Laura M. Suz

Environmental drivers of ectomycorrhizal communities in Europe's temperate oak forests

Ectomycorrhizal fungi are major ecological players in temperate forests, but they are rarely used in measures of forest condition because large‐scale, high‐resolution, standardized and replicated belowground data are scarce. We carried out an analysis of ectomycorrhizas at 22 intensively monitored long‐term oak plots, across nine European countries, covering complex natural and anthropogenic environmental gradients. We found that at large scales, mycorrhizal richness and evenness declined with decreasing soil pH and root density, and with increasing atmospheric nitrogen deposition. Shifts in mycorrhizas with different functional traits were detected; mycorrhizas with structures specialized for long‐distance transport related differently to most environmental variables than those without. The dominant oak‐specialist Lactarius quietus, with limited soil exploration abilities, responds positively to increasing nitrogen inputs and decreasing pH. In contrast, Tricholoma, Cortinarius and Piloderma species, with medium‐distance soil exploration abilities, show a consistently negative response. We also determined nitrogen critical loads for moderate (9.5–13.5 kg N/ha/year) and drastic (17 kg N/ha/year) changes in belowground mycorrhizal root communities in temperate oak forests. Overall, we generated the first baseline data for ectomycorrhizal fungi in the oak forests sampled, identified nitrogen pollution as one of their major drivers at large scales and revealed fungi that individually and/or in combination with others can be used as belowground indicators of environmental characteristics.

Mycelial abundance and other factors related to truffle productivity in Tuber melanosporum–Quercus ilex orchards

Relative quantification of DNA from Tuber melanosporum mycelia was performed by conventional and real-time PCR in soil from trees in three truffle orchards of different ages to determine: (1) whether burn appearance is related to the amount of T. melanosporum mycelium in soil, and (2) whether productivity onset and truffle production are related to (a) the amount of T. melanosporum mycelium in soil, (b) tree height and diameter, (c) burn extension and (d) surface rock cover. The burn seems to appear only after a certain amount of mycelium has formed. Precociously productive trees presented higher quantities of mycelium than nonproductive trees in the productivity onset study, while highly productive trees presented less quantities of mycelium than nonproductive trees in the productivity study. Trees with high but not excessive surface rock cover showed greater truffle production. Larger trees tended to display a burn earlier than smaller trees.

What’s for dinner?: Undescribed species of porcini in a commercial packet

Accurate diagnosis of the components of our food and a standard lexicon for clear communication is essential for regulating global food trade and identifying food frauds. Reliable identification of wild collected foods can be particularly difficult, especially when they originate in under-documented regions or belong to poorly known groups such as Fungi. Porcini, one of the most widely traded wild edible mushrooms in the world, are large and conspicuous and they are used as a food both on their own and in processed food products. China is a major exporter of porcini, most of it ending up in Europe. We used DNA-sequencing to identify three species of mushroom contained within a commercial packet of dried Chinese porcini purchased in London. Surprisingly, all three have never been formally described by science and required new scientific names. This demonstrates the ubiquity of unknown fungal diversity even in widely traded commercial food products from one of the most charismatic and least overlooked groups of mushrooms. Our rapid analysis and description makes it possible to reliably identify these species, allowing their harvest to be monitored and their presence tracked in the food chain.

Monitoring ectomycorrhizal fungi at large scales for science, forest management, fungal conservation and environmental policy

Key message The ICP Forests network can be a platform for large-scale mycorrhizal studies. Mapping and monitoring of mycorrhizas have untapped potential to inform science, management, conservation and policy regarding distributions, diversity hotspots, dominance and rarity, and indicators of forest changes.ContextA dearth of information about fungi at large scales has severely constrained scientific, forest management, fungal conservation and environmental policy efforts worldwide. Nonetheless, fungi fulfil critical functional roles in our changing environments and represent a considerable proportion of terrestrial biodiversity. Mycorrhizal fungi are increasingly viewed as a major functional guild across forest ecosystems, and our ability to study them is expanding rapidly.AimsThis study aimed to discuss the potential for starting a mycorrhizal monitoring programme built upon the existing forest monitoring network, raise questions, propose hypotheses and stimulate further discussion.ResultsAn overview of the state-of-the-art regarding forest ectomycorrhizal ecology raises questions and recommendations for scaling up mycorrhizal assessments aimed at informing a variety of stakeholders, with a new focus on conservation and policy.ConclusionFungal research and conservation are areas that can be informed by ICP Forests and may lead to useful spin-offs; research linked to long-term forest monitoring plots will enhance the relevance of science and conservation.

First Report of Cladobotryum mycophilum Causing Cobweb on Cultivated King Oyster Mushroom in Spain

In 2010, symptoms of cobweb were observed on cultivated king oyster mushroom (Pleurotus eryngii) in Castilla-La Mancha (Spain) affecting 16% of the blocks of substrate cultivated. Cobweb appeared at the end of the crop cycle, first as small, white patches on the casing soil, subsequently spreading to the nearest king oyster mushroom by means of a fine gray-white mycelium, and eventually sporulating to produce masses of dry spores. The mycelium can quickly cover pinheads, stalks, pileus, and gills, eventually resulting in decomposition of the entire fruit body. Infected tissues of P. eryngii were plated onto potato dextrose agar (PDA) and the parasitic fungus was isolated. Fungal colonies consisted of abundant and cottony aerial mycelium spreading rapidly on PDA and red pigment spreading in the agar. Conidiogenous cells were 24 to 35 μm long, 3.5 to 5 μm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 17 to 25 (-28) × 8 to 10 μm, and zero to three septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified for one isolate using ITS1F/ITS4 primers (1,3). The amplicon was sequenced (GenBank Accession No. JF505112). BLAST analysis showed 100% similarity of the obtained ITS sequence with two sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. Y17096 and Y17095) (2). Pathogenicity tests were performed using 24 blocks containing sterilized, spawned, and incubated P. eryngii substrate (3.6 kg, 352 cm2 in area). The blocks were placed in a mushroom-growing room and cased with a 40-mm layer of a casing soil (0.7 liter block-1) made with mineral soil + Sphagnum peat 4:1 (vol/vol). Five days after casing, a conidial suspension (7 × 103 conidia ml-1) of one isolate of C. mycophilum was sprayed (5 ml per block) onto the surface of the casing layer at a rate of 106 conidia m-2. Twenty-two blocks were sprayed with sterile distilled water as a control. A temperature of 17 to 18°C and 85 to 90% relative humidity were maintained throughout cropping. The first cobweb symptoms developed 23 days after inoculation and C. mycophilum was consistently reisolated from nine (37.5%) of the inoculated blocks. Noninoculated blocks remained healthy. In a second test, conidial suspensions (3.4 × 105 conidia ml-1) of one isolate of C. mycophilum were inoculated onto 20 P. eryngii fruit bodies. Ten fruit bodies were inoculated externally while the other 10 fruit bodies were cut in half and inoculated internally with 50 μl of conidial suspension per fruit body. Sterilized distilled water was used as a control. All fruit bodies were then incubated at 22°C in a moist chamber. Assays were conducted twice and the results were recorded after 7 days. C. mycophilum grew on 85% of the internally inoculated fruit bodies and on 40% of those inoculated superficially, while the control mushrooms remained symptomless. To our knowledge, this is the first report of C. mycophilum causing cobweb in king oyster mushroom in Spain. This finding will have a potentially significant impact on button mushroom farms where cobweb is one of the most common diseases. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) G. J. McKay et al. Appl. Environ. Microbiol. 65:606, 1999. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First report of cobweb on white button mushroom (Agaricus bisporus) in Spain caused by Cladobotryum mycophilum.

Between 2008 and 2011, symptoms of cobweb were observed in commercial white button mushroom (Agaricus bisporus) crops in Castilla-La Mancha (Spain). Typical symptoms started as white, cobweb-like mycelial growth over the surface of the casing soils and fruiting bodies. Later, the mycelium changed to a grayish white, dense powder and the affected fruiting bodies turned pale yellow or reddish brown before rotting. Two types of cap spotting were observed, dark brown spots with a poorly defined edge and light brown spots. The first symptoms were commonly seen in the second or third break (flush) of mushrooms. Infected tissues of A. bisporus were plated onto potato dextrose agar (PDA) and a parasitic fungus was isolated. Fungal colonies consisted of abundant, cottony, aerial mycelium spreading rapidly over the PDA, and red pigment spreading into the agar. The cultures lacked a camphor odor. Conidiogenous cells were 24 to 45 μm long, 3 to 6 μm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 15 to 28 × 8 to 11 μm, and zero- to three-septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA amplified for one mycelial isolate using ITS1F/ITS4 primers (2,4). The amplicon was sequenced (GenBank Accession No. JQ004732). BLAST analysis showed highest similarity (99 and 100%) of the ITS sequence to four ITS sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. AB527074, JF505112, Y17095, and Y17096) (1,3) among other sequences of the same species. Two pathogenicity trials (A and B) were performed in mushroom-growing rooms, with 24 blocks in each assay containing pasteurized, spawned, and incubated A. bisporus substrate (10 kg, 0.15 m2). The blocks were cased with a 35-mm layer of a peat-based casing soil (5.5 liter/block). Nine days after casing, a conidial suspension (7.5 × 103 conidia/ml) of one isolate of C. mycophilum was sprayed (20 ml/block) onto the surface of the casing layer of 12 blocks at 106 conidia/m2. Twelve blocks were sprayed with sterile distilled water as a control treatment. Blocks were maintained at 17.5°C and 90% relative humidity. The first cobweb symptoms developed 25 days after inoculation, between the second and third breaks in trial A; and after 11 days, between the first and second breaks in trial B. C. mycophilum was consistently reisolated from eight inoculated blocks (67%) in trial A, and 11 inoculated blocks (92%) in trial B. The total area of the crop affected by cobweb was 30% in inoculated blocks in trial A and 45% in trial B. The noninoculated blocks remained healthy. Compared with the noninoculated control blocks, a 10.7% decrease in yield of mushrooms was observed in trial A and 9.1% in trial B. Previously, C. dendroides was the only known causal agent of cobweb in Spain. To our knowledge, this is the first report of C. mycophilum causing cobweb in white button mushroom in Spain, although the disease and causal agent were previously reported on cultivated king oyster mushroom (Pleurotus eryngii) in Spain (3). References: (3) C.-G. Back et al. J. Gen. Plant Pathol. 76:232, 2010. (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (4) F. J. Gea et al. Plant Dis. 95:1030, 2011. (2) T. J. White et al. PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Morphotyping and Molecular Methods to Characterize Ectomycorrhizal Roots and Hyphae in Soil

At the interface between plants and soils, ectomycorrhizal (ECM) fungi explore soils, acquire resources, transfer resources to plants, and acquire carbon from plants. Mycorrhizas enhance plant survival, nutrition and growth and play key roles in ecosystems processes such as decomposition, nutrient cycling, soil carbon storage, productivity and sustainability. Mycorrhizas are critical for plant colonization of new soils (e.g. mine spoils, volcanic deposits, glacial moraines). ECM diversity ensures plant reestablishment after disturbance and can enhance survival and growth of trees in reforestation. ECM fungi can promote fine root development as well as produce antibiotics, hormones and vitamins. Mycorrhizal associations may help protect roots from pathogens and moderate effects of heavy metals and toxins. Many environmental problems may be alleviated by mycorrhizas – problems such as pollution, erosion, soil degradation, climate change, degradation of natural resources, and poor land use management. Mycorrhizal abilities to carry out important functions are linked to diversity. ECM diversity is large and documented in many ecosystems, particularly coniferous ecosystems (Gehring et al. 1998; Goodman and Trofymow 1998; Kranabetter and Wylie 1998; van der Heijden et al. 1998; Stendell et al. 1999; Bidartondo et al. 2000). This ECM diversity has been based on surveys of fruiting bodies, but is now based on more recent methods – morphotyping (microscopic observations) and phylotyping (molecular characterization). An advantage of fruiting body surveys is ease of collection and identification based on morphology; a disadvantage is the assumption that fungi fruiting in an area also form ectomycorrhizas on nearby roots. Clearly identification of ectomycorrhizas on roots is preferable. However there are difficulties in ECM identification – complex sampling design and

Orchidioid fungi of the form-genus Rhizoctonia associated with the roots of Chloraea cuneata Lindl. from Araucanía, Chile

Chloraea cuneata es una orquidea endemica de Chile en Peligro Critico de Extincion. Se aislaron e identificaron hongos orquidioides a partir de pelotones ubicados en celulas corticales de sus raices pertenecientes al genero-forma Rhizoctonia, teleomorfo Thanatephorus (multinucleado), los que podrian ser usados en iniciativas de conservacion.

Environment and host as large-scale controls of ectomycorrhizal fungi

Explaining the large-scale diversity of soil organisms that drive biogeochemical processes—and their responses to environmental change—is critical. However, identifying consistent drivers of belowground diversity and abundance for some soil organisms at large spatial scales remains problematic. Here we investigate a major guild, the ectomycorrhizal fungi, across European forests at a spatial scale and resolution that is—to our knowledge—unprecedented, to explore key biotic and abiotic predictors of ectomycorrhizal diversity and to identify dominant responses and thresholds for change across complex environmental gradients. We show the effect of 38 host, environment, climate and geographical variables on ectomycorrhizal diversity, and define thresholds of community change for key variables. We quantify host specificity and reveal plasticity in functional traits involved in soil foraging across gradients. We conclude that environmental and host factors explain most of the variation in ectomycorrhizal diversity, that the environmental thresholds used as major ecosystem assessment tools need adjustment and that the importance of belowground specificity and plasticity has previously been underappreciated.Analyses of data from 137 forest plots across 20 European countries show that ectomycorrhizal fungal diversity is strongly influenced by environmental and host species factors and provide thresholds to inform ecosystem assessment tools

Multi-cropping edible truffles and sweet chestnuts: production of high-quality Castanea sativa seedlings inoculated with Tuber aestivum , its ecotype T. uncinatum , T. brumale , and T. macrosporum

The plantation and management of sweet chestnut (Castanea sativa Mill.) orchards is a common and traditional land use system in many areas of Europe that offers the advantage of simultaneous production of nuts and timber. During the last decades, sweet chestnut has declined dramatically in many regions because of the profound social changes in rural areas coupled with pathogen attacks. Truffles, the hypogeous ascocarps of the ectomycorrhizal genus Tuber, are currently cultivated using host trees inoculated with these fungi for improving production in truffle orchards. The production of good forestry quality chestnut seedlings inoculated with European truffles in nurseries is essential for multi-cropping plantation establishment, but so far, it has not been implemented in agroforestry practices. Moreover, it is necessary to assess the physiological condition of the seedlings due to the high calcium amendment needed for the growth of Tuber spp. mycelium that can become toxic for the host plants. In this study, seedlings of C. sativa were inoculated with Tuber aestivum and its ecotypes T. uncinatum, T. brumale, and T. macrosporum and were grown in a greenhouse using culture conditions favorable for the production of high-quality plants for forestry purposes. At the end of the assay, levels of root colonization and morphological and physiological parameters of the seedlings were measured. The colonization of C. sativa with T. aestivum, its ecotype T. uncinatum, and T. brumale was successful, and the seedlings showed normal growth. Inoculation protocols with T. macrosporum need to be improved. Tuber species formed well-developed ectomycorrhizae on C. sativa in nursery conditions.