Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species‐specific PCR primers from the ribosomal ITS region

Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species‐specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries.     

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.     

Pathogenicity of three Lophodermium species on Pinus sylvestris L.

The pathogenicity of Lophodermium pinastri, L. conigenum and L. seditiosum on 14-month old transplants was investigated in Scotland. While L. seditiosum was found to be a serious pathogen, L. conigenum and L. pinastri were not. Under outdoor conditions, L. seditiosum was found to kill needles five months after inoculation.     

Genetic diversity of Lophodermium piceae in South Finland

Lophodermium piceae, the dominant needle endophyte of Norway spruce (Picea abies), is known to be extremely diverse. This work aimed to test the possible occurrence of cryptic species within the morphological species L. piceae. Genetic variation in 36 South Finnish L. piceae isolates originating from six localities was investigated by comparing DNA sequences of three genetic markers. One of the markers was the internal transcribed sequence (ITS) of the ribosomal DNA and the other two (LP1 and LP2) were based on sequence characterized amplified regions designed for L. piceae. The LP2 marker could be detected only from isolates of L. piceae but not from 20 other ascomycete species tested. This sequence, therefore, is considered as a species‐specific marker for L. piceae. For comparison, ITS sequences of isolates representing two other Lophodermium species, L. pinastri and L. seditiosum, were also investigated. In a neighbor‐joining analysis of ITS sequences all L. piceae isolates fell into one cluster, which was clearly separate from those of L. pinastri and L. seditiosum. Dendrograms of the three markers were incongruent indicating that the L. piceae population examined consisted of a single phylogenetic species. No geographical differentiation was observed. Our results confirm that L. piceae is a genetically highly diverse endophytic species.     

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.     

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.     

黄山松上的散斑壳

对安徽省岳西县妙道山国家森林公园黄山松上的散斑壳进行了调查研究，共发现６个种，其中岳西散斑壳系一新种；南方散斑壳，扰乱散斑壳为黄山松上的新记录；针叶树散斑壳，库曼散斑壳，松针散斑壳为国内已记载的种。对新种作了汉文和拉丁文描述，记载了已知种的戽性。     

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.     

Physiological studies on variation in resistance of Pinus sylvestris to Lophodermium pinastri

Buffering capacity and pH values of extracts of pine needles varied with age in the course of the ontogeny of needles. Significant differences between a resistant and a susceptible pine clone group in relation to resistance against Lophodermium pinastri could be shown in autumn and winter. Also the variability of the parasite influences the host-parasite interaction as shown by alteration of pH values by 3 L. pinastri strains in cultural trials on needle extract medium.     

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.     

Fungicidal Control of Lophodermium seditiosum on Pinus sylvestris Seedlings in Swedish Forest Nurseries

During the 1990s, there were serious outbreaks of the pathogen Lophodermium seditiosum on pine seedlings in Swedish forest nurseries, even though the seedlings had been treated with the fungicide propiconazole. The present experiment was carried out to evaluate two other fungicides, fluazinam and azoxystrobin, as possible alternatives to propiconazole. In the tests, which were all carried out in the same forest nursery, seedlings were treated with either propiconazole, fluazinam or azoxystrobin, and the proportion of needles with ascocarps of L. seditiosum and the number of ascocarps per needle were recorded over the following 2 yrs. Seedlings treated with azoxystrobin already appeared healthier than control seedlings in September of the first year, and by November all azoxystrobin-treated seedlings had fewer ascocarps per needle compared with control seedlings. In autumn of the second year, there were no ascocarps on seedlings treated with fluazinam or azoxystrobin, whereas seedlings treated with propiconazole had similar numbers of ascocarps to non-treated control seedlings.     

Seasonal patterns of colonization of Norway spruce needles by Lophodermium piceae

Summary Colonization of living Norway spruce needles by Lophodermium piceae was studied in southern Sweden. One‐month to 3‐year‐old needles were assessed for frequency of infection. Needles appearing in the growing season of 1996 were monitored and sampled at 1 to 4 week intervals over a period of 14 months. Infection frequency of the current‐year needles increased from around 20% in July to approximately 40% in August 1996. This level had then doubled to approximately 80% after another growing season in August 1997. Multiple infections were rare in the needles during the first growing season, but after the second growing season at least 70% of the needles had multiple infections. The average number of infections in 9‐, 21‐ and 33‐month‐old needles were 1.3, 6.3 and 8.4, respectively. The size of the individual mycelia was estimated to be small in all needles, none of the individuals could be isolated from more than two adjacent needle sections, about 1.5 mm long. In conclusion, the number of L. piceae infections per needle increased during ageing of needles but mycelial growth is probably postponed until needle senescence and death.     

Veranderung der Krankheitsdisposition von Forstpflanzen durch Herbizide

Modification in disease-sensibility of forest plants by herbicide treatment. II. Laboratory studies on Lophodermium pinastri. The effect of TOP-KH (2, 4, 5-T) and Dichlobenil on the growth of Lophodermium pinastri in culture was studied in the laboratory. The effect of aqueous solutions of TOP-KH on germination of Lophodermium spores was also tested. Assessments were also made of the number of apothecia produced on herbicide-treated plants, their size and the number of spores emitted. A final discussion of results is given.     

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.     

Über Sukzession und pathogene Eigenschaften Kiefernnadeln bewohnender Pilze

Succession and pathogenic properties of fungi inhabiting pine needles . Isolates from healthy and dying pine needles of various age permit conclusions about the relationship of some needle fungi to their host. Of the six most prevalent fungi, only Lopbodermium seditiosum has a pathogenic nature.     

Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Co‐occurrence of the ascomycete Lophodermium piceae and the rust fungus Chrysomyxa abietis in Norway spruce needles

The frequency of needles and the proportion of needle segments infected by Lophodermium piceae were compared in symptomless and Chrysomyxa abietis‐infected, 1‐year‐old needles of Picea abies. In late spring, symptomless needles from both rust‐infected and healthy saplings were sampled. In addition, rust‐infected, totally chlorotic needles and needles with chlorosis along about half their length from the diseased trees were examined. In all three stands, the proportion of segments infected by L. piceae was larger in the rust‐infected half of the needle than in the symptomless half; but the difference was statistically significant in only one of the stands. The proportion of L. piceae‐infected segments among the nonrust‐infected needles was the same as that found for the uninfected half of rust‐infected needles (after correction for size differences). No differences in the proportion of L. piceae‐infected segments were found between the totally chlorotic, rust‐infected needles and the green needles of diseased or healthy trees.     

Endophytische Pilze in Nadeln von gesunden und geschädigten Fichten (Picea abies [L.] Karsten)

Endophytic fungi in needles of healthy-looking and diseased Norway spruce (Picea abies [L.] Karsten). Endophytic fungi were isolated from surface-sterilized, 5 to 6 year old needles of healthy-looking and diseased trees. Two fungal species could be isolated frequently: Lophodermium piceae and Tiarosporella parca. At any site neither L. piceae nor T. parca could be isolated more frequently from needles of diseased trees. The possible types of relationship between these two fungi and Norway spruce are discussed.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.