Decomposition and Fungi of Needle Litter from Slow- and Fast-growing Norway Spruce (Picea abies) Clones

The fungal species involved in the decomposition of needle litter and their response to intraspecific genetic variation of trees are poorly known. First, we compared the needle decomposition and fungal decomposers underneath eight different Norway spruce clones in situ. This experiment revealed 60-70% loss of needle mass in two years. Although spruce clones differed considerably in growth (twofold height difference) and their needles differed in chemical composition, no significant difference was found for loss of needle mass under the spruce clones. Furthermore, the spruce clones did not affect the community structure of the fungal decomposers. Fungi inhabiting needle litter were identified by extracting ribosomal RNA (rRNA) and sequencing complementary DNA (cDNA) of internal trascribed spacer 1 (ITS1) region. The most frequent identifications were Lophodermium, Pezizales, Mycena, and Marasmius, suggesting that endophytic fungi were involved in the decomposition process. Second, we evaluated the potential of endophytes to decompose needle material in a microcosm experiment in which all other fungi than endophytes were excluded. Within 2 years, the endophytes had decomposed 35-45% of the needle mass. Sequences of Mollisia, Lophodermium, Lachnum, and Phialocephala were most frequently found in rRNA and rDNA extracted from the needles at the end of the microcosm experiment. The dominant needle endophyte in fresh, green needles was Lophodermium piceae, and this species was also found frequently in the needle material after 2 years of decay both in the field and laboratory experiments. Moreover, the relative abundance of Lophodermium-derived denaturing gradient gel electrophoresis (DGGE) bands correlated positively with the decomposition in the microcosm experiment. Hence, our results suggest a significant role of endophytic fungi, and particularly L. piceae, in the process of needle decomposition in boreal forests.     

Fungal Diversity of Norway Spruce Litter: Effects of Site Conditions and Premature Leaf Fall Caused By Bark Beetle Outbreak

Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocellulose matrix, which other organisms are unable to digest. However, little is known regarding the factors affecting components of fungal diversity. Here, we quantified richness of internal fungi in relation to litter nutrient and phenolic concentrations, sampling season (spring or fall), and premature leaf shedding due to low precipitation and infestation of bark beetles (mainly Ips typographus and Ips duplicatus). The study was conducted in 37-year-old Norway spruce [Picea abies (L.) Karst.] stands, with three plots each in mixed forest (MF) and coniferous forest (CF) site conditions in south-central Poland. Fifty-four species of sporulating fungi were identified in 2,330 freshly fallen needles sampled during 2003-2005, including 45 species in MF and 31 in CF. The significantly higher number of species in MF was likely related to moister conditions at that site. Among isolated fungi, 22% (12 species) were identified as endophytes of Norway spruce in prior studies. During spring of 2005, we found less than half the number of isolates and fungal species at each forest site as compared to fall for the two prior years. This pattern was observed in typical soil fungi (e.g., Penicillium daleae, Penicillium purpurogenum) and endophytes/epiphytes (e.g., Aureobasidium pullulans, Alternaria alternata, Cladosporium spp., and Lophodermium piceae). Premature shedding of needles was the most likely cause of this decline because it shortened the time period for fungi to infect green needles while on the tree. For all sites and sampling periods, richness of internal fungi was strongly and positively related to the age of freshly fallen litter (assessed using needle Ca concentration as a needle age tracer) and was also negatively related to litter phenolic concentration. Richness of internal fungi in freshly fallen litter may be adversely affected by low soil moisture status, natural inhibitors slowing fungal colonization (e.g., phenolics) and biotic (e.g., insect infestation) and abiotic (e.g., drought) factors that shorten leaf life span.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent

Root endophytes are common and genetically highly diverse suggesting important ecological roles. Yet, relative to above-ground endophytes, little is known about them. Dark septate endophytic fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are ubiquitous root colonizers of conifers and Ericaceae, but their ecological function is largely unknown. Responses of Norway spruce seedlings of two seed provenances to inoculations with isolates of four PAC species were studied in vitro. In addition, isolates of Phialocephala subalpina from two populations within and one outside the natural range of Norway spruce were also included to study the effect of the geographic origin of P. subalpina on host response. The interaction of PAC with Norway spruce ranged from neutral to highly virulent and was primarily isolate-dependent. Variation in virulence was much higher within than among species, nonetheless only isolates of P. subalpina were highly virulent. Disease caused by P. subalpina genotypes from the native range of Norway spruce was more severe than that induced by genotypes from outside the natural distribution of Norway spruce. Virulence was not correlated with the phylogenetic relatedness of the isolates but was positively correlated with the extent of fungal colonization as measured by quantitative real-time PCR.     

Endophytic fungi in forest trees: are they mutualists?

Forest trees form symbiotic associations with endophytic fungi which live inside healthy tissues as quiescent microthalli. All forest trees in temperate zones host endophytic fungi. The species diversity of endophyte communities can be high. Some tree species host more than 100 species in one tissue type, but communities are usually dominated by a few host-specific species. The endophyte communities in angiosperms are frequently dominated by species of Diaporthales and those in gymnosperms by species of Helotiales. Divergence of angiosperms and gymnosperms coincides with the divergence of the Diaporthales and the Helotiales in the late Carboniferous about 300 million years (Ma) ago, indicating that the Diaporthalean and Helotialean ancestors of tree endophytes had been associated, respectively, with angiosperms and gymnosperms since ≥300 Ma. Consequently, dominant tree endophytes have been evolving with their hosts for millions of years. High virulence of such endophytes can be excluded. Some are, however, opportunists and can cause disease after the host has been weakened by some other factor. Mutualism of tree endophytes is often assumed, but evidence is mostly circumstantial. The sheer impossibility of producing endophyte-free control trees impedes proof of mutualism. Some tree endophytes exhibit either a pathogenic or a putatively mutualistic behaviour depending on the situation. The lifestyle (mutualism, commensalism, parasitism) of most tree endophytes is, however, not known. They are just there in the tissue and resume growth at the onset of natural senescence of the host tissue on which they eventually sporulate. Density of colonization of conifer needles by endophytes increases with needle age. It is postulated that the needles die as soon as colonization density reaches a threshold value. Normally, the threshold is not reached before the onset of natural senescence. The threshold value may, however, be reached earlier under some adverse conditions, e.g. lack of light in dense stands. As a consequence, endophytes kill the needles prematurely. Needle endophytes could, thus, be useful to eliminate “parasitic” needle mass, i.e. needles which consume more than they produce.     

Interaction of soil filamentous fungi affects needle composition and nutrition of Norway spruce seedlings

The effects of the interactions of soil filamentous fungi (including saprotrophic, mycorrhizal and endophytic fungi) on several morphological and physiological parameters of Norway spruce seedlings [Picea abies (L.) Karst.] were studied in a pot experiment. Both mycorrhizal variants (Hebeloma bryogenes and Cadophora finlandica) were slightly inhibited with respect to the accumulation of aboveground biomass. However, these variants exhibited significantly improved foliar content of N, P, and Ca (H. bryogenes variant also K), compared to controls. The presence of the saprotrophic fungus Setulipes androsaceus attenuated the positive effect of mycorrhizal fungi on Ca, P, and K content, but did not reduce the positive effect of mycorrhizal fungi on the N content of the seedlings’ needles. Raman spectroscopy revealed that both mycorrhizal fungi increased the foliar content of carotenoids compared to the controls, but the effect diminished in the presence of S. androsaceus. In contrast, in the presence of the dark septate endophyte Phialocephala fortinii, needles exhibited significantly higher wax content and lower carotenoid content compared to the mycorrhizal variants. The presence of the saprotrophic isolate Serpula himantoides resulted in a decrease in needle waxes in comparison to controls. The needles’ carotenoid concentration was positively correlated with the levels of needle nutrients (N, P, K, Ca, Mg), while correlation of needle nutrients (N, P) with the needle wax concentration was negative. We conclude that not only individual fungi but also their interactions profoundly affect the nutrition and needle composition of Norway spruce seedlings.     

Damage caused by the Green Spruce Aphid to Norway and Sitka spruce needles

Three-month-old needles of Sitka spruce were less susceptible to Elatobium abietinutn than 15-month-old needles. Symptoms appeared after longer aphid feeding times but only a proportion of damaged needles fell. After short feeding periods symptoms appeared in more Norway spruce needles than in Sitka spruce, whereas longer feeding periods resulted in more needles producing symptoms in the Sitka spruce. The symptoms took 4—6 days longer to appear in Norway spruce, and needle fall followed a longer feeding period than on Sitka spruce. Following 72 h feeding, needle fall occurred more quickly on Sitka spruce than on Norway spruce. The time taken for needle fall to occur was inversely related to the feeding time in Sitka spruce but such a response was not evident in Norway spruce. The results are discussed in relation to the differences exhibited in the probing behaviour of the aphid on the two spruces.     

The community of needle endophytes reflects the current physiological state of Norway spruce

This study investigated fungal endophytes in the needles of Norway spruce (Picea abies) cuttings in relation to host tree growth. We also determined the prevalence of endophytes in needles incubated for six months. The cuttings originated from clonal origins showing slow- and fast-growth in long-term field trials but the heritable differences in growth rate were not yet detected among the studied cutting. Endophytes were isolated from surface-sterilized needles with culture-free DNA techniques. No significant differences were observed between endophyte communities of slow- and fast-growing clonal origins. However, the endophyte community correlated with the current growth rate of cuttings suggesting that endophytes reflect short- rather than long-term performance of a host. The concentration of condensed tannins was similar in slow- and fast-growing clonal origins but it showed a negative relationship with endophyte species richness, implying that these secondary compounds may play an important role in spruce tolerance against fungal infections. More than a third of endophyte species were detected in both fresh and decomposing needles, indicating that many needle endophytes are facultative saprotrophs. Several potentially pathogenic fungal species were also found within the community of saprotrophic endophytes.     

Diversity and pathogenicity of ophiostomatoid fungi associated with <Emphasis Type="Italic">Tetropium</Emphasis> species colonizing <Emphasis Type="Italic">Picea abies</Emphasis> in Poland

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.     

Fungal endophytes in seeds and needles of Pinus monticola

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95 % of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (χ² = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17 % nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.     

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae)

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.     

Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity

Calotropis procera has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its fungal endophytes. This study analyzed, for the first time, the community of endophytic fungi of C. procera outside its region of origin. A total of 156 fungal isolates distributed across 19 taxa were obtained from 468 fragments of C. procera leaves at different stages of maturation. The rate of endophyte colonization increased with the leaf age/development. The dominant species of endophytic fungi of C. procera introduced in Northeast Brazil were different from those found in studies on the same species and other species of the same genus in native regions. The dominant endophyte was Phaeoramularia calotropidis (63.5 %), followed by Guignardia bidwellii (21.1 %). Six isolates of endophytic fungi showed antimicrobial activity against human pathogenic micro-organisms and one plant pathogenic fungus. The antibacterial activity was more intense than the antifungal activity. The endophytic Curvularia pallescens (URM 6048) stood out inhibited Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes, the plant pathogenic fungus Colletotrichum dematium. Ecological and biotechnological aspects of endophytic mycota are discussed.     

湖北烟草内生真菌生物多样性和种群结构分析

【目的】研究传统药用植物烟草(Nicotiana tabacum L.)内生真菌的丰富度,揭示其种群多样性和群落结构,为烟草内生真菌资源的有效利用奠定基础。【方法】采用组织分离法进行烟草内生真菌的分离,通过形态学和分子生物学相结合的方法进行菌株分类鉴定,以香农多样性指数及相对分离频率反映内生真菌物种多样性及分布规律。【结果】从不同组织部位、不同生长时期、不同海拔样地的健康烟草中共分离获得539株内生真菌,根据r DNA-ITS系统发育分析鉴定为31属73种,香农多样性指数为2.78,曲霉属Aspergillus和镰孢属Fusarium为优势菌群,其相对分离频率分别为22.63%和12.99%。其分布规律表现为茎部内生真菌的多样性高于叶部和根部;随着生育期的延长,内生真菌多样性逐步增多;随着海拔高度升高,内生真菌的种类和数量呈现降低的趋势。【结论】烟草内生真菌具有丰富的生物多样性,其分布表现出组织、生长时期、海拔高度专化性。阐明内生真菌在烟草中的分布规律,可以为烟草内生真菌在农业生产领域的开发应用提供科学依据。     

Fungal decomposition of Abies needle and Betula leaf litter

The effect of litter type and incubation temperature on the ability of fungi to decompose leaf litter of subalpine trees was examined by a pure-culture test. Mass loss of Abies needle and Betula leaf litter and utilization patterns of lignin and carbohydrates were investigated under two temperature conditions (20 C and 10 C) and compared for 29 species in basidiomycetes, ascomycetes and zygomycetes. The decomposing ability was generally higher in basidiomycetes than in ascomycetes and zygomycetes. Mass loss (% original mass) of litter was higher in Betula than in Abies and higher at 20 C than at 10 C. The 29 fungi were divided into lignocellulose decomposers, cellulose decomposers and sugar fungi based on their substrate utilization in Abies and Betula litter. Mass loss of lignin and carbohydrates by lignocellulose and cellulose decomposers was higher in Betula than in Abies. Mass loss of carbohydrates was higher at 20 C than at 10 C, but the temperature did not influence mass loss of lignin, indicating lignin decomposition by fungi was less sensitive to temperature than carbohydrate decomposition. Lignin/carbohydrate loss ratio (L/C) of Collybia spp. that caused selective delignification was lower at 20 C than at 10 C. These results indicate that the decomposability of litter, lignin and carbohydrate was different between Abies and Betula and that temperature affected not only the rate at which fungi decompose litter but also the ability of fungi to use lignin and carbohydrates.     

The effect of previously infested spruce needles on the growth of the green spruce aphid, Elatobium abietinum, and the effect of the aphid on the amino acid balance of the host plant

The effect of chlorosis induced in needles of Sitka and Norway spruce by the green spruce aphid on growth of the aphid is investigated, and the effect of infestation of the aphid on amino acid levels in Sitka spruce foliage is reported. On both Sitka and Norway spruce green spruce aphids were heavier when reared on chlorotic (previously infested) needles than when reared on green (previously uninfested) needles. The effect was more pronounced on Sitka than on Norway spruce. Infestation of the aphid altered the amino acid balance of Sitka spruce foliage but not the concentration of total amino acids. Possible causes of chlorosis, the influence of individual amino acids on aphid growth, the potential effect of chlorosis on outbreaks of the aphid and the differences in susceptibility of Sitka and Norway spruce to damage by the aphid are discussed.     

赤松枯梢病叶内生真菌多样性研究

在微生物层面研究枯梢病与植物针叶内生微生物的互作关系,分析内生真菌的多样性差异,为松枯梢病的防控提供基础数据。利用高通量测序技术测定赤松(Pinus densiflora)不同染病程度的针叶内生真菌的多样性。结果显示,随着病害的加重,P. densiflora针叶内生真菌丰富度呈现出上升的趋势,多样性指数表现为先下降后上升的趋势。无病斑针叶内生真菌中,子囊菌门(Ascomycota)与担子菌门(Basidiomycota)相对丰度最高,优势属为Lapidomyces和Selenophoma,病害导致优势菌相对丰度的降低。通过对不同染病情况的P. densiflora针叶内生真菌的测定,明确了枯梢病不同发病程度的P. densiflora针叶内生真菌的多样性及群落结构组成。     

Spatial and temporal variation of fungal endophytic richness and diversity associated to the phyllosphere of olive cultivars

Fungal endophytes are micro-organisms that colonize healthy plant tissues without causing disease symptoms. They are described as plant growth and disease resistance promoters and have shown antimicrobial activity. The spatial-temporal distribution of endophytic communities in olive cultivars has been poorly explored. This study aims to investigate the richness and diversity of endophytic fungi in different seasons and sites, within the Alentejo region, Portugal. Additionally, and because the impact of some pathogenic fungi (e.g. Colletotrichum spp.) varies according to olive cultivars; three cultivars, Galega vulgar, Cobrançosa and Azeiteira, were sampled. 1868 fungal isolates were identified as belonging to 26 OTUs; 13 OTUs were identified to the genera level and 13 to species level. Cultivar Galega vulgar and season autumn showed significant higher values in terms of endophytic richness and diversity. At site level, Elvas showed the lowest fungal richness and diversity of fungal endophytes. This study reinforces the importance of exploring the combined spatio-temporal distribution of the endophytic biodiversity in different olive cultivars. Knowledge about endophytic communities may help to better understand their functions in plants hosts, such as their ecological dynamics with pathogenic fungi, which can be explored for their use as biocontrol agents.     

Diversity of endophytic fungal community associated with Piper hispidum (Piperaceae) leaves

Tropical and subtropical plants are rich in endophytic community diversity. Endophytes, mainly fungi and bacteria, inhabit the healthy plant tissues without causing any damage to the hosts. These fungi can be useful for biological control of pathogens and plant growth promotion. Some plants of the genus Piper are hosts of endophytic microorganisms; however, there is little information about endophytes on Piper hispidum, a medicinal shrub used as an insecticide, astringent, diuretic, stimulant, liver treatment, and for stopping hemorrhages. We isolated the fungal endophyte community associated with P. hispidum leaves from plants in a Brazilian forest remnant. The endophytic diversity was examined based on sequencing of the ITS1-5.8S-ITS2 region of rDNA. A high colonization frequency was obtained, as expected for tropical angiosperms. Isolated endophytes were divided into 66 morphogroups, demonstrating considerable diversity. We identified 21 isolates, belonging to 11 genera (Alternaria, Bipolaris, Colletotrichum, Glomerella, Guignardia, Lasiodiplodia, Marasmius, Phlebia, Phoma, Phomopsis, and Schizophyllum); one isolate was identified only to the order level (Diaporthales). Bipolaris was the most frequent genus among the identified endophytes. Phylogenetic analysis confirmed the molecular identification of some isolates to genus level while for others it was confirmed at the species level.     

Seasonal variation in the monoterpenes in needles of Picea abies (L.) Karst.

Summary This investigation was conducted to obtain information about the fluctuations in composition and amount of needle monoterpenes during the development of spruce needles. Studies conducted with two Norway spruce clones clearly revealed the existence of fluctuations. In juvenile needles, the amounts of the oxygenated terpenes increase constantly with age during the first 2 months of needle growth. The hydrocarbon terpenes dominate within the first weeks, some of them even showing a very distinct first maximum within the first 3 weeks after bud burst. All terpenes, including the oxygenated ones, have a maximum in June/July, which favours the hypothesis of a substitution of the hydrocarbons later on. There are significant changes even in mature needles of Norway spruce. The terpene level of 1-year-old needles of the clonal trees increased from spring to early summer and then dropped again towards winter. In addition, fluctuations in mature needles were shown for a set of ten wild trees. Needles of the same age class, which emerged in 1986, were sampled 4 times from 1986 to 1988. The needle terpene concentrations of the 1 -year-old needles were considerably lower in spring at the time of bud burst than in autumn. The terpene level of older needles thus seems to be influenced by biosynthetic and catabolic activities.     

Richness of cultivable endophytic fungi along an altitudinal gradient in wet forests of Costa Rica

We collected various plant species along an altitudinal gradient ranging from 400 to 2900 m in tropical wet forests of Costa Rica, isolated the associated endophytic fungi, and performed bioinformatic analyses to determine whether changes in altitude are related to changes in their richness and community structure. We showed that the richness of endophytic fungi varied along the altitudinal gradient, being higher in the lowest stratum and decreasing as elevation increases. Each stratum presented a particular composition and diversity of endophytes, although the whole population was characterized by the presence of a few dominant and apparently ubiquitous species, coexisting with a number of less abundant species that presented a more limited host range. These results have important implications for better understanding the role of altitude on the distribution and composition of endophytic fungal populations in tropical forests, but also for maximizing the number and diversity of endophytic isolates in bioprospecting campaigns.