Quantification of Phaeocryptopus gaeumannii colonization in Douglas‐fir needles by ergosterol analysis

Current assessments of infection levels of Phaeocryptopus gaeumannii, the incitant pathogen of Swiss needle cast disease on Pseudotsuga menziesii, typically rely on surveys of abundance of fruit bodies on diseased needles. The relationship between this measure and internal fungal colonization is unknown. In this article, a series of experiments to determine whether ergosterol can be used to quantify P. gaeumannii internal colonization within Douglas‐fir needles is reported. It was found that ergosterol content in seven commonly occurring Douglas‐fir foliar fungi is proportionally related to biomass, and in P. gaeumannii this relationship is not affected by age of the culture. Furthermore, at four sites tested, P. gaeumannii was the most common fungus species isolated from Douglas‐fir needles, accounting for approximately 50% of the isolations. Ergosterol content in these needles was best related to P. gaeumannii despite the presence of other fungi. The strong relationship between ergosterol and P. gaeumannii is attributed to its greater contribution to total fungal biomass compared with all other fungi present within Douglas‐fir needles.     

Association of Tiarosporella parca with needle reddening and needle cast in Norway spruce

Needles of Norway spruce turned red-brown in late fall and were consequently shed. On these needles fungal fruit bodies of Tiarosporella parca were found. This fungus could also be isolated from green needles of all ages.     

Nutrient uptake by intact mycorrhizal Pinus sylvestris seedlings: a diagnostic tool to detect copper toxicity

We developed a nondestructive method for detecting early toxic effects of inflethal copper (Cu) concentrations on ectomycorrhizal and non-mycorrhizal (NM) Scots pine (Pinus sylvestris L.) seedlings. The fungal symbionts examined were Paxillus involutus (Fr.) Fr., Suillus luteus (Fr.) S.F. Gray and Thelephora terrestris (Ehrh.) Fr. The accumulation of Cu in needles and fungal development (ergosterol) in roots and infstrate were assessed. Inorganic phosphate (P(i)) and ammonium (NH(4) (+)) uptake capacities were determined in a semi-hydroponic cultivation system on intact P-limited plants that were exposed for 3 weeks to 0.32 (control), 8 or 16 &mgr;moles Cu(2+). Short-term effects of a 1-hour exposure to 32 &mgr;moles Cu(2+) on nutrient uptake rates were also determined. None of the Cu(2+) treatments affected plant growth or root ergosterol concentrations. The active fungal biomass in infstrate invaded by S. luteus was reduced by 50% in the 16 &mgr;M Cu(2+) treatment compared with the control treatment; however, colonization by S. luteus prevented an increased accumulation of Cu in the needles. In contrast, the 16 &mgr;M Cu(2+) treatment caused a 2.2-fold increase in needle Cu concentration in NM plants. Ergosterol concentrations in the infstrate colonized by P. involutus and T. terrestris were not affected by 16 &mgr;molar Cu(2+). Although P. involutus and T. terrestris were less sensitive to Cu(2+) than S. luteus, T. terrestris did not prevent the accumulation of Cu in needles of its host plant in the 16 &mgr;molar Cu(2+) treatment. Mycorrhizal plants consistently had higher P(i) and NH(4) (+) uptake capacities than NM plants. In the control treatment, specific P(i) uptake rates were almost 10, 4 and 3 times higher in plants associated with P. involutus, S. luteus and T. terrestris, respectively, than in NM plants, and specific NH(4) (+) uptake rates were about 2, 2 and 5 times higher, respectively, than those of NM seedlings. Compared with the corresponding control plants, a 3-week exposure to 8 &mgr;M Cu(2+) had no effect on the nutrient uptake potential of plants. In contrast, the 16 &mgr;M Cu(2+) treatment significantly reduced P(i) uptake capacity of all plants and decreased NH(4) (+) uptake capacity of seedlings colonized by S. luteus or T. terrestris. The 32 &mgr;M Cu(2+) 1-h shock treatment reduced specific NH(4) (+) and P(i) uptake rates of roots colonized by S. luteus to 39 and 77%, respectively, of the original rates. The Cu(2+) 1-h shock treatment reduced the NH(4) (+) uptake rate of NM plants by 51%.     

High performance liquid chromatography determination of ergosterolas a measure of ectomycorrhiza infection in Scots pine

A technique for determining the ergosterol content in mycorrhizal pine roots using HPLC was developed. Pure cultures of different my corrhizal fungi contained very similar and constant amounts of ergosterol, and calculations of fungal biomass in mycorrhizal roots based on ergosterol readings agreed well with results using other methods. The extraction and sample purification were simple and reliable. Consequently, the technique is considered practical wherever accurate estimates of the intensity of mycorrhizal infection are required.     

Presence of fungi in Scots pine needles found to correlate with air quality as measured by bioindicators in northern Spain

Atmospheric pollution has increased worldwide during the last decades affecting forest ecosystems. Thermal power plants give off high levels of contaminants, which can damage forest health. Bioindicators can be helpful tools used for evaluating environment changes by giving an accurate measure of the extent of pollution. The focus of this study was to analyse fungal mycoflora in Pinus sylvestris stands near a thermal power plant and the possible correlation between the presence of the fungi and the air quality as measured by lichens and mosses. Fungi associated with pine needles were selected as subjects of this study because of their phytopathological importance and association with the pine trees' decline. Samples for this study were selected from eight plots in close proximity to a thermal power station in northern Spain. Symptoms of decline were previously observed in all the plots before sampling was performed. Lichens were used as bioindicators of environmental quality in two ways, first using the Index of Atmospheric Purity and second through categorization of lichen species based on their sensitivity. In addition, botanical quality was measured regarding the presence or absence of mosses. With two seasonal samplings (autumn and spring) and the use of four types of pine needle plant material (green needles, dried needles, half–green, half‐dried needles and fallen needles), both endophytes and epiphytes present in the needles were isolated and observed. Thirty fungal species in total were identified of the 1095 isolates obtained. Furthermore, significant differences in fungal isolates were observed between seasons and among the different plant material. Results showed that both environmental air quality and botanical quality were negatively correlated with the relative isolation frequency of fungi. The higher number of isolates was attributed to a possible infection produced by fungi, which could be a leading factor in the trees' decline.     

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.     

Differential effects of plant ontogeny and damage type on phloem and foliage monoterpenes in jack pine (Pinus banksiana)

Coniferous trees have both constitutive and inducible defences that deter or kill herbivores and pathogens. We investigated constitutive and induced monoterpene responses of jack pine (Pinus banksiana Lamb.) to a number of damage types: a fungal associate of the mountain pine beetle (Dendroctonus ponderosae Hopkins), Grosmannia clavigera (Robinson-Jeffrey & R.W. Davidson); two phytohormones, methyl jasmonate (MJ) and methyl salicylate (MS); simulated herbivory; and mechanical wounding. We only included the fungal, MJ and mechanical wounding treatments in the field experiments while all treatments were part of the greenhouse studies. We focused on both constitutive and induced responses between juvenile and mature jack pine trees and differences in defences between phloem and needles. We found that phytohormone applications and fungal inoculation resulted in the greatest increase in monoterpenes in both juvenile and mature trees. Additionally, damage types differentially affected the proportions of individual monoterpenes: MJ-treated mature trees had higher myrcene and β-pinene than fungal-inoculated mature trees, while needles of juveniles inoculated with the fungus contained higher limonene than MJ- or MS-treated juveniles. Although the constitutive monoterpenes were higher in the phloem of juveniles than mature jack pine trees, the phloem of mature trees had a much higher magnitude of induction. Further, induced monoterpene concentrations in juveniles were higher in phloem than in needles. There was no difference in monoterpene concentration between phytohormone applications and G. clavigera inoculation in mature trees, while in juvenile trees MJ was different from both G. clavigera and simulated herbivory in needle monoterpenes, but there was no difference between phytohormone applications and simulated herbivory in the phloem.     

马尾松叶表微生物多样性

为发掘对林木生长发育有利的优良微生物资源,并筛选适合叶表微生物的收集方法,以马尾松针叶为试验材料,分别用悬摇法和超声波法收集马尾松叶表微生物,用扩增子高通量测序技术、MUSCLE和Qiime软件研究马尾松叶表微生物的多样性。结果表明:扫描电镜观测结果显示,马尾松针叶表面定殖有大量微生物,包括真菌(菌丝及孢子)和细菌。扩增子高通量测序结果表明,马尾松叶表微生物物种丰富,包含细菌运算分类单位(OTUs)490个,真菌OTUs 1273个。马尾松叶表细菌以未分类的蓝细菌属(unidentified_Cyanobacteria)(36.53%)、未分类的拜叶林克氏菌属(unidentified_Beijerinckia)(28.60%)、鞘氨醇单胞菌属(Sphingomonas)(2.35%)为优势属;叶表真菌以枝孢属(Cladosporium)(2.45%)、拟盘多毛孢属(Pestalotiopsis)(0.92%)、无头孢菌属(Capnobotryella)(0.91%)为优势属。针对叶表细菌多样性的研究表明,悬摇法和超声波法均有较高的物种检出度;在叶表真菌多样性的研究中超声波法优于悬摇法,但超声波法样品间数据变异性较大,测定结果不稳定。     

The relationship between Swiss needle cast symptom severity and level of Phaeocryptopus gaeumannii colonization in coastal Douglas‐fir (Pseudotsuga menziesii var. menziesii)*

This study examined 108 15‐year‐old Douglas‐fir (Pseudotsuga menziesii var. menziesii) trees to investigate whether trees exhibiting less severe Swiss needle cast (SNC) symptoms were more resistant (had less fungal colonization) or more tolerant (maintained healthy foliage under similar infection levels). Trees were sampled from six open pollinated families that were categorized into three disease severity groups (two families for each group; mild, moderate and severe disease symptoms). The amount of retained foliage and level of discoloration were visually assessed on trees in the field. Fungal colonization (as determined by proportion of stomata occluded with pseudothecia and by amount of Phaeocryptopus gaeumannii DNA in sampled needles) was measured on 1‐ and 2‐year‐old needles in the laboratory. Trees in the different disease severity groups were similar with respect to amount of fungus in their needles, yet the trees in the mild symptom group retained higher proportions of needles and maintained greener foliage. The relationship between amount of P. gaeumannii in needles and SNC symptom severity was statistically significant (p < 0.05) for amount of fungal DNA in 1‐year‐old needles and average needle retention (NR) over the last four growing seasons. Average NR decreased with increased amount of pathogen DNA in the mild disease symptom families. This relationship was reversed in the severe disease symptom group and there was no relationship in the moderate disease symptom group. Because the amount of P. gaeumannii DNA in foliage did not differ significantly among the groups, differences in symptom severity were attributed to tolerance, not resistance. Visual scoring of individual trees for average NR over the past four growing seasons could be used to effectively assess for SNC tolerance in Douglas‐fir.     

洋葱挥发油的抗真菌作用及其机理

采用砂堡培养基液体稀释培养法和薄层色谱分析法研究了洋葱挥发油抗真菌效果及机理,以期发现天然抗真菌药物,开发洋葱资源．结果表明,洋葱挥发油提取物对酿酒酵母、白假丝酵母、新生(?)球菌、黑曲霉、黄曲霉、红曲霉、青霉等真菌的生长有较强的抑制作用;其最低抑菌浓度酿酒酵母和白假丝酵母为0．25％,新生隐球菌为0．50％．薄层色谱分析表明:洋葱挥发油处理后的白假丝酵母的羊毛固醇、麦角固醇的含量降低,而角鲨烯的含量增高,说明洋葱挥发油可能抑制白假丝酵母麦角固醇的早期合成．     

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.     

Lophodermium piceae and Rhizosphaera kalkhoffii in fallen needles of Norway spruce (Picea abies)

Falling needles collected from under individual 70-year-old Norway spruce (Picea abies) trees for about four years were checked for fungal fruitbodies. The most common fungi were Lophodermium piceae and Rhizosphaera kalkhoffii. Maximum frequencies of fruitbodies occurred in late autumn for L. piceae (ca. 80% of the needles), in summer for R. kalkhoffii (ca. 60%) and in winter for the third most common fungus, Tiarosporella parca (ca. 10%). The frequencies of needles with fruiting fungi varied greatly within and between years. This is the first report of T. parca from Sweden.     

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.     

FDA‐active fungal mycelium and lignin concentrations in some needle and leaf litter types

The concentration of live fungal biomass (FDA mycelium) was investigated in seven chemically very different litter types, i.e. grey alder leaves, green and brown leaves of white birch, green and brown needles of both Scots pine and lodgepole pine. Concentration of FDA mycelium in the litters changed with the season, being low during the dry late summer and relatively higher during the more rainy, wet autumn. During autumns, when moisture was not limiting, lignin concentration and live fungal biomass were negatively related. It was concluded that the concentration of live fungal biomass in litter decreased linearly as the lignin concentration increased and thus also decreased linearly to decomposition level of the litter.     

Influence of ectomycorrhization and cesium/potassium ratio on uptake and localization of cesium in Norway spruce seedlings

Norway spruce (Picea abies (L.) Karst.) seedlings growing in a growth pouch system were used to investigate the effects of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex St. Amans) Quél. and various Cs/K ratios on the uptake of (134)Cs, expressed as a percentage of the total amount of (134)Cs supplied. The amount of (134)Cs taken up by seedlings increased with increasing Cs/K ratio. At a Cs/K ratio of 0.1, uptake of (134)Cs ranged between 7.2 and 7.3% and was independent of ectomycorrhizal status, whereas at Cs/K ratios >/= 1 uptake of (134)Cs varied from 8.1 to 11.1% for ectomycorrhizal and from 10.4 to 14.4% for non-inoculated plants. Ectomycorrhizal seedlings contained a lower concentration of (134)Cs than non-inoculated seedlings. Among plant parts, the amount of (134)Cs was significantly lower in needles and lateral roots of ectomycorrhizal seedlings compared with non-inoculated seedlings. Among fungal and seedling tissues, highest X-ray net counts of (133)Cs were measured in fungal hyphae of ectomycorrhizal mantles. X-Ray net counts of (133)Cs in lateral roots of ectomycorrhizal and non-inoculated plants were similar, but 5 to 10 times higher than in main roots and needles, suggesting an accumulation of (133)Cs in lateral roots and slow translocation to other plant parts. In contrast, X-ray net counts of K indicated that K was readily mobilized from lateral roots to main roots and needles. Elemental mapping showed a relatively homogeneous distribution of (133)Cs within the root.     

Fungi infecting Pinus sylvestris needles of various ages

The studies were carried out in three 8–11 year old stands of Pinus sylvestris in Southwestern Poland. They included the isolation of fungi from infection spots which were sampled after 1, 1, 5 and 2, 5 years on living needles developed in 1978 on lower and upper branches of tree crowns. There were 28 fungal species isolated from such needles. An attempt was made to evaluate the role of the 5 most numerous species in causing needle cast of Pinus sylvestris.     

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.     

Ecological interactions between fungi, other biota and forest litter composition in a unique Scottish woodland

The composition of forest litter and understorey layer, andfungal biomass (in terms of ergosterol) were measured in eightsubplots over a winter–spring period (January to April).The sampling site was positioned in a range of woodland habitats(variously dominated by beech, Fagus sylvatica; birch, Betulapendula x pubescens, and oak Quercus petraea) and a clear areacovered with grass (dominated by Holcus lanatus). The resultswere analysed together with data on bacteria and microinvertebratesavailable from parallel research. Levels of ergosterol in individualsubplots ranged between 50 and 160 µg g^–1 DW. Fungalbiomass decreased in March, and then increased significantlyin April. Stepwise regression models for ergosterol indicatedpositive relationships with moisture content (February), bacteria(all but February and March), flagellates (February) and plant-feedingnematodes and flies (January, overall). The relationships withroots, seeds, the collective variable ‘other microinvertebrates’(all March), amoebae (February) and fragments (March, overall)were negative, while the relationship between fungi- and microbial-feedingnematodes changed sign between February (–) and March(+). Results of analysis of covariance for fungal ergosterolwere significant only for January and the combined dataset.In January, fungi were shown to be significantly related toamoebae, bacteria and a collembolan Folsomia candida, whilethe only significant predictor returned by the overall modelwas bacteria. Correlation analysis confirmed some effects alreadynoted, and revealed a number of further interactions. The resultshighlighted the complexity of factors influencing temporal dynamicsand spatial variability of fungal biomass in forest litter.Most of the registered interactions appeared to be transient,and this should be taken into account while interpreting environmentalobservations. Interpretation of the specific relationships isgiven and implications for further research and overall ecosystemfunctioning 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.     

Foliar nitrogen concentrations and natural abundance of (15)N suggest nitrogen allocation patterns of Douglas-fir and mycorrhizal fungi during development in elevated carbon dioxide concentration and temperature

Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) seedlings were grown in a 2 x 2 factorial design in enclosed mesocosms at ambient temperature or 3.5 degrees C above ambient, and at ambient CO2 concentration ([CO2]) or 179 ppm above ambient. Two additional mesocosms were maintained as open controls. We measured the extent of mycorrhizal infection, foliar nitrogen (N) concentrations on both a weight basis (%N) and area basis (Narea), and foliar delta15N signatures (15N/14N ratios) from summer 1993 through summer 1997. Mycorrhizal fungi had colonized nearly all root tips across all treatments by spring 1994. Elevated [CO2] lowered foliar %N but did not affect N(area), whereas elevated temperature increased both foliar %N and Narea. Foliar delta15N was initially -1 per thousand and dropped by the final harvest to between -4 and -5 per thousand in the enclosed mesocosms, probably because of transfer of isotopically depleted N from mycorrhizal fungi. Based on the similarity in foliar delta15N among treatments, we conclude that mycorrhizal fungi had similar N allocation patterns across CO2 and temperature treatments. We combined isotopic and Narea data for 1993-94 to calculate fluxes of N for second- and third-year needles. Yearly N influxes were higher in second-year needles than in third-year needles (about 160 and 50% of initial leaf N, respectively), indicating greater sink strength in the younger needles. Influxes of N in second-year needles increased in response to elevated temperature, suggesting increased N supply from soil relative to plant N demands. In the elevated temperature treatments, N effluxes from third-year needles were higher in seedlings in elevated [CO2] than in ambient [CO2], probably because of increased N allocation below ground. We conclude that N allocation patterns shifted in response to the elevated temperature and [CO2] treatments in the seedlings but not in their fungal symbionts.