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.     

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).     

Fungi associated with Cyclaneusma needle cast in Scots pine in the west of Poland

Needles of Pinus sylvestris with and without symptoms of Cyclaneusma needle cast, from the west of Poland, were examined for abundance and diversity of fungi using Illumina sequencing. Fungal communities were dominated by Ascomycota (93.6%–98.6% of OTUs). Basidiomycota and non‐culturable fungi were less frequent. Needles were colonized by 260 taxa. Ascomycota, Basidiomycota and Glomeromycota were represented by 149, 39 and 1 taxa. Abundance of fungi was least in 1.5‐year‐old needles attached to twigs and greatest in fallen 2‐year‐old needles. Fungal communities had least diversity in 1.5‐year‐old needles and most diversity in 0.5‐year‐old needles of current growth. It was found that (a) the most common fungi were the needle pathogens Cyclaneusma minus, Lophodermium spp. and Sydowia polyspora; (b) less common potential pathogens were Cenangium ferruginosum, Coniothyrium complex, Desmazierella acicola, Neocatenulostroma germanicum and species in the genera Neodidymelliopsis, Pestalotiopsis, Phoma, Pleurophoma and Pyrenochaeta; (c) common primary or secondary saprotrophs included species of Alternaria, Aureobasidium, Beauveria, Cladophialophora, Cladosporium, Epicoccum, Exophiala, Lecanicillium, Penicillium, Cryptococcus and Kwoniella; (d) Lophodermium was represented mostly by Lophodermium pinastri which occurred 4–72 times more frequently than Lophodermium seditiosum; (e) frequencies of C. minus and C. ferruginosum were lower in the 0.5‐year‐old symptomless needles, increased in the symptomatic and symptomless 1.5‐year‐old needles and decreased after needle fall; (f) frequency of L. seditiosum was highest in 0.5‐year‐old needles; (g) frequency of L. pinastri increased with needle age whereas S. polyspora increased after needle fall; (h) lower frequency of L. pinastrii was associated with higher frequency of S. polyspora. It was concluded that Cyclaneusa needle cast in Poland may be caused by C. minus accompanied by C. ferruginosum, L. seditiosum, L. pinastrii and S. polyspora. Participation of Coniothyrium spp., Epicoccum nigrum, Pestalotiopsis spp. and Phoma spp. in the disease progress cannot be excluded.     

Infection approach of Diplodia sapinea,the causal agent of tip blight of Pinus tabulaeformis in China

To clarify the infection approach of Diplodia sapinea, a pathogen that causes tip blight of Pinus tabulaeformis, the infection process of the pathogen in needles was observed using scanning electron microscopy (SEM). In addition, the disease incidence on branches damaged by Aphrophora flavipes (Hemiptera: Cercopidae) and Dioryctria splendidella (Lepidoptera: Pyralidae) in the forest was also investigated. Then, branches and needles of P. tabulaeformis were inoculated using the D. sapinea spore suspension under indoor and field conditions. The results showed that the damage caused by A. flavipes could aggravate the occurrence of tip blight of P. tabulaeformis to some extent. Moreover, the pathogen could also penetrate 1‐, 2‐ and 3‐year‐old pine needles through stomata in the field. The pathogen infected the 1‐year‐old branches first and then gradually spread to 2‐ and 3‐year‐old branches.     

Invasive brown spot needle blight caused by Lecanosticta acicola in Estonia

Lecanosticta acicola (Thüm.) Syd., a serious foliage pathogen of pines in many regions of the world, is an emerging invasive species in northern Europe. After the first record of L. acicola on Pinus ponderosa Dougl. ex Laws. in northern Estonia in 2008, monitoring was started to investigate the spread and host range of the fungus in the country. By the beginning of 2015, L. acicola was also recorded on P. uncinata Mill. ex Mirb., P. mugo Turra and on P. mugo var. pumilio (Haenke) Zenari, being the northernmost records of the fungus in Europe. So far, the single native pine species Pinus sylvestris L. has not been found to be infected. Molecular analysis proved infection of L. acicola on pines in five different localities of Estonia: in Tallinn (mainly in the Botanic Garden, northern Estonia), in Tori and Kärdla (western Estonia), in Vasula and Kärevere (east-central Estonia). The sexual state (teleomorph) of the fungus was not found, but the existence of the both mating types, MAT1-1 and MAT1-2, was confirmed.     

Hendersonia acicola on pine needles infected by Lophodermella sulcigena

Hendersonia acicola normally infects only needles already infected, in July-August of their first year, by Lophodermella sulcigena. Secondary infection, by H. acicola, occurs at the base of the L. sulcigena lesion either in August-October of the first year (early secondary infection) or after fruiting of L. sulcigena in June-July of the second year (late secondary infection). Early secondary infection prevents fruiting of L. sulcigena. An intercellular matrix forms at the base of the L. sulcigena lesion in response to both early and late secondary infection and rapid tissue disorganisation and disintegration ensues. Secondary infection decreases with increase in height up the tree and often results in needle breakage.     

First report of Mycosphaerella dearnessii in Switzerland

Brown-spot needle blight caused by Mycosphaerella dearnessii [syn. Scirrbia acicola; anamorphic state: Lecanosticta acicola (syn. Septoria acicola)] was observed for the first time in Switzerland. So far, the disease seems to be confined to planted Pinus mugo and Pinus uncinata near Zurich. Pinus sylvestris adjacent to diseased P. uncinata did not show any disease symptoms.     

Intraspecific response of Pinus taeda L. to Grosmannia huntii and Leptographium terebrantis infection

We examined intraspecific and inter‐year variation in tolerance of Pinus taeda to two ophiostomatoid fungi, Leptographium terebrantis and Grosmannia huntii. Containerized seedlings of P. taeda from 27, 32, 17 and 23 different elite genetic families were artificially inoculated with L. terebrantis and G. huntii in years 2013, 2014, 2016 and 2017, respectively. Six connector families were inoculated every year. Eight weeks post‐inoculation, lesion and occlusion were measured on each seedling to determine the relative susceptibility/tolerance of families to these fungi. Pinus taeda families widely differed in these parameters suggesting intraspecific variation in the susceptibility/tolerance to the inoculated pathogens. The overall tolerance of the connector families to these fungi varied among the experimental years. These results showed that intraspecific variation to L. terebrantis and G. huntii exists among P. taeda families and it could be possible to select tolerant families to minimize the potential impact due to these fungi.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.     

Morphological and molecular characterization of Leptomelanconium allescheri associated with necrotic lesions on Pinus mugo needles in the Polish Tatra Mountains

Leptomelanconium allescheri was identified for the first time in Poland in 2016 in the Tatra Mts. on Pinus mugo needles. It produced numerous conidiomata on necrotic lesions on live needles. In our study, we provide detailed characteristics of disease symptoms and describe morphology of the structures of the fungus. The shape and size of macroconidia are proven to be more variable in vitro than in situ. The malt extract agar colonies of the fungus obtained from germinating macroconidia are described for the first time. Our observations prove also that, apart from macroconidia, L. allescheri produces in vitro unicellular, hyaline microconidia, 3.5–10 × 1.8–5.0 μm in size, the previously unknown aspect in the development cycle of this species. The BLAST search with ITS and LSU sequences of L. allescheri resulted in the closest match with Cenangium acuum accessions (94%–95% and 99%, respectively). Two gene based, LSU plus ITS, phylogenetic positioning of L. allescheri proves that it belongs to Pezizomycotina, Helotiales. Further, on family level, placement of L. allescheri, as well as its two closest species C. acuum and Piceomphale bulgarioides, remains undefined. The L. allescheri barcode sequences (accession numbers: ITS— MF573935 , LSU— MF573936 , IGS— MF573937 ) and cultures were submitted to NCBI GenBank and CBS Utrecht, The Netherlands culture collection.     

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.     

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.     

Phosphorus supply and cycling at long-term forest monitoring sites in Germany

In this study, the supply and input–output balances of phosphorus (P) were investigated for a 10-year-period at 85 long-term monitoring sites in German forest ecosystems under the European Level II programme. These sites encompass 23 European beech (Fagus sylvatica L.) stands, 9 oak stands comprised of common oak (Quercus robur L.) and/or sessile oak (Quercus petraea Liebl.), 20 Scots pine (Pinus sylvestris L.) and 33 Norway spruce (Picea abies H.Karst.) stands. We quantified P concentrations in needles and leaves, P inputs from the atmosphere, P outputs through leaching and harvesting, and total P in the soil and humus layers. The P concentrations in European beech leaves from two sites (>1 mg P g⁻¹ dry weight), and in Norway spruce needles from four sites (>1.2 mg P g⁻¹ dry weight), were deficient over several years. In contrast, the oak and Scots pine sites were well supplied with P. When P removal through harvesting was disregarded, P balances were positive or stable (median 0.21 kg P ha⁻¹ a⁻¹). With harvesting, balances were mostly negative (median −0.35 kg P ha⁻¹ a⁻¹), with long-term P removal from the forest ecosystems.     

Study on biological characteristics ofHendersornia acicola

The morphological and cultural characteristics ofHendersonia acicola Munch & Tub., isolated from the needles ofPinus sylvestris var.mongolica Litvin, were described. The fungi was cultured on three kinds of media and the speed of mycelial growth and the required time to produce conidia were different. On the medium of needle decoction agar, the fungi grew fast and produced conidia after 5 days culturing. On PDA came second and it needed 7 days to produce conidia. On the medium of maize flour agar, it grew slowly and produces conidia after 27 days incubating. The fungi cannot produce conidia under darkness on all three kinds of media. The conidia can germinate at 8°C–28°C. The optimum conditions for conidia to germinate are 25°C, RH 100% and pH 4 and under these conditions it only takes four hours for conidia to germinate. The conidia germination is not affected by light     

Pinus taeda L. response to differential inoculum density of Leptographium terebrantis colonized toothpicks

Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm³ per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.     

Pseudomonas associated with Bursaphelenchus xylophilus,its insect vector and the host tree: A role in pine wilt disease?

In this study, we characterized the diversity of Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector (Monochamus galloprovincialis) and its host (Pinus pinaster), by a culture‐independent approach using rpoD clone libraries. Clone libraries of Pseudomonas rpoD were obtained from B. xylophilus, M. galloprovincialis and infected P. pinaster. Most M. galloprovincialis and B. xylophilus sequences grouped together in the P. fluorescens group. Genes related to xenobiotics degradation and phenylacetate synthesis were present in the genomes of the type strains closely related to sequences retrieved from the nematode libraries. Results demonstrated that the nematode, during its life stages inside the tree, maintains a diverse Pseudomonas community that is closely related to the one associated with the insect vector. These bacteria might contribute to degradation of xenobiotics and tree weakening during the nematode tree infection.     

Sources of inoculum of Lophodermium seditiosum on Pinus sylvestris1

A survey of the incidence of Lophodermium seditiosum, L. pinastri and L. conigenum in plantations of Pinus sylvestris in north-east Scotland showed that L. seditiosum predominated on fallen cones and on young needles which tell prematurely in early summer. L. pinastri predominated on old senescent needles which fell in autumn. The incidence of L. conigenum was low on fallen cones and needles and it is not thought that tins species could effectively replace L. seditiosum. It is concluded that the main source of L. seditiosum inoculum for infections in plantations and nearby nurseries is young infected fallen needles on which ascocarps form in late summer and autumn.     

The influence of soil scarification on the turn‐over rate of slash needles and nutrient release

The effect of soil scarification on decomposition of green Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) needles and mineral nutrient release was investigated. The treatments studied were ploughing, disk trenching, mound on inverted humus and untreated control. The study was conducted on two clear‐felled sites with different ground moisture conditions, located in south Sweden. The needles decomposed and released their contents of N and Ca considerably faster on scarified areas than on untreated ones. For P, Mg and especially K, only minor differences between scarified and untreated areas were established. At the wet site, needle decomposition and nutrient mineralization were generally affected to the same extent by all three scarification methods studied. At the dry to fresh site, mounding and ploughing enhanced needle decomposition and nutrient mineralization more than disk trenching. The effect of different scarification methods on the long‐term production capacity of soils is discussed.     

Colonization and lignin decomposition of pine needle litter by Lophodermium pinastri

The colonization and lignin decomposition of pine (Pinus thunbergii and Pinus densiflora) needle litter by an endophytic fungus, Lophodermium pinastri, was examined with field observations, a field experiment and laboratory experiments. In pine needles collected from the field, needle mass per length and lignin content were lower in needle portions bearing Lophodermium fruiting bodies than in the remaining needle portions, whereas total carbohydrate content was not different between them. Total and live hyphal lengths were greater in needle portions bearing Lophodermium fruiting bodies than in the remaining needle portions. Lophodermium fruiting bodies were not formed on sterilized needles after a 6‐month incubation on the forest floor, whereas they formed on 20% of non‐sterilized needles, indicating that this fungus can only colonize live needles on the branch. In pure culture decomposition tests, mass loss of lignin was detected for several isolates of L. pinastri, but was variable among isolates and between the needles of two pine species. A comparison of the rDNA‐internal transcribed spacer sequences between L. pinastri isolates of P. thunbergii and P. densiflora indicated that the two groups were phylogenetically separated. This study is the first to show that L. pinastri has the ability to decompose lignin in dead pine needles.