Antoni Werner

Altered root growth and plant chemistry of Pinus sylvestris seedlings subjected to aluminum in nutrient solution

One-year-old Scots pine (Pinus sylvestris L.) seedlings were grown for 9 weeks in nutrient solutions containing 0, 0.5, 1, 2 and 4 mM aluminum nitrate (Al(NO3)3) at pH 4.2. Nine weeks exposure to Al significantly reduced total plant, shoot and root mass and caused a linear decline in proportional allocation of biomass to roots. Relative growth rate of roots declined to as low as zero. Aluminum treatment decreased calcium and magnesium uptake and increased Al content in roots and needles. After 3 weeks of exposure a 10–60% increase in total phenols in roots and a 20–40% increase in o-diphenols in roots and needles were noted. Roots affected by Al showed degeneration of meristematic cells, fewer cell divisions, deformation in cell walls and higher lignification and suberization. The majority of root apices were structurally similar to dormant roots, and a premature senescence of the entire root system was observed. Net photosynthetic rate after 6 weeks of treatment was negatively correlated with needle Al content and Al/Ca ratio (r < -0.9, P < 0.1). The results suggest that Scots pine may be more susceptible to Al than was expected based on previous experiments.

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.

Behaviour of the hyphae of Laccaria laccata in the presence of Trichoderma harzianum in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.

Intraspecific variation in Heterobasidion annosum for mortality rate on Pinus sylvestris and Picea abies seedlings grown in pure culture

One-month-old Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings were inoculated in vitro with Heterobasidion annosum strains, four each of the P-, S- and F-intersterility groups. Variation among strains and between the IS groups in virulence, expressed in mortality rate, was detected during twelve months after inoculation. Most of the strains were more virulent on spruce than on pine, and mortality of spruce seedlings was significantly higher. The P strains displayed similar virulence on both hosts, while S strains caused higher mortality of spruce seedlings and significantly lower mortality of pine seedlings. Strains of the F group were less virulent, but killed significantly (P < 0.05) more spruce than pine seedlings. In the interspecific analyses with two hosts, the isolates and IS groups accounted for most of the explained variation in the host mortality

Concentrations of terpenes in mycorrhizal roots of Scots pine (Pinus sylvestris L.) seedlings grown in vitro

The effect of Paxillus involutus, Laccaria laccata, Suillus luteus, S. bovinus, Hebeloma crustuliniforme and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) on the content of volatile organic compounds in roots of Pinus sylvestris seedlings grown in vitro was investigated. Volatile compounds extracted with a supercritical fluid extraction were primarily terpenes and sesquiterpenes and qualitatively were the same in roots of mycorrhizal and nonmycorrhizal plants. The major monoterpenes were α-pinene, Δ3-carene and β-pinene. Inoculation of plants with the fungi resulted in statistically non-significant increases in the total amount of the volatiles. The mycorrhizal fungi showed diversified effect on the concentrations of several terpenoids.

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.

Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi

The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

The effects of heavy metals, content of nutrients and inoculation with mycorrhizal fungi on the level of terpenoids in roots of Pinus sylvestris seedlings

The effect of two substrates differing in the amount of toxic metals and nutrients on the content of volatile organic compounds in roots of Pinus sylvestris seedlings inoculated with Paxillus involutus, Laccaria laccata, Suillus luteus and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) in pot experiment was investigated. Volatile compounds extracted with a supercritical fluid extraction were primarily terpenes and sesquiterpenes, and they were qualitatively the same in roots of mycorrhizal and nonmycorrhizal plants grown on both substrates. The major monoterpenes were α-pinene, Δ3-carene and β-pinene. On the unpolluted substrate, inoculation of plants with the fungi resulted in a similar amount of the volatiles, while on the polluted substrate the mycorrhizal roots showed significantly lower content of terpenoids than nonmycorrhizal roots. The mycorrhizal fungi showed different effects on the concentrations of several terpenoids on both substrates.

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Abstract Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid–gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the hosts cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.

The effects of toxic metals, content of nutrients and inoculation with mycorrhizal fungi on the level of phenolics in roots and growth of Scots pine seedlings

The effects of two substrates and several strains of mycorrhizal fungi on the content of soluble phenolics in roots and growth of Scots pine seedlings was investigated. The first substrate was fertile and contaminated with copper, zinc and lead, whereas the second one displayed nutrient deficiency. The dry weights of needles, trunks, roots and the total biomass were higher in groups of seedlings inoculated with mycorrhizal fungi on fertile and polluted substrate. Inoculation of pine seedlings on this substrate resulted in a decrease in concentration of phenolics in roots and except for seedlings inoculated with Laccaria laccata negatively influenced the above-ground part: root ratio.