Bioassay-guided isolation of three new alkaloids from Suillus bovinus and preliminary mechanism against ginseng root rot.

In order to control the occurrence of ginseng root rot caused by Fusarium solani (Mart.) Sacc., the antifungal compounds of the mushroom Suillus bovinus were investigated. And three new alkaloids (1-3), named bovinalkaloid A-C, along with one known analog (4), were isolated and identified by bioassay-guided isolation and spectroscopic analyses. Compound 1 strongly inhibited the mycelial growth and spore germination of F. solani with minimum inhibitory concentration of 2.08 mM. Increases in electrical conductivity, nucleic acid, and protein contents, and decreases in lipid content showed that the membrane permeability and integrity were damaged by compound 1. Compound 1 also increased the contents of malondialdehyde and hydrogen peroxide and the activities of antioxidant enzymes, indicating that lipid peroxidation had taken place in F. solani. Compound 1 may serve as a natural alternative to synthetic fungicides for the control of ginseng root rot.

Chromosome-Level Assembly and Comparative Genomic Analysis of Suillus bovinus Provides Insights into the Mechanism of Mycorrhizal Symbiosis.

Suillus bovinus is a wild edible ectomycorrhizal fungus with important economic and ecological value, which often forms an ectomycorrhiza with pine trees. We know little about the mechanisms associated with the metabolism and symbiosis of S. bovinus and its effects on the nutritional value. In this study, the whole-genome sequencing of S. bovinus was performed using Illumina, HiFi, and Hi-C technologies, and the sequencing data were subjected to genome assembly, gene prediction, and functional annotation to obtain a high-quality chromosome-level genome of S. bovinus. The final assembly of the S. bovinus genome includes 12 chromosomes, with a total length of 43.03 Mb, a GC content of 46.58%, and a contig N50 size of 3.78 Mb. A total of 11,199 coding protein sequences were predicted from genome annotation. The S. bovinus genome contains a large number of small secreted proteins (SSPs) and genes that encode enzymes related to carbohydrates, as well as genes related to terpenoids, auxin, and lipochitooligosaccharides. These genes may contribute to symbiotic processes. The whole-genome sequencing and genetic information provide a theoretical basis for a deeper understanding of the mechanism of the mycorrhizal symbiosis of S. bovinus and can serve as a reference for comparative genomics of ectomycorrhizal fungi.

Effects of four bolete species on ectomycorrhizae formation and development in Pinus thunbergii and Quercus acutissima.

BACKGROUND: Bolete cultivation is economically and ecologically valuable. Ectomycorrhizae are advantageous for plant development and productivity. This study investigated how boletes affect the formation of Pinus thunbergii and Quercus acutissima ectomycorrhizae using greenhouse-based mycorrhizal experiments, inoculating P. thunbergii and Q. acutissima with four species of boletes (Suillus bovinus, Suillus luteus, Suillus grevillei, and Retiboletus sinensis). RESULTS: Three months after inoculation, morphological and molecular analyses identified S. bovinus, S. luteus, S. grevillei and R. sinensis ectomycorrhizae formation on the roots of both tree species. The mycorrhizal infection rate ranged from 40 to 55%. The host plant species determined the mycorrhiza morphology, which was independent of the bolete species. Differences in plant growth, photosynthesis, and endogenous hormone secretion primarily correlated with the host plant species. Infection with all four bolete species significantly promoted the host plants' growth and photosynthesis rates; indole-3-acetic acid, zeatin, and gibberellic acid secretion increased, and the abscisic acid level significantly decreased. Indole-3-acetic acid was also detected in the fermentation broths of all bolete species. CONCLUSIONS: Inoculation with bolete and subsequent mycorrhizae formation significantly altered the morphology and hormone content in the host seedlings, indicating growth promotion. These findings have practical implications for culturing pine and oak tree species.

Mycorrhizosphere Bacterial Communities and their Sensitivity to Antibacterial Activity of Ectomycorrhizal Fungi.

We investigated whether ectomycorrhizal (ECM) fungal species exhibit antibacterial activity towards culturable bacterial communities in mycorrhizospheres. Four hundred and thirty bacterial strains were isolated from the ECM root tips of Pinus densiflora and bulk soil, and 21 were co-cultured with six ECM fungal species. Three hundred and twenty-nine bacterial 16S rDNA sequences were identified in ECM roots (n=185) and bulk soil (n=144). Mycorrhizosphere isolates were dominated by Gram-negative Proteobacteria from 16 genera, including Burkholderia, Collimonas, Paraburkholderia, and Rhizobium. Paraburkholderia accounted for approximately 60%. In contrast, bulk soil isolates contained a high number of Gram-positive Firmicutes, particularly from Bacillus. Paraburkholderia accounted for ≤20% of the bacterial isolates from bulk soil, which was significantly lower than its percentage in ECM root tips. Co-cultures of six ECM fungal species with the 21 bacterial strains revealed that eight strains of three Gram-positive genera-Arthrobacter, Bacillus, and Lysinibacillus-were sensitive to the antibacterial activity of the fungi. In contrast, the Gram-negative strains, including five Paraburkholderia strains, two Burkholderia strains, and a Rhizobium sp., were not sensitive. The strength of fungal antibacterial activity varied in a species-dependent manner, but consistently affected Gram-positive bacteria. These results suggest that Gram-positive bacteria are excluded from the mycorrhizosphere by the antibacterial activity of ECM fungi, which develops specific soil bacterial communities in the mycorrhizosphere.

Population structure and dynamics in Suillus bovinus as indicated by spatial distribution of fungal clones.

The spatial distribution of clones in the ectomycorrhizal fungus Suillus bovinus (L. ex Fr.) O. Kuntze was studied from somatic incompatibility pairings of isolates in four Scots pine (Pinus sylvestris L.) stands with different forest history. A pattern of increasing clonal size, decreasing clonal number, and decreasing fruiting per unit area, was found with increased forest age and relatively little disturbance. Establishment by spores was important on disturbed sites, while mycelial spread increased in importance with decreased disturbance. The number of clones was found to be 800 ha-1 in younger forest stands and decreased to between 25-130 ha-1 in mature stands. At the same time the diameter of the area occupied by a single clone increased from 1-3 m to 30 m. Implications of the observations are discussed in terms of ecological strategies and population dynamics.

Size, distribution and biomass of genets in populations of Suillus bovinus (L.: Fr.) Roussel revealed by somatic incompatibility.

The spatial distribution of genets in the ectomycorrhizal fungus Suillus bovinus (L.: Fr.) Roussel were studied in somatic incompatibility pairings of isolates from five Scots pine (Pinus sylvestris L.) stands differing in forest history and age, With increasing forest age, the size of genets increased while the number of genets and production of sporocarps per unit area decreased. There was an estimated 700-5700 genets ha-1 in younger forests and 30-120 ha-1 in older ones. The maximum size of genets was 1.7-5.3 m in the younger forest and to 17.5 m in the older ones. The production of sporocarps per unit area decreased with increasing forest age. Furthermore, production of sporocarps increased nonlinearly with the size of the genet, suggesting that genets become fragmented. Ergosterol measurements indicated that the fungal biomass of one genet consisted of 20-45% sporocarps and 55-80% mycorrhiza, not including extramatrical mycelia. Mycorrhizal aggregations in soil were mapped in two 5 m2 areas and, based on somatic incompatibility tests, all were round CO belong to the same genet as sporocarps present above ground. Production of spores per sporocarp was estimated to be 1.1-12.8 × 108 . The observations are discussed in terms of population ecology.

A zinc-adapted fungus protects pines from zinc stress.

• Here we investigated zinc tolerance of ectomycorrhizal Scots pine (Pinus sylvestris) seedlings. An ectomycorrhizal genotype of Suillus bovinus, collected from a Zn-contaminated site and showing adaptive Zn tolerance in vitro, was compared with a nonadapted isolate from a nonpolluted area. • A dose-response experiment was performed. Dynamics of plant and fungal development, and phosphate and ammonium uptake capacity, were assessed under increasing Zn stress. Effects of Zn on transpiration, nutrient content and Zn accumulation were analysed. • Significant Zn-inoculation interaction effects were observed for several responses measured, including uptake rates of phosphate and ammonium; phosphorus, iron and Zn content in shoots; transpiration; biomass of external mycelia; and fungal biomass in roots. • The Zn-tolerant S. bovinus genotype was particularly efficient in protecting pines from Zn stress. The growth of a Zn-sensitive genotype from a normal wild-type population was inhibited at high Zn concentrations, and this isolate could not sustain the pines' acquisition of nutrients. This study shows that well adapted microbial root symbionts are a major component of the survival strategy of trees that colonize contaminated soils.

Multitrophic interactions between a Rhizoctonia sp. and mycorrhizal fungi affect Scots pine seedling performance in nursery soil.

• Interspecific variation in ectomycorrhizal fungal (ECMF) control of a root pathogenic uninucleate Rhizoctonia sp. (UnR) was identified in vitro and in planta. • Fungal-fungal and host-fungal interactions were assessed in direct confrontation and cell-free assays, the rhizosphere of Scots pine (Pinus sylvestris) seedling radicles and seedling mycorrhizospheres developed in N-limited nursery soil. • Isolates of Suillus bovinus inhibited UnR growth although no agar-diffusable fungicidal activity was detected. Presence of nonsymbiotic ECMF mycelia did not prevent UnR colonization of radicle apices and the onset of damping-off symptoms. Seedlings hosting S. bovinus mycorrhizas and extensive extramatrical mycelium showed vigorous and healthy shoot growth after a 168-d UnR challenge. Root biomass of Wilcoxina mikolae and Paxillus involutus colonized seedlings were negatively affected by both low soil nutrient status and UnR exposure. However, UnR was isolated from long and mycorrhizal short roots in all ECMF coinoculation treatments. • The differential responses highlight multitrophic host-fungal interaction dynamics that require further characterization in the development of 'efficient' UnR biological control solutions utilizing mycorrhizal fungal inoculants.

Short-term phosphorus uptake rates in mycorrhizal and non-mycorrhizal roots of intact Pinus sylvestris seedlings.

Short-term phosphate uptake rates were measured on intact ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings using a new, non-destructive method. Uptake was quantified in semihydroponics from the depletion of Pi in a nutrient solution percolating through plant containers. Plants were grown for 1 or 2 months after inoculation at a low relative nutrient addition rate of 3% d-1 and under P limitation. Four ectomycorrhizal fungi were studied: Paxillus involutus, Suillus luteus, Suillus bovinus and Thelephora terrestris. The Pi -uptake capacity of mycorrhizal plants increased sharply in the month after inoculation. The increase was dependent on the development of the mycobionts. A positive correlation was found between the Pi -uptake rates of the seedlings and the active fungal biomass in the substrate as measured by the ergosterol assay. The highest Pi -uptake rates were found in seedlings associated with fungi producing abundant external mycelia. At an external Pi concentration of 10 µM, mycorrhizal seedlings reached uptake rates that were 2.5 (T. terrestris) to 8.7 (P. involutus) times higher than those of non-mycorrhizal plants. The increased uptake rates did not result in an increased transfer of nutrients to the plant tissues. Nutrient depletion was ultimately similar between mycorrhizal and non-mycorrhizal plants in the semihydroponic system. Net Pi absorption followed Michaelis-Menten kinetics: uptake rates declined with decreasing Pi concentrations in the nutrient solution. This reduction was most pronounced in non- mycorrhizal seedlings and plants colonized by T. terrestris. The results confirm that there is considerable heterogeneity in affinity for Pi uptake among the different mycobionts. It is concluded that the external mycelia of ectomycorrhizal fungi strongly influence the Pi -uptake capacity of the pine seedlings, and that some mycobionts are well equipped to compete with other soil microorganisms for Pi present at low concentrations in soil solution.