An improved method for extraction of soil fungal mycelium.

Fungal mycelium is a major component of the soil microbiome. The soil hyphosphere represents a complex and dynamic niche for specific microorganisms, where multitrophic interactions occur, affecting ecosystem processes. However, extracting fungal mycelium from the soil to enable its taxonomical, chemical, and structural characterisation is challenging in the absence of a fast, efficient, and low-cost procedure. In this study, an old method (Bingle and Paul 1985), based on successive soil wet filtrations and density gradient centrifugation, was improved and tested in three different soil types (silty clay, silty clay loam, and loamy sand). The improved method reduced the number of filtrations by about five times and the centrifugation time from 40 min to 1 min. It avoided using any chemical substance which may impair further chemical analyses or DNA isolation and amplification. The method efficiency was about 50 % in the clay and 23 % in the sandy soils. However, a pre-step consisting of removing the fine-root fragments and other debris under the stereomicroscope may increase the method efficiency to more than 65 %, independent of the soil type.•A simple, efficient, and low-cost method suitable for extracting soil mycelium from a large number of samples.•The protocol includes successive soil wet filtrations and sucrose gradient centrifugation.•The method efficiency increases if the fine-root fragments and other debris are previously removed from the soil.

Selective Quantification of Chemotropic Responses of Fusarium graminearum.

Chemotropism refers to the directional growth of a living organism toward a chemical stimulus. Molecular mechanisms underlying chemotropism of fungal pathogens have recently been enabled by advancements in biological chemotropic assays, with a particular focus on the roles of G-protein-coupled receptors and their plant-derived ligands in chemotropism. Here we describe in detail an assay that enables quantification of chemotropic responses of Fusarium graminearum, with variations recently reported for Fusarium oxysporum and Trichoderma atroviride.

Arbuscular mycorrhizal hyphal respiration makes a large contribution to soil respiration in a subtropical forest under various N input rates.

Arbuscular mycorrhizal fungi (AMF) are widespread in subtropical forests and play a crucial role in belowground carbon (C) dynamics. Nitrogen (N) deposition or fertilization may affect AMF and thus the flux of plant-derived C back to the atmosphere via AMF hyphae. However, the contribution of AMF hyphal respiration to soil respiration and the response AMF hyphal respiration to increased soil N availability remain unknown. We studied the effect of N fertilization (0, 50, 100 and 200 kg N ha-1 yr-1) on AMF hyphal respiration, root respiration and heterotrophic (microbial) respiration in a subtropical Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) plantation. We found that short-term N addition did not affect root, AMF hyphal and soil microbial respiration, because soil N availability and extraradical hyphae were not affected by N addition. The AMF hyphal respiration contributed 12 % of total soil respiration and 25 % of the autotrophic respiration. Root, AMF hyphal and soil microbial respiration were positively correlated with soil moisture content but not with soil temperature. Our results indicate that AMF hyphal respiration is a large source of soil respiration, and should be considered in partitioning soil respiration into different components in future studies to better understand the response of soil respiration to N addition.

HyLength: a semi-automated digital image analysis tool for measuring the length of roots and fungal hyphae of dense mycelia.

In plant-fungus phenotyping, determining fungal hyphal and plant root lengths by digital image analysis can reduce labour and increase data reproducibility. However, the degree of software sophistication is often prohibitive and manual measuring is still used, despite being very time-consuming. We developed the HyLength tool for measuring the lengths of hyphae and roots in in vivo and in vitro systems. The HyLength was successfully validated against manual measures of roots and fungal hyphae obtained from all systems. Compared with manual methods, the HyLength underestimated Medicago sativa roots in the in vivo system and Rhizophagus irregularis hyphae in the in vitro system by about 12 cm per m and allowed to save about 1 h for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, the HyLength overestimated the length by about 21 cm per m compared with manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae, the underestimation was about 8 cm per m with a time saving of about 10 min for a single germinating spore. By benchmarking the HyLength against the AnaMorf plugin of the ImageJ/Fiji, we found that the HyLength performed better for dense fungal hyphae, also strongly reducing the measuring time. The HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples. Therefore, we propose the HyLength as a useful freeware tool for measuring fungal hyphae of dense mycelia.

Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola.

Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

Mycorrhizal symbiosis down-regulates or does not change root aquaporin expression in trifoliate orange under drought stress.

Arbuscular mycorrhizas absorb water from soil to host plants, while the relationship between mycorrhizas and aquaporins (AQPs, membrane water channel proteins, which function in water transport) in mycorrhizal plants is unclear. In this study, Funneliformis mosseae-colonized trifoliate orange (Poncirus trifoliata) seedlings were grown in pots fitted with 37-µm nylon meshes at the bottom of each pot to allow mycorrhizal hyphae absorb water from an outer beaker. The expression of seven plasma membrane intrinsic proteins (PIPs) genes, six tonoplast intrinsic proteins (TIPs) genes, and four nodulin-26 like intrinsic proteins (NIPs) genes were analyzed in roots of both well-watered (WW) and drought stressed (DS) plants. The six-week DS plants dramatically increased hyphal water absorption rate by 1.4 times, as compared with WW plants. Mycorrhizal plants exhibited greater plant growth performance, leaf water status (water potential and relative water content), and gas exchange under both WW and DS conditions. Mycorrhizal inoculation induced diverse expression patterns in these AQPs under WW: up-regulation of PtNIP1;1, PtPIP2;1, and PtPIP2;5, down-regulation of PtNIP1;2, PtNIP6;1, PtPIP1;2, PtPIP1;5, PtPIP2;8, PtTIP1;1, PtTIP1;2, PtTIP1;3, and PtTIP5;1, and no changes in other AQPs. However, the expression of PtPIPs and PtNIPs was down-regulated by mycorrhizal inoculation under DS, and PtTIPs was not induced by mycorrhizal colonization under DS. The expression pattern of AQPs in response to mycorrhizas under DS is a way of mycorrhizal plants to minimize water loss.

Long-term effects of grazing and topography on extra-radical hyphae of arbuscular mycorrhizal fungi in semi-arid grasslands.

Grazing and topography have drastic effects on plant communities and soil properties. These effects are thought to influence arbuscular mycorrhizal (AM) fungi. However, the simultaneous impacts of grazing pressure (sheep ha-1) and topography on plant and soil factors and their relationship to the production of extra-radical AM hyphae are not well understood. Our 10-year study assessed relationships between grazing, plant species richness, aboveground plant productivity, soil nutrients, edaphic properties, and AM hyphal length density (HLD) in different topographic areas (flat or sloped). We found HLD linearly declined with increasing grazing pressure (1.5-9.0 sheep ha-1) in sloped areas, but HLD was greatest at moderate grazing pressure (4.5 sheep ha-1) in flat areas. Structural equation modeling indicates grazing reduces HLD by altering soil nutrient dynamics in sloped areas, but non-linearly influences HLD through plant community and edaphic changes in flat areas. Our findings highlight how topography influences key plant and soil factors, thus regulating the effects of grazing pressure on extra-radical hyphal production of AM fungi in grasslands. Understanding how grazing and topography influence AM fungi in semi-arid grasslands is vital, as globally, severe human population pressure and increasing demand for food aggravate the grazing intensity in grasslands.

Optimization of Herbicidin A Production in Submerged Culture of Streptomyces scopuliridis M40.

Herbicidin A is a potent herbicide against dicotyledonous plants as well as an antibiotic against phytopathogens. In this study, fermentation parameters for herbicidin A production in submerged culture of Streptomyces scopuliridis M40 were investigated. The herbicidin A concentration varied with the C/N ratio. High C/N ratios (>4) resulted in a herbicidin A production of more than 900 mg/l, whereas maximally 600 mg/l was obtained at ratios between 1 and 3.5. In 5-L batch fermentation, there was a positive correlation between the oxygen uptake rate (OUR) and herbicidin A production. Once the OUR increased, the substrate consumption rate increased, leading to an increase in volumetric productivity. Mechanical shear force affected the hyphal morphology and OUR. When the medium value of hyphal size ranged from 150 to 180 µm, high volumetric production of herbicidin A was obtained with OUR values >137 mg O2/l·h. The highest herbicidin A concentration of 956.6 mg/l was obtained at 500 rpm, and coincided with the highest relative abundance of hyphae of 100-200 µm length and the highest OUR during cultivation. Based on a constant impeller tip speed, which affects hyphal morphology, herbicidin A production was successfully scaled up from a 5-L jar to a 500-L pilot vessel.

Effects of a mycophagous Collembola on the symbioses between Trifolium subterraneum and three arbuscular mycorrhizal fungi.

We examined the effect of the mycophagous Collembola Folsomia Candida Willem on the symbioses between Trifolium subterraneum L. and three arbuscular mycorrhizal (AM) fungi. Mycorrhizal and non-mycorrhizal plants were grown in pots with a central mesh bag which constituted a root-free soil compartment (RFSC) where the soil was labelled with 32 P. This allowed us to study hyphal P transport as affected by the presence of Collembola in the soil with roots. The hyphal P transport was monitored by measuring the 32 P content in leaflets sampled from the latest fully developed trifoliate leaf after 3, 4, 5 and 6 wk. Measurements on each plant after 6 wk furthermore included total contents of 32 P and 31 P, root length and length of hyphae inside the mesh bag. The presence of F, Candida reduced the average hyphal 32 P transport from the RFSC by 12% and the average AM hyphal length in the RFSC was reduced by 30%. The root dry weights of mycorrhizal plants were also reduced by the presence of Collembola. The reduction in total hyphal 32 P transport was not revealed by the data from the time-course study using leaflet samples, which demonstrates the uncertainty of analyses of AM fungal activity based solely on measurements of leaf materials. The results from this work suggest that F. Candida grazed on roots and/or hyphae of AM; however, shoot yields were unaffected by this grazing. In conclusion, Collembola seemed to have little effect on the functioning of AM fungi.