Ectomycorrhizal Fungus Suillus Bovinus Research Articles

Morphogenesis and metabolomics reveal the compatible relationship among Suillus bovinus, Phialocephala fortinii, and their co-host, Pinus massoniana.

Ectomycorrhizal (ECM) fungi and dark septate endophytes (DSEs) can both form a symbiotic relationship with the same host plant. However, the interactions that occur among these two types of fungi and their co-hosts are largely unknown. Here, we investigated interactions that occur among the ECM fungus Suillus bovinus, the DSE Phialocephala fortinii, and their co-host Pinus massoniana. We used both scanning electron microscopy and optical microscopy to characterize the morphogenesis of the two symbionts and employed the ultra-high-performance liquid chromatography-tandem mass spectrometry technique to assess the effects of fungal inoculation on the root metabolome. Under pure culture conditions, no synergistic or antagonistic effects were observed between Phi. fortinii and S. bovinus. Generally, S. bovinus and Phi. fortinii can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus can colonize the root locus where Phi. fortinii has already invaded but not vice versa, which may be due to the physical barrier effect of the mantle. Both fungi can significantly promote the growth of P. massoniana, and they have a synergistic effect on host N and K uptake. Metabolite accumulation patterns in roots inoculated with Phi. fortinii and/or S. bovinus were greatly altered, especially with respect to organic acids, flavonoids, lipids, and phenolic acids. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. These findings reveal compatible relationships among P. massoniana, S. bovinus, and Phi. fortinii, and suggest a theoretical basis for ECM fungi and DSE co-application when cultivating seedlings. IMPORTANCE The prevalence of both ectomycorrhizal fungi and dark septate endophytes in the roots of a wide spectrum of tree species is well recognized. In this study, we investigated the interactions that occur among the ECM fungus S. bovinus, the DSE Phi. fortinii, and their co-host, P. massoniana. The two fungi can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus appears to be superior to Phi. fortinii in microniche competition, which may be due to the physical barrier effect of the mantle. The two fungi have different effects on root metabolite accumulation patterns. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. This is the first study revealing the morphological and metabolic mechanisms that contribute to the compatible relationship among ECM fungi, DSEs, and their co-host.

Preliminary studies on genetic diversity of ectomycorrhizal fungus Suillus bovinus in Lithuania

Abstract Motiejūnaitė J., Kačergius A., Kasparavičius J., 2013: Preliminary studies on genetic diversity of ectomycorrhizal fungus Suillus bovinus in Lithuania [Pradiniai ektomikorizinio grybo Suillus bovinus genetinio kintamumo Lietuvoje tyrimai]. - Bot. Lith., 19(2): 91-98. Genetic diversity of some Lithuanian populations of Suillus bovinus was assessed basing on nucleotide sequence of ITS1 and 2 regions, and the 5.8S RNA gene obtained from 42 samples. Four haplotypes were defined, each representing isolate groups of varying abundance and distribution. The most abundant and widely distributed was haplotype 2. All Lithuanian haplotypes nested in one, the largest clade of European isolates of S. bovinus.

Response of somatic embryos of Scots pine to fungal cell wall elicitors

SummaryFungal cell wall elicitors have notable effects on germination of pine seeds, defence response and many other aspects of metabolic processes. In this study, we investigated morphological, physiological and molecular response of somatic embryos of Scots pine to fungal cell wall elicitors from ectomycorrhizal fungus Suillus bovinus, pathogenic fungus Heterobasidion annosum or weak Scots pine pathogen (Heterobasidion parviporum). Results from the embryo rooting experiment indicated that the genotypic origin of the conifer tree and the source of the elicitors affected the root development. Elicitors from either S. bovinus or H. parviporum increased the root formation but increased concentration of elicitors from H. annosum decreased the rooting and survival rate of the embryos. At the molecular level, altered transcript changes were observed among genes encoding proteins involved in cell division, cell wall modification, and stress following exposure of embryos to elicitors. Increased levels of actin and peroxidase gene expression were detected at 7 days after exposure to S. bovinus or H. parviporum elicitors. H. annosum elicitors provoked significant induction of the ethylene synthesis gene PsACS‐1. Generally the gene expression patterns at early stages observed in the subset of genes investigated after treatment with H. parviporum elicitor were found to be similar to that documented for S. bovinus. In contrast, somatic embryos exposed to H. annosum elicitor had different patterns of regulation marked by delayed response, and in some cases gradual degradation and death of the embryos were observed.

Cytoskeleton and mitochondrial morphology of saprotrophs and the pathogen Heterobasidion annosum in the presence of Suillus bovinus metabolites

Ectomycorrhizal fungi are known to synthesize antifungal compounds both in vitro and in symbiosis with the host-plants. Culture filtrates of the ectomycorrhizal fungus Suillus bovinus (at pHs of 2.5–6) showed antifungal activity towards saprotrophs Trichoderma harzianum, and Trichoderma virens and the pathogen Heterobasidion annosum, by significantly suppressing their growth relative to sterile liquid medium at the same pHs. In the presence of the culture filtrates, hyphae of the saprotrophs and the pathogen were characterized by distensions, irregular and frequent branching, tip damage and cytoplasm coagulation. Since hyphal abnormalities may be evoked by disruptions in the cytoskeleton and mitochondria, their structural changes were also examined. Depolymerization of microtubules was confirmed for all of the fungi. Serious damage to mitochondria morphology may cause significant functional impairment. Growth of mycelia was inhibited in the lower pH S. bovinus culture filtrate, and the mitochondrial morphology was altered. This suggests that the activity of antifungal compounds synthesized by ectomycorrhizal fungus is significantly affected by pH.

Antagonistic activity of the ectomycorrhizal fungus Suillus bovinus challenged by saprotrophic fungi from different soils

Antagonistic activity of the ectomycorrhizal fungus Suillus bovinus challenged by saprotrophic fungi from different soils

A knight of symbiosis

A knight of symbiosis

Development of SSR markers from an ectomycorrhizal fungus, Suillus bovinus

Nine simple sequence repeat (SSR) loci were isolated from an ectomycorrhizal fungus Suillus bovinus by dual-suppression PCR. Three of the SSR loci isolated were polymorphic. The number of alleles per locus was between two and seven, and expected heterozygosities ranged from 0.087 to 0.740. One of these was confirmed to be species specific and codominant, suggesting applicability for the analysis of belowground population structure and gene flow of S. bovinus.

Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus Suillus bovinus

Under laboratory conditions, spores of ectomycorrhizal fungi usually germinate very poorly or not at all. In a previous study, we showed that spores of the ectomycorrhizal fungus Suillus bovinus germinated through the combination of activated charcoal treatment of media and co-culture with seedlings of Pinus densiflora, which suggested that some substances contained in root exudates induced the germination. Among the compounds reported from root exudates, flavonoids have been elucidated to play various and substantial roles in plant-microbe interactions; we therefore investigated the effects of flavonoids on basidiospore germination of S. bovinus by the diffusion gradient assay on water agar plates pretreated with charcoal powder. Seven out of the 11 flavonoids tested, hesperidin, morin, rutin, quercitrin, naringenin, genistein, and chrysin, had greater effects than controls, whereas flavone, biochanin A, luteolin, and quercetin showed no positive effects. The effective concentration presumably corresponded to several micromolar levels, which was equivalent to those effective for pollen development, nod gene induction, and spore germination of F. solani f. sp. pisi and AM fungi. The results suggest that flavonoids play a role as signaling molecules in symbiotic relationships between woody plants and ectomycorrhizal fungi.

Development of SSR markers from an ectomycorrhizal fungus, <i>Suillus bovinus</i>

Nine simple sequence repeat (SSR) loci were isolated from an ectomycorrhizal fungus Suillus bovinus by dual-suppression PCR. Three of the SSR loci isolated were polymorphic. The number of alleles per locus was between two and seven, and expected heterozygosities ranged from 0.087 to 0.740. One of these was confirmed to be species specific and codominant, suggesting applicability for the analysis of belowground population structure and gene flow of S. bovinus.

Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 muM Zn(2+) for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.

Ectomycorrhizas impede phytoremediation of polycyclic aromatic hydrocarbons (PAHs) both within and beyond the rhizosphere

Exploitation of mycorrhizas to enhance phytoremediation of organic pollutants has received attention recently due to their positive effects on establishment of plants in polluted soils. Some evidence exist that ectomycorrhizas enhance the degradation of pollutants of low recalcitrance, while less easily degradable polyaromatic molecules have been degraded only by some of these fungi in vitro. Natural polyaromatic (humic) substances are degraded more slowly in soil where ectomycorrhizal fungi are present, thus phytoremediation of recalcitrant pollutants may not benefit from the presence of these fungi. Using a soil spiked with three polycyclic aromatic hydrocarbons (PAHs) and an industrially polluted soil (1 g kg −1 of ∑12 PAHs), we show that the ectomycorrhizal fungus Suillus bovinus, forming hydrophobic mycelium in soil that would easily enter into contact with hydrophobic pollutants, impedes rather than promotes PAH degradation. This result is likely to be a nutrient depletion effect caused by fungal scavenging of mineral nutrients.

T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker.

The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells. Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph)and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV35S terminator. Transformation resulted in hygromycin B-resistant clones, which were mitotically stable. Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis. The latter analysis proved both multiple- and single-copy integrations of the genes in the S. bovinus genome. A. tumeficiens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S. bovinus.

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus.

Comparative in vivo (31)P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li+, Na+, K+, or Rb+ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li+, Na+, or Rb+ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K+, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb+ < K+ < Na+ < Li+ inhibited the net P(i) accumulation. Furthermore, it is postulated that the Pi accumulation was also regulated by the intracellular content in polyP.

Scots pine expresses short-root-specific peroxidases during development.

Nine short-root-specific proteins from Scots pine (Pinus sylvestris L.) detected and isolated as individual spots by 2D-PAGE were identified. The similar peptide mass maps obtained for all nine polypeptide spots together with lectin-blotting results suggest that they represent forms of the same modified protein. N-Terminal sequence analysis of two of the peptides showed high similarity to peroxidases. RT-PCR with oligonucleotide primers corresponding to determined peptide sequences and conserved regions in plant peroxidases led to isolation of Psyp1 cDNA which is most abundantly expressed in short roots. Psyp1 is the first peroxidase cDNA to be isolated from the genus Pinus. It encodes a 363-amino-acid class-III peroxidase with a calculated molecular mass of 35.7 kDa and theoretical pI of 4.74. The predicted PSYP1 amino-acid sequence is grouped with other class-III peroxidases in phylogenetic analyses, but it has a unique amino-acid sequence which may be associated with its function in short roots or with its phylogenetic group. The presence of a signal sequence for extracellular transport indicates that PSYP1 belongs to the group of secreted class-III peroxidases. The presence of 10 tyrosine residues and putative auxin-binding regions in PSYP1 suggests that the function of the enzyme is associated with cell-wall formation in short roots. The downregulation of Psyp1 expression in symbiotic short roots hosting the ectomycorrhizal fungus Suillus bovinus is perhaps related to the change in cell-wall structure necessary for ectomycorrhizal development.

Developmentally regulated proteins during differentiation of root system and ectomycorrhiza in Scots pine (Pinus sylvestris) with Suillus bovinus

Total and radiolabelled proteins from Pinus sylvestris roots, ectomycorrhiza and the ectomycorrhizal fungus Suillus bovinus, were analyzed after separation by 2‐D gel electrophoresis. The purpose was to address the contribution of individual proteins to the development of ectomycorrhiza. In Pinus sylvestris, ectomycorrhiza forms only in the short roots and therefore special attention was paid to the proteins of short roots before formation of ectomycorrhiza. A main result, from comparisons of protein amounts in main and lateral root tips and short roots, was that the specialized growth pattern of short roots is associated with the production of five short root‐specific proteins, and that several proteins of the main and lateral roots are repressed in short roots. At the morphologically different stages of the ectomycorrhiza, only few changes appeared in the amounts of the host and the symbiont proteins. Only one ectomycorrhiza‐specific protein could be distinguished.

The biology of mycorrhiza in the Ericaceae: XIX. Fungal mycelium as a nitrogen source for the ericoid mycorrhizal fungus Hymenoscyphus ericae and its host plants.

Measurements of the chitin content of the rooting horizons of a typical mor-humus heathland soil, indicate that chitin can contain in excess of 20% of the total nitrogen in the litter (L) horizon and 30% in the fermentation (F) horizon. Much of this chitin-nitrogen is thought to be contained in the mycelial walls of soil fungi. Experiments were therefore designed to test the hypothesis that such sources of N could be rendered accessible to the ericaceous plants by their fungal endophytes. Mycelium of the ericoid endophyte Hymenoscyphus ericae (Read) Korf & Kernan and of the ectomycorrhizal fungus Suillus bovinus (Fr.) O. Krantze were grown in liquid culture before being killed and added either in the intact condition, or after fractionation, as sole sources of N to sterile media upon which were grown H. ericae in pure culture, or mycorrhizal and non-mycorrhizal plants of Vaccinium macrocarpon Ait. and Calluna vulgaris L. The abilities of the test organisms to utilize the nitrogen contained in the intact mycelial necromass, or in its fractions, were assessed by determining their yields and nitrogen concentrations of their tissues. It was revealed that H. ericae was able to produce significantly higher yield when grown on intact fungal necromass than when provided with equivalent concentrations of N in the form of ammonium. Its yields on mycelial fractions were lower, but still significantly greater than those obtained in the controls lacking N. Significantly greater yields and N contents were also found in the ericaceous plants grown with these nitrogenous substrates in the mycorrhizal condition. Without H. ericae they had no access to the substrates. The possible ecological implications of these results are discussed.

Effects of aluminium on polyphosphate mobilization of the ectomycorrhizal fungus Suillus bovinus

Aluminium toxicity may be an important factor in the decline in vitality of many forest trees and the associated ectomycorrhizal fungal flora. In this study, comparative in vivo 31P NMR investigations on Al-adapted and non-Aladapted fungus of Suillus bovinus in pure culture have produced interesting new data. With respect to intracellular compartments, 31P NMR spectroscopy showed the spectra to differ in a peak-6 ppm appearing in the spectra of the A1-adapted fungus indicating terminal phosphate groups of mobile polyphosphate. Thus, in the Al-adapted fungus the average chain length of mobile polyphosphate is considerably shorter than in the non-Al-adapted fungus. A special method of cyclic phosphate supply followed by block averaging of the NMR spectra was used to determine the kinetic behaviour of phosphate uptake, storage and incorporation into polyphosphate at a constant external pH 3.5. While the Al-adapted fungus showed resistance to Al, an irreversible break-down in phosphate metabolism of the non-Al-adapted fungus by exposure to Al was caused. In comparison with the non-Al-adapted fungus supplied by nutrient solutions omitting Al, the Al-adapted fungus showed higher levels in both phosphate uptake and mobile polyphosphate concentration. As a consequence of these results a de-toxification of freely mobile Al-ions into a stable and insoluble complex in the Al-adapted fungus is considered to be due to a capture of intracellular Al by mobile polyphosphate of shorter chain length.

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.

Effects of aluminium on growth and nutrient uptake of small Picea abies and Pinus sylvestris plants

The effects of aluminium concentrations between 0.2 and 30 mM at pH 3.8 ±0.2 on small plants of Norway spruce [(Picea abies (L.) Karst], Scots pine (Pinus sylvestris L.), and Scots pine infected with the ectomycorrhizal fungus Suillus bovinus (L. ex Fr.) O. Kuntze were investigated. The plants were grown at maximum relative growth rate (RG % day−1) with free access but very low external concentrations of nutrients. Steady-state conditions with respect to relative growth rate (RG) and internal nutrient concentrations were achieved before addition of aluminium, which was added as AlCl3 and/or Al(NO3)3. There were reductions in rg at aluminium concentrations of 0.3 mM in spruce, 6 mM in pine and 10 mM in ectomycorrhizal pine, i. e. at aluminium concentrations considerably higher than those normally occurring in the top layer of the mineral soil where most fine roots are found. Nutrient uptake rate per unit root growth rate was calculated for different nutrient elements. The uptake rate of calcium and magnesium was reduced at aluminium concentrations of 0.2 mM (spruce), 1 mM (pine) and 3 mM (ectomycorrhizal pine), without influencing Rg. The results question the validity of the hypothesis of aluminium toxicity to forest tree species at low external concentrations.

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.