. septosporum isolates from Pinus nigra in Hungary, microsatellite markers revealed the presence of a second Dothistroma species. The aim of this study was to identify the DNB pathogens occurring in Hungary using four different molecular techniques. These included sequencing of the rDNA ITS region, a species‐specific ITS‐RFLP, mating type primers and a diagnostic microsatellite marker, Doth_A. Results showed that both D. septosporum and D. pini occur in Hungary and that both DNB pathogens were present on the same trees and in some cases, the same needles. Mating types MAT1 and MAT2 of D. septosporum were shown to be present in Hungary, but only the MAT2 of D. pini was found. In addition, examination of needles with DNB symptoms from Russia revealed the presence of D. pini on Pinus pallasiana and P. nigra as well as on a new host, Pinus mugo. The molecular markers applied in this study were sufficiently robust to identify and differentiate between the two DNB pathogens, both in culture and directly from needles. They will consequently be useful to document the geographical range and to monitor the spread of D. septosporum and D. pini in future studies.     

Dothistroma rhabdoclinis sp. nov. associated with Rhabdocline pseudotsugae on Douglas fir

Dothistroma rhabdoclinis, a new coelomycete on needles of Pseudotsuga menziesii is described. The fungus is associated, possibly as a hyperparasite, with Rhabdocline pseudotsugae, the causal agent of Rhabdocline needle‐cast of Douglas fir. The presence of D. rhabdoclinis interferes with and sometimes completely inhibits the production of ascomata of R. pseudotsugae. The cultural and morphological characters of the new fungus are compared with the only other known member of the genus, Dothistroma septospora.     

New hosts for Lecanosticta acicola and Dothistroma septosporum in newly established arboreta in Spain

A historical outbreak of needle blight disease was recorded during 2018 to 2019 in plantations of Pinus radiata and Pinus nigra in the North of Spain. The main pathogens involved in this historical outbreak were identified as Lecanosticta acicola and Dothistroma septosporum. Recently, a variety of tree species in three arboreta planted between 2011 and 2013 in the Basque Country as part of the European project REINFFORCE were showing symptoms of needle blight and defoliation. The aim of this study was to determine which pine species were affected with these pathogens. Tree species sampled included several provenances of P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda. Using molecular identification methods, Lecanosticta acicola was confirmed infecting Pinus brutia (Provenance: Alexandropolis, Greece and var. eldarica, Crimea) and represents a new host species for this pathogen. Pinus elliottii (Provenance: Georgia, USA) and P. ponderosa (Provenance: Central California, USA) are new host reports of L. acicola for Spain. Dothistroma septosporum was found for the first time on P. brutia (Provenance: Marmaris, Turkey) and P. ponderosa (Provenance: Oregon, USA) in Spain and was also detected infecting P. nigra (Provenance: Sologne Vayrières, France).     

Artificial inoculation and susceptibility of Pinus armandii to Dothistroma septosporum

Dothistroma needle blight (DNB) is a serious needle disease of conifers that primarily affects pine species (Pinus spp.). Dothistroma septosporum is one of the DNB pathogens that has a diverse range of host species excluding Pinus armandii. In 15 inoculated P. armandii seedlings, D. septosporum acervuli were observed in 43 infected needles of ten seedlings with a mean disease severity of 1.11% at 25 weeks after inoculations, demonstrating the potential of D. septosporum to cause symptoms on the needles of P. armandii via artificial inoculation. The disease severity of P. armandii was similar to the positive control, Pinus nigra (median 0.75 for P. armandii to 0.70 for P. nigra), thus, P. armandii acts under artificial conditions as a susceptible host species.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

Modelling of the spread of Dothistroma septosporum in Europe

Dothistroma needle blight (DNB), a disease affecting several pine species, is currently generating great concern in Europe. Caused by Dothistroma pini and Dothistroma septosporum, DNB affects pine needles and causes premature defoliation, which results in growth reduction and, in extreme cases, mortality. The disease has increased in importance in Europe over the last 20 years, with an increase in the number of observations of DNB in regions with large areas of Pinus sylvestris in northern Europe. This article presents a cell‐based spatiotemporal model for predicting the likelihood and intensity of the future spread of D. septosporum in Europe. Here, “spread” includes both invasion of new regions and infection of healthy stands within already‐colonized regions. Predicted spread depends on the availability of host species, climatic suitability of different regions to D. septosporum and dispersal of sexual and asexual spores from infected trees to surrounding forests via water splash, mist and wind. Long‐distance spread through transport of infected seedlings is also included in the model. Simulations of spread until 2007 and 2015 were used to validate the model. These simulations produced similar patterns of spread to those observed in Europe. Simulations for 2030 suggested that additional and new outbreaks are likely to occur in Scotland, southern Norway, southern and central Sweden, northern parts of Germany and Poland, Estonia, Latvia and south‐west Finland. Preventing the delivery of infected seedlings would be an effective method for reducing the spread of D. septosporum in the Nordic countries, Scotland and Ireland, the Baltic countries, and parts of Germany, Poland and Belarus. In these states, prevention of transport of infected seedlings can reduce the probability of additional spread by 15%–40%.     

Fungicide sensitivity of Dothistroma septosporum isolates in the UK

Dothistroma septosporum, a causal agent of Dothistroma needle blight (DNB), is a damaging fungal pathogen of pines that has recently started to affect native Scots pine woodlands in the UK. In addition to silvicultural methods, fungicide spraying of forest nursery stock can help prevent the spread of DNB. However, the effectiveness of modern single‐site fungicides against D. septosporum and the risk of fungicide resistance evolution remain largely unknown. In this project, we aimed to establish sensitivity profiles of D. septosporum to some widely used single‐site fungicide classes in vitro, and to determine whether fungicide resistance is already present, as this could increase the spread of D. septosporum genotypes on planting stock in native woodlands. For this purpose, we compared isolates of D. septosporum, originating from pine stands unexposed to fungicides, with isolates from nursery outbreaks, for sensitivity to a range of commonly applied fungicides. Most of the fungicides we tested were effective in vitro and we observed no significant shifts in sensitivity in forest nurseries. Although further tests in planta are required to confirm effectiveness of single‐site fungicides against D. septosporum, our results suggest that they can be successfully used in DNB control, although appropriate measures to prevent the evolution of fungicide resistance are strongly recommended.     

Microspatial genetic diversity of Dothistroma septosporum

The distribution of genetic diversity in a local population of Dothistroma septosporum was determined on a microspatial geographical scale. The study was conducted in a seed orchard of Pinus nigra divided into four plots, each of 0.84 ha in area. Ninety‐two isolates were obtained from needles with red band needle blight symptoms. Molecular variance, Mantel test and autocorrelation spatial analyses were performed with the use of random amplified microsatellites markers to test the null hypothesis of a random distribution of genotypes. The groups of isolates from individual plots demonstrated small differences as regards intrapopulation variation. The mean contribution of polymorphic loci for isolate groups was almost 77%, genetic diversity 0.270, while the Shannon coefficient was 0.389. The Mantel test demonstrated a significant, positive correlation between Huff's genetic distance and geographical distance. Significant spatial genetic structure was detected with autocorrelation coefficients being significant in the first two distance classes up to about 8–12 m.     

Dothistromin biosynthesis genes allow inter‐ and intraspecific differentiation between Dothistroma pine needle blight fungi

Dothistroma septosporum and D. pini are the causal agents of Dothistroma needle blight (DNB) of Pinus spp. in natural forests and plantations. The main aim of this study was to develop molecular diagnostic procedures to distinguish between isolates within D. septosporum, for use in biosecurity and forest health surveillance programmes. This is of particular interest for New Zealand where the population is clonal and introduction of a new isolate of the opposite mating type could have serious consequences. Areas of diversity in the dothistromin toxin gene clusters were identified in D. septosporum (51 isolates) and D. pini (6 isolates) and used as the basis of two types of diagnostic tests. PCR‐restriction fragment length polymorphism (RFLP) of part of the dothistromin polyketide synthase gene (pksA) enabled distinction between two groups of D. septosporum isolates (A and B) as well as distinguishing D. septosporum and D. pini. The intergenic region between the epoA and avfA genes allowed further resolution between some of the A group isolates in RFLP assays. These regions were analysed further to develop a rapid real‐time PCR method for diagnosis by high‐resolution melting (HRM) curve analysis. The pksA gene enabled rapid discrimination between D. septosporum and D. pini, whilst the epoA–avfA region distinguished the New Zealand isolate from most other isolates in the collection, including some isolates from DNB epidemics in Canada and Europe. Although this study is focused on differences between the New Zealand isolate and other global isolates, this type of diagnostic system could be used more generally for high‐throughput screening of D. septosporum isolates.     

Dothistroma Needle Blight in Slovenia is caused by two cryptic species: Dothistroma pini and Dothistroma septosporum

Dothistroma Needle Blight (DNB) has been known in Slovenia since 1971, but the disease symptoms have intensified in recent years. With this study, the DNB symptoms in Slovenia are for the first time connected to both phytopathogenic Dothistroma species: D. pini and D. septosporum. Based on the ITS‐rDNA comparisons, the Slovenian isolates of D. pini group with the D. pini isolates from France, Hungary and the USA and not with the D. pini isolates from Russia and Ukraine. Both mating types MAT1 and MAT2 of D. septosporum are present in Slovenia, while for D. pini, only MAT2 was found. The current widespread occurrence of D. pini in native forest stands indicates the prolonged presence of this species in Slovenia.     

The hosts and geographic range of Dothistroma needle blight in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) were studied in 2014–2017 around Slovakia. A total of 84 localities, both native and planted, were investigated, and the presence of DNB was confirmed in 73 of them. In all positive locations, symptoms typical of DNB were observed and the Dothistroma species was confirmed using species‐specific primers either from fungal cultures or directly from needles. Both Dothistroma species—D. septosporum and D. pini—were identified. Both species occurred together in 29 locations, only D. septosporum in 42 and only D. pini in two locations. The host range of D. septosporum included 10 pine species and two spruce species. The host range of D. pini comprised the same number of pine hosts but only one spruce species. Five pine hosts, P. aristata, P. coulteri, P. densiflora, P. jeffreyi, P. × schwerinii, and one spruce host P. abies are new hosts species of D. pini. P. densiflora and Picea pungens have earlier been reported to be susceptible for DNB. In this study, D. septosporum was found from both tree species.     

The hosts and potential geographic range of Dothistroma needle blight

Dothistroma needle blight, one of the most important foliar diseases of Pinus spp., is caused primarily by the fungus Dothistroma septosporum (Dorog.) Morelet, and to a lesser extent by Dothistroma pini Hulbary. The potential distribution and abundance of Dothistroma spp. was determined by (i) developing a process-oriented model of potential distribution of Dothistroma spp. from known locations, (ii) compiling a comprehensive list of susceptible host species from existing scientific literature and (iii) determining the distribution of susceptible hosts in areas predicted to be suitable for range expansion of Dothistroma spp. Using these three sources of information regions at risk were identified as those that were predicted to be suitable for range expansion by Dothistroma spp. and included significant areas of susceptible host species.     

First molecular detection of Lecanosticta acicola from Poland on Pinus mugo

The presence of quarantine pathogen Lecanosticta acicola, the causal agent of brown spot needle blight, was confirmed using molecular methods for the first time in northern part of Poland on the coastal area adjacent to the Baltic Sea. This area includes sandy beaches, where Pinus mugo and P. sylvestris were planted. Symptomatic needles were collected in 2017 from 20 P. mugo trees growing in one stand in Ustka. Typical symptoms of brown spot needle blight infection, including dead needle tips and central zones with yellow or reddish brown, circular spots in green tissue, were observed on all samples. Only, the asexual stage of L. acicola was obtained during this work. The pathogen species identity was confirmed using classical morphological methods (microscopic examination of the infected needles), real‐time and species‐specific priming (SSPP) PCR, and ITS sequencing. Analysis of mating‐type (MAT) genes showed the presence of both mating types in northern Poland.     

Dothistroma pini—Eine Gefahr fürPinus sylvestris?

Zusammenfassung Dothistroma pini wurde auf einer ganzen Reihe von Kiefernarten festgestellt. Sporenl?nge und das gelegentliche Auftreten der HauptfruchtformScirrhia pini belegen das Vorhandensein der var.linearis. Konidien wurden von M?rz bis in den November gefunden, besonders zahlreich von April bis Juni. Erstmals, festgestellt wurde das Pathogen aufPinus cembra, P. aristata, P. koraiensis undP. tabuliformis, allerdings ist anzunehmen, da? die Infektion dieser Arten nur unter starkem Infektionsdruck stattfindet und die wirtschaftliche Bedeutung des Pilzes in diesen F?llen sehr begrenzt ist. Gleiches gilt offensichtlich auch fürPinus sylvestris. Diese Kiefernart wird nach unseren Beobachtungen auch bei engem Kontakt mit sehr stark infizierten Schwarzkiefern nur unbedeutend oder überhaupt nicht befallen. In diese Richtung deuten auch die ersten Ergebnisse eines Infektionsversuches, bei demP. sylvestris symptomfrei blieb, w?hrendP. nigra z. T. sehr starke Symptome einer Infektion zeigte. Eine nennenswerte Sch?digung vonP. sylvestris konnte bei Freilandbeobachtungen in keinem Falle festgestellt werden. Gegenteilige Hinweise in der Literatur sind auf Verwechselungen (des Pathogens oder des Wirtes) zurückzuführen.

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Dothistroma pini—a danger for Pinus sylvestris?

Summary Dothistroma pini was found on several different pine species. The length of the conidia and the existence of the teleomorph, which sometimes could be noticed, prove the presence of the var.linearis of the fungus. Conidia were detected between March and November, in high quantities especially from April to June. For the first time, the pathogen has been mentioned onPinus cembra, P. aristata, P. koraiensis andP. tabuliformis. It must be assumed, however, that these species only become infected under high infection pressure. That means, that the economic importance of the pathogen is very limited in these cases. Obviously the same is true withPinus sylvestris. After our observations this pine species was little or not at all infested even when in direct contact with heavily attackedP. nigra-plantations. The result of an infection experiment, in whichP. sylvestris did not show any symptoms whereasP. nigra was partly heavily attacked, points in the same direction. Damages toPinus sylvestris worth mentioning could not be found during observations in the field. Opposite indications from the literature can be explained by mistaking pathogens or host species.

     

New host and country records of the Dothistroma needle blight pathogens from Europe and Asia

Dothistroma needle blight (DNB) is a serious disease of pines (Pinus spp.), with a worldwide distribution. It is caused by the ascomycete fungi Dothistroma septosporum (teleomorph: Mycosphaerella pini) and Dothistroma pini (teleomorph unknown). Recently, DNB was found on Pinus peuce in Austria, Pinus pallasiana in Ukraine and the European part of south-western Russia, as well as on Pinus radiata and Pinus wallichiana in Bhutan. Based on DNA sequence comparisons of the internal transcribed spacer and β-tubulin gene regions, isolates from Austria and Bhutan were identified as D. septosporum, while isolates from Ukraine and south-western Russia were identified as D. pini. Additional isolates studied from Pinus mugo in Hungary confirmed the presence of D. septosporum in this country. The record of D. septosporum on exotic P. peuce in Austria represents a new host report of this needle blight pathogen in Europe. Likewise, DNB and the associated pathogen, D. septosporum are reported from Bhutan, eastern Himalayas, for the first time. In addition, D. pini was found in two European countries and on a new host, P. pallasiana. These European records represent the only reports of D. pini from outside the north-central USA. Morphological examination of selected specimens from different hosts and countries showed that D. septosporum and D. pini overlap in the length of their conidia, while the width is slightly wider in D. pini than in D. septosporum. The differences in conidial width are so small, however, that identification of the two Dothistroma species solely based on morphology is virtually impossible. The new host and country records provided here are consistent with the continuing trend of reports of the DNB pathogens from new hosts and new geographical areas during the last two decades, particularly in the northern hemisphere.     

Needle blight caused by Dothistroma pini in Slovakia: distribution,host range and mating types

Dothistroma needle blight (DNB) has been observed in Slovakia during the last two decades. Up until 2017, Dothistroma septosporum has only been detected and molecularly confirmed to cause DNB in Slovakia. Here, we report the detection of Dothistroma pini at six localities around Slovakia, representing different plantation types. Four pine species (Pinus sylvestris, P. nigra, P. mugo and P. jeffreyi) were confirmed as hosts of D. pini in Slovakia, of which only P. mugo has been previously reported as host in Slovakia. Three gene regions (ITS, EF1 –α, and ß-tubulin) of each of the 13 isolates were sequenced and assigned as D. pini. Based on ITS sequences, the studied isolates represent the haplotypes Dp_HAP.1, Dp_HAP.2. Both mating types were detected but at different localities. Our results suggest that in addition to D. septosporum, D. pini may contribute to DNB also in Slovakia.     

First report of Lecanosticta acicola on non‐native Pinus mugo in southern Sweden

In 2017, severe symptoms of brown spot needle blight, similar to those caused by Lecanosticta acicola, were observed on needles of non‐native Pinus mugo var. Hesse planted in an arboretum in southern Sweden. Microscopic characterization and molecular diagnostics of isolates obtained from diseased needles confirmed the identity of L. acicola. This is the first report of the quarantine pathogen L. acicola in Sweden. Subsequent surveys are urgently needed to assess the presence and extent of the L. acicola invasion in Scots pine (Pinus sylvestris) stands in Sweden and other Scandinavian countries.     

Lophodermium pinastri and an unknown species of Teratosphaeriaceae are associated with needle cast in a Pinus radiata selection trial

Spring needle cast (SNC) in Tasmania is defined by the early casting of pine needles and appears in 6‐ to 7‐year‐old pine plantations after canopy closure. The severity of SNC can vary significantly among trees growing within the same plantation. Fungal communities were surveyed in a Pinus radiata family trial, using DNA extraction from needles, PCR with fungal‐specific primers and sequencing to detect the presence of fungal species. Samples were taken at the same time as the trees were scored for disease severity. Trees with contrasting levels of SNC disease severity have significantly different needle fungal communities, but family pedigree and different ages of needle are not clearly distinguished by their fungal communities. All common fungal pathogens previously implicated in causing SNC were identified from the study, but of these, only Lophodermium pinastri was correlated with high levels of disease. Several species of Teratosphaeriaceae were detected, and one of these was also strongly associated with needle cast.     

The hemibiotrophic lifestyle of the fungal pine pathogen Dothistroma septosporum

Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non‐host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi‐biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic‐type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100‐fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.