Phytophthora species isolated from ash stands in Białowieża Forest nature reserve

Five Phytophthora species were isolated from the rhizosphere of ash‐dominated areas of the Bia?owieza European lowland forest nature reserve area. Morphological and DNA analyses identified Phytophthora fragariaefolia, P. plurivora, P. cactorum, P. lacustris and P. pseudosyringae. This is the first record of P. fragariaefolia in ash forests and demonstrates that several species in the potentially highly damaging Phytophthora genus are present within a near‐pristine ecosystem.     

Association of seed‐borne Fusarium species on Pinus ponderosa with germination and seedling viability in Argentina

Pinus ponderosa seedlots from eight seed orchards in Western Patagonia, Argentina, were evaluated for the presence of Fusarium species, and isolates obtained were subjected to pathogenicity tests. The following species were isolated: Fusariumoxysporum, Fusarium equiseti, Fusarium verticillioides, Fusarium incarnatum, Fusarium acuminatum, Fusarium graminearum and Fusarium proliferatum. With the exception of F. proliferatum and F. graminearum all Fusarium species were recovered from P. ponderosa seedlings with symptoms of damping‐off. Five Fusarium species were re‐isolated from necrotic roots of seedlings showing chlorosis (F. oxysporum, F. equiseti, F. incarnatum, F. acuminatum and F. verticillioides). High damping‐off losses occurred in Fusarium‐contaminated seed of P. ponderosa and low disease incidence occurred on less contaminated seed. The role of seed‐borne inoculum in disease spread within forest nurseries is discussed.     

Pathogenicity of three Phytophthora spp. causing late seedling rot of Quercus ilex ssp. ballota

Within a research project on quality of plants for forestation of agricultural lands, we studied the aetiology of a late seedling rot affecting holm oak (Quercus ilex ssp. ballota) in two forest nurseries in southern Spain. Major disease symptoms were foliage wilting and necrosis of feeder roots. Phytophthora cinnamomi, Phytophthora cryptogea and Phytophthora drechsleri were isolated from necrotic roots of holm oaks. Selected isolates of the three Phytophthora species were pathogenic to Quercus ilex ssp. ballota and Quercus suber seedlings in artificial inoculations. Soil flooding conditions were essential for infection and root rot development. There was no host specificity among the species, the isolates of P. cinnamomi being the most virulent in all inoculated plants. In these inoculations, Q. ilex ssp. ballota plants were more susceptible than those of Q. suber. This work is the first report of P. cinnamomi, P. drechsleri and P. cryptogea affecting Q. ilex ssp. ballota in forest nurseries.     

Fungi Isolated from Diseased Nursery Seedlings in Spain

Fungal endophytes in container-grown seedlings in forest nurseries located in the North Plateau of Spain have been studied. Influences of nursery, season (spring and autumn), host genus and plant part on those fungal assemblages have been also evaluated. Four nurseries were sampled in spring and three in autumn. Analysed material consisted of stems, roots and rhizosphere from five host species, Quercus ilex, Q. pyrenaica, Pinus pinea, P. nigra and P. sylvestris. A total of 581 isolates corresponding to 13 taxa were found in this study. Fusarium moniliforme, F. oxysporum and Trichoderma viride were the most frequently isolated fungi. Number of fungal species per observation differed significantly among the sampled parts. Multivariate Correspondence Analysis allowed the discrimination of samples in five groups based on nursery, season, host genus and plant part. Isolation frequencies of F. moniliforme and F. oxysporum were higher in the roots and in spring, whereas those of T. viride were higher in the rhizosphere and in autumn. A relationship was observed between presence of T. viride and absence of Fusarium spp. in forest nurseries. T. viride, could serve as a biological control agent against Fusarium spp. in forest nurseries.     

First report of Calonectria montana causing damping‐off disease on pine and spruce seedlings in Europe

In 2015–2016, damping‐off symptoms were observed on Pinus sylvestris and Picea abies seedlings in four forest nurseries in Poland. Some Calonectria isolates were obtained from symptomatic seedlings by culturing pieces of necrotic tissue on malt extract agar.Morphological and molecular analyses revealed that isolates represent Calonectria montana, a recently described species from natural forests in China. The isolates represent the first detection of this species outside of Asia. Pathogenicity tests demonstrated that C. montana is a severe pathogen of P. sylvestris seedlings.     

Slow sand filtration for elimination of phytopathogens in water used in forest nurseries

Irrigation water from natural sources can be contaminated by fungi, Oomycota, or bacteria capable of causing serious diseases in seedlings in forest nurseries. Slow sand filtration (SSF) is a cost-effective technique for water decontamination prior to irrigation. The aim of the current study was to assess the effectiveness of SSF as a means of eliminating plant pathogens including Pythium sterilum, Fusarium solani, Xanthomonas campestris, Pseudomonas syringae pv. Syringae and Rhizobium radiobacter from the lake water source used for irrigation. The applied SSF was effective in removing 80–90% of fungal and Oomycota inoculum as well as 70% of the bacteria in the investigated water. Therefore, SSF represents a practical step to lowering the use of pesticides during production of planting stocks in forest nurseries and a valuable element in an integrated plant protection system.     

Characterization of Cylindrocarpon‐like species associated with litter in the old‐growth beech forests of Central Europe

Cylindrocarpon‐like fungi are globally distributed plant pathogens and have a wide range of host species. However, very little is known about the species that live in the topsoil of forests and their potential role in reducing the natural regeneration of tree species, particularly of forest trees that produce abundant fruit only once every few years. To enhance our understanding of the species that inhabit the topsoil, we studied the diversity and pathogenicity of Cylindrocarpon‐like fungi inhabiting the litter in old‐growth mixed‐beech forests in the Carpathians (Poland) and in the Alps (Austria), and in a managed beech stand in the Krakowsko‐Cz?stochowska Highland (Poland). The fungi inhabiting the beech litter were investigated using beechnuts and pine seedlings as bait. Isolates were identified based on morphology and DNA sequencing. The pathogenicity of the most common species was investigated by inoculating beech germinants. A wide range of Cylindrocarpon‐like fungi were associated with the beech litter: 718 cultures representing 12 species were isolated. Five taxa were identified down to species level: namely Ilyonectria crassa, I. pseudodestructans, I. rufa, Neonectria candida and N. obtusispora, and seven species were identified to genus level (Neonectria or Ilyonectria species). Ilyonectria destructans, which is considered to be the sexual morph of ‘Cylindrocarpon destructans’, was not found. There were qualitative and quantitative differences between the different forest sites in terms of Neonectria and Ilyonectria species composition in beech litter. The isolation frequency and species richness of Cylindrocarpon‐like fungi were greatest in beech litter taken from old‐growth mixed‐beech forests. Neonectria and Ilyonectria species were capable of killing beech germinants, suggesting that they may play a negative role in natural beech regeneration.     

Pathogenic fungi associated with pre‐ and post‐emergence seedling blight of pine and cypress in Fars Province,Iran

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and corn meal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization, respectively, and incubated at 25°C for 3–5 days. Decaying seeds of all plant species were also collected from nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies. Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia‐like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified as Fusarium proliferatum also caused pre‐emergence seed decay of pine and cypress. Isolates of Phytophthora and Pythium were the most virulent of these fungal isolates.     

Microbial communities in roots of Pinus sylvestris seedlings with damping‐off symptoms in two forest nurseries as determined by ITS1/2 rDNA sequencing

A methodological molecular procedure, which included extraction and cloning of the ITS1/2 rDNA of root‐associated organisms with subsequent transformation and sequencing of representative clones, was effective for detection, discrimination and determination of the frequency of the main damping‐off pathogens in roots of Pinus sylvestris seedlings growing in different forest‐tree nursery soils and exhibiting different rates of disease progress. Roots exhibiting slower damping‐off progression were colonized by Fusarium oxysporum, Neonectria radicicola (Ascomycota) and Pythium spp. (Oomycota), which comprised 50% of the microbial community. Roots exhibiting faster damping‐off progression were dominated by Thanatephorus cucumeris (Basidiomycota), which comprised 80% of the microbial community. The microbial community was more diverse in roots with slower damping‐off progression (14 species) than in roots with faster disease progression (seven species).     

Virulence of Fusarium oxysporum and F. commune to Douglas‐fir (Pseudotsuga menziesii) seedlings

Fusarium species can cause damping‐off and root rot of young conifer seedlings, resulting in severe crop and economic losses in forest nurseries. Disease control within tree nurseries is difficult because of the inability to characterize and quantify Fusarium spp. populations with regard to disease potential because of high variability in isolate virulence. Fusarium isolates were collected from healthy and diseased seedlings of Douglas‐fir (Pseudotsuga menziesii) and western white pine (Pinus monticola) from a nursery in Idaho, USA. Molecular markers such as DNA sequences (mitochondrial small subunit and nuclear translation elongation factor 1‐alpha) and amplified fragment length polymorphism were used to identify isolates as either F. oxysporum or F. commune. In addition, diagnostic primers were developed to detect and distinguish F. commune from F. oxysporum. In vitro and greenhouse virulence tests were completed on Douglas‐fir germinants and seedlings. For Douglas‐fir germinants and seedlings, F. oxysporum isolates generally caused less severe symptoms, whereas most F. commune isolates caused mortality through damping‐off. This is the first report of direct evidence that F. commune can cause damping‐off disease on Douglas‐fir seedlings under greenhouse conditions.     

Potential use of visible and near-infrared spectroscopy for pine species discrimination by examination of needles

The correct identification of pine species is necessary for proper application of wood in forest?based industries, since the quality of each species’ wood depends on factors intrinsic to the material. The aim of this study was to evaluate the potential use of near?infrared and visible spectroscopy in the discrimination of pine species planted in southern Brazil. Needles of Pinus clausa, P. glabra, P. kesiya, P. oocarpa, P. palustris, P. pseudostrobus, P. rigida, P. roxburghii and P. serotina were collected from experimental plantations located in the region of Rio Negro, Paraná, Brazil. The needles were dried and milled for analysis. The evaluation was performed with a spectral range of 400–750?nm (visible) and 1 000–2 500?nm (near?infrared). Analysis using the visible spectra resulted in two principal components explaining 95% of the variation between the needle samples. In the near?infrared analysis, it was possible to discriminate between all nine pine species studied using only two principal components, where the first explained 99% of the variation between species. Spectroscopy based on needles can be used for pine species discrimination, using the original data without mathematical treatment, in southern Brazil.     

Fungi isolated from Picea abies infested by the bark beetle Ips typographus in the Białowieża forest in north‐eastern Poland

The assemblage of fungi occurring in the sapwood of Norway spruce (Picea abies) and in bark beetle galleries following attack by the Eurasian spruce bark beetle Ips typographus was investigated in the Bia?owie?a forest in north‐eastern Poland. Fungi were isolated from blue‐stained sapwood of beetle‐infested spruce trees in June 2002, and a few isolates were also obtained from ascospores and conidia taken from perithecia and asexual structures occurring in the gallery systems of the insects. The mycobiota of I. typographus in the Bia?owie?a forest was dominated by ophiostomatoid fungi, which were represented by seven species. Four species, including Ceratocystis polonica, Grosmannia penicillata, Ophiostoma ainoae and Ophiostoma bicolor were isolated at high frequencies, whereas three other taxa, Ceratocystiopsis minuta, Ceratocystiopsis alba and a Pesotum sp. were rare. The anamorphic fungus Graphium fimbriisporum and yeasts also occurred occasionally. In addition, the basidiomycete Gloeocystidium ipidophilum was relatively common. The pathogenic blue‐stain fungus C. polonica was the dominant fungal associate of I. typographus in the Bia?owie?a forest, which is consistent with a previous study at this area in the 1930s. Ceratocystis polonica was the most frequently isolated species at the leading edge of fungal colonization in the sapwood and had on an average penetrated deeper into the wood than other fungal associates. This suggests that it acts as a primary invader into the sapwood after attack by I. typographus in the Bia?owie?a forest, followed by O. bicolor, O. ainoae, G. ipidophilum and G. penicillata. Thus far, the Bia?owie?a forest is one of the few areas in Europe, where C. polonica has been reported as a dominate fungal associate of I. typographus.     

Involvement of Phytophthora spp. in chestnut decline in the Black Sea region of Turkey

Chestnut blight caused by Cryphonectria parasitica is a serious disease of Castanea sativa in the Black Sea region of Turkey. During disease surveys, dieback and decline symptoms were observed on trees without apparent blight and ink disease symptoms. Black necroses, similar to those caused by Phytophthora infections, were noted on some of the chestnut coppices and saplings in one nursery in Ordu and led to an investigation into this disease complex. Only symptomatic plants showing dieback symptoms were investigated. Soil samples together with fine roots were collected from two directions, north and north‐east, approximately 150 cm away from the main stems. Phytophthora spp. were baited with young chestnut leaves. Three Phytophthora spp., P. cambivora, P. cinnamomi and P. plurivora, were identified from 12 soil samples collected from 73 locations, while from the nurseries, only P. cinnamomi was obtained. Phytophthora cinnamomi was the most common species, obtained from seven locations in five provinces and from four nurseries having similar symptoms mentioned above in different locations. Phytophthora cambivora and P. plurivora were less frequently obtained, from three to two stands, respectively. Phytophthora cinnamomi and P. cambivora were the most aggressive species when inoculated at the stem base on 3‐year‐old chestnut saplings, killing six saplings of eight inoculated in 2 months. The three Phytophthora species were first recorded on chestnut in Black sea region of Turkey with the limited samples investigated in a large area about 150 000 ha chestnut forest.     

Studies on anastomosis groups of Rhizoctonia solani isolates causing disease in two forest nurseries in Poland

Thirty‐eight isolates of Rhizoctonia spp. were isolated from Scots pine (Pinus sylvestris) seedlings with damping‐off symptoms, originating from two forest nurseries in central‐west Poland (Wronczyn and Jarocin) and from diseased seedlings grown in soil from Wronczyn nursery. Majority of these isolates (79%) had multinucleate cells and were identified as Rhizoctonia solani. The remaining isolates were recognized as binucleate Rhizoctonia spp. R. solani isolates were characterized using hyphal anastomosis and were divided into five anastomosis groups (AG). The most prevalent was AG5 (37% of isolates), followed by AG2‐1 (30%) and 27% of the isolates were identified as AG4. Groups AG1‐IB and AG2‐2 were only represented by single isolates. The virulence recorded as mortality (in percentage) was comparatively high for binucleate and multinucleate isolates of Rhizoctonia spp. Sequence analysis of the polymerase chain reaction (PCR)‐amplified internal transcribed spacer (ITS) rDNA region was used for phylogenetic analysis. The dendrogram showed that isolates were distinctly separated based on their AG types and there was no relationship between pathogenicity on Scots pine seedlings and the AG to which the isolates belong to. The results are discussed with respect to pathogenic potential of the various AG groups.     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.     

Identification of Phytophthora species baited and isolated from forest soil and streams in northwestern Yunnan province,China

Phytophthora species were surveyed by collecting soil samples and placing bait leaves in selected streams during June–October in the years 2005, 2006 and 2010 at three sites in oak forests in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, China. Seventy‐three isolates of Phytophthora spp. were recovered from 135 baited leaf samples and 81 soil samples. Eight Phytophthora species were identified by observation of morphological features and ITS1‐5.8S‐ITS2 rDNA sequence analysis. The eight taxa included two well‐known species P. gonapodyides and P. cryptogea, two recently described species P. gregata and P. plurivora, two named but as yet undescribed taxa, P. taxon PgChlamydo and P. taxon Salixsoil, and two previously unrecognized species, Phytophthora sp.1 and P. sp.2. The most numerous species, P. taxon PgChlamydo, and the second most abundant species, P. taxon Salixsoil, were recovered at all three sites. Phytophthora cryptogea was detected only once at site Nixi. Phytophthora gregata and P. sp.2 were isolated from a stream only at site Bitahai, while the other three species were each found at two sites. Phylogenetic analysis revealed that the isolates belonged to three ITS clades, one species including six isolates in clade 2, six species including 66 isolates in clade 6 and one species in clade 8. There was a relatively rich species and genetic diversity of Phytophthora detected in the investigated regions where the forest biotic and abiotic factors affecting the growth and evolution of Phytophthora populations were diverse.     

Rapid diagnosis of pathogenic Phytophthora species in soil by real‐time PCR

Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands.     

Aggressiveness of Fusarium oxysporum and F. verticillioides isolates on stone and scots pine under greenhouse conditions

Scots pine (Pinus sylvestris) and Stone pine (Pinus pinea) are two important species used in re-forestation that are subject to damage by damping-off fungi in forest nurseries. Twenty-two isolates of Fusarium oxysporum and F. verticillioides from diseased seedlings of eight different hosts were tested for aggressiveness on seeds and seedlings of both pine species, including the effects on seedling emergence and mortality. Scots pine was more susceptible to damping-off than Stone pine, as indicated both by reduced seedling emergence and elevated seedling mortality. The impact of F. oxysporum and F. verticillioides on seeds and seedlings did not differ significantly for either pine species. Our findings support previous studies that found that these are damping-off pathogens on the studied pines. Whereas most isolates proved to be pathogenic, some isolates of both Fusarium species showed to be non-pathogenic.     

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.