The role of low-level ozone exposure and mycorrhizas in chemical quality and insect herbivore performance on Scots pine seedlings

The effects of low‐level ozone exposure and suppression of natural mycorrhizas on the above‐ground chemical quality of Scots pine (Pinus sylvestris L.) needles and insect herbivore performance were studied in a two‐year field experiment. Seedlings were fumigated with the ozone doses 1.5–1.7 times the ambient, and natural mycorrhizal infection level was about 35% reduced in roots with fungicide propiconazole. On ozone‐exposed seedlings the mean relative growth rate (MRGR) of Lygus rugulipennis Popp. nymphs was lower than on ambient ozone seedlings, but Gilpinia pallida Klug sawfly larvae grew better on elevated ozone seedlings than on ambient ozone seedlings. MRGR of Schizolachnus pineti Fabr. and Cinara pinea L. aphid nymphs or Neodiprion sertifer Geoffr. sawfly larvae or the oviposition of L. rugulipennis and N. sertifer were not affected by ozone exposure. Although ozone exposure did not affect total phenolics, total terpene, total or individual resin acid, total free amino acid, nutrient or sugar concentrations in needles, MRGR of L. rugulipennis positively correlated with total terpenes and MRGR of G. pallida positively with total amino acids. In addition, ozone exposure increased serine and proline concentration and marginally also starch concentration in needles. When mycorrhizas were reduced with fungicide, only MRGR of L. rugulipennis nymphs increased, but performance of other insect herbivores studied was not changed. However, number of L. rugulipennis eggs correlated positively with mycorrhizal infection level and also with total sugars. Reduction of mycorrhizas did not strongly affect the concentrations of analysed compounds in needles, because only phosphorus and potassium and some individual resin acids were reduced by fungicide treatment. These results suggest that low‐level ozone exposure and moderately declined mycorrhizal infection do not drastically affect either the above‐ground chemical quality of Scots pine seedlings or performance of studied insect herbivores.     

The influence of elevated CO2 and O3 on fine roots and mycorrhizas of naturally growing young Scots pine trees during three exposure years

Young Scots pine trees naturally established at a pine heath were exposed to two concentrations of CO₂ (ambient and doubled ambient) and two O₃ regimes (ambient and doubled ambient) and their combination in open-top field chambers during growing seasons 1994, 1995 and 1996 (late May to 15 September). Filtered ozone treatment and chamberless control trees were also included in the treatment comparisons. Root ingrowth cores were inserted to the undisturbed soil below the branch projection of each tree at the beginning of the fumigation period in 1994 and were harvested at the end of the fumigation periods in 1995 and 1996. Root biomasses were determined from different soil layers in the ingrowth cores, and the infection levels of different mycorrhizal types were calculated. Elevated O₃ and CO₂ did not have significant effects on the biomass production of Scots pine coarse (Ø > 2 mm) or fine roots (Ø < 2 mm) and roots of grasses and dwarf shrubs. Elevated O₃ caused a transient stimulation, observable in 1995, in the proportion of tuber-like mycorrhizas, total mycorrhizas and total short roots but this stimulation disappeared during the last study year. Elevated CO₂ did not enhance carbon allocation to root growth or mycorrhiza formation, although a diminishing trend in the mycorrhiza formation was observed. In the combination treatment increased CO₂ inhibited the transient stimulating effect of ozone, and a significant increase of old mycorrhizas was observed. Our conclusion is that doubled CO₂ is not able to increase carbon allocation to growth of fine roots or mycorrhizas in nutrient poor forest sites and realistically elevated ozone does not cause a measurable limitation to roots within a period of three exposure years.     

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.     

Fatty acids,starch and biomass of Scots pine needles and roots in open-air ozone exposure

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone (cumulative exposure) over 2 seasons in an open-air experiment. Starch and fatty acid concentrations were analyzed in needle and root tissue in the summer, autumn and early winter. Seedling growth was determined by measuring the height of the stem and the total shoot and root biomass. Significant decreases in growth were found in exposed seedlings, even though visible symptoms were lacking. Almost significant reductions in needle and root starch concentrations were found. In the ozone treated foliage, significant increases in myristic acid (140) were detected, but the major fatty acids remained unchanged. Fatty acid ratios showed that the degree of unsaturation decreased in treated needles in the summer. In the roots of ozone treated seedlings, changes in fatty acids were different from those in the foliage. Decreases of the main root fatty acids (160, 180, 181, 18:2, 183) were detected in the summer. These results show that Scots pine is susceptible to enhanced levels of ozone. If the tropospheric ozone levels continue to increase it may have deleterious effects on Scots pine forests in Finland.     

Mycorrhizal growth in pure cultures in the presence of pesticides

The effects of pesticides on 64 ectomycorrhizal fungi of boreal forest trees were studied in vitro. The pesticides (fungicides: benomyl, chlorothalonil, copper oxychloride, maneb and propiconazole; herbicides: chlorthiamid, glyphosate, hexazinone, linuron and terbuthylazine; insecticide: cypermethrin) were selected as those commonly used in Nordic forest nurseries and afforestation sites. In general, the fungicides proved to be more toxic to ectomycorrhizal fungi than the herbicides and cypermethrin. The fungicides, chlorothalonil and propiconazole, had the clearest inhibitory effect on growth of mycorrhizal fungi. Conversely, maneb, glyphosate and terbuthylazine stimulated the growth of some mycorrhizal fungi. Leccinum versipelle and L. scabrum, Paxillus involutus and Cenococcum geophilum were the most sensitive ectomycorrhizal fungi to the various pesticides.     

Studies on Ectomycorrhizae of Pine II. Growth Inhibition of Mycorrhizal Fungi by Volatile Organic Constituents of Pinus silvestris (Scots Pine) Roots

The effects of several monoterpenes and the sesquiterpene longifolene on the vegetative growth of two mycorrhizal fungi, Boletus variegatus and Rhizopogon roseolus, were studied in petri plate experiments on an agar medium. The test substances are natural constituents of Pinus silvestris (Scots pine) roots. Vapors from 20 μl of all the test substances, upon a 5 day exposure inhibited the growth of both the mycorrhizal fungi 55% to 86%. The two fungi, however, differed in their tolerance to individual compounds. It is suggested that the terpenes and the sesquiterpenes play an important role in the development of the symbiotic condition in ectomycorrhizae by restricting the growth of the mycorrhizal fungi within the host root.     

Direct application of carbendazim and propiconazole at field rates to the external mycelium of three arbuscular mycorrhizal fungi species: effect on 32P transport and succinate dehydrogenase activity

 The influence of the systemic fungicides propiconazole (Tilt 250E) and carbendazim (Bavistin) at field application rates on the functioning of three arbuscular mycorrhizal fungi was studied. Short-term fungal ³²P transport and succinate dehydrogenase (SDH) activity in external hyphae of Glomus intraradices Schenck and Smith, G. claroideum Schenck and Smith and G. invermaium Hall in symbiosis with pea (Pisum sativum L.) were measured. In the experimental system used, the hyphae grew into two root-free hyphal compartments (HCs). The fungicides were applied to each HC 24 days after sowing and ³²P was added to one HC of each pot. Four days later, the fungicide effect on fungal P transport was measured as the difference in ³²P content of treated and untreated plants. SDH activity in fungal hyphae was determined in the HCs given no ³²P. Carbendazim severely inhibited ³²P transport and SDH activity in external hyphae at an application rate of 0.5 μg g^–1 soil. The ergosterol inhibitor propiconazole affected none of these parameters. The fungicides had similar effects on all three fungal species, although P transport efficiency and SDH activity differed markedly between the fungi. Accepted: 12 December 1996     

Slightly elevated ozone exposure causes cell structural changes in needles and roots of Scots pine

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone over 2 seasons in an open-air experiment. Fumigation started in the early spring and continued into late autumn during both years. Needle and root cell structures were analyzed in the summer, autumn and early winter following the second fumigation period. Under the light microscope an increase in the intercellular space and disintegrating cells in the mesophyll tissue near the stomata and stomatal cavities were observed in the ozone-exposed needles. Darkening of chloroplast stroma, increased plastoglobulus size and decreased chloroplast size were characteristic ultrastructural changes associated with ozone exposure. In addition, less dense grouping of the chloroplasts in the needles of elevated ozone-exposed seedlings as compared to the controls (background ozone) was observed in the early winter. Fewer starch grains and an increased accumulation of tannin-like substances were detected in both mycorrhizal and uninfected roots of ozone-exposed seedlings as compared to the control seedlings. For the first time, we were able to show that the ozone-induced darkening of needle chloroplast stroma is a reversible symptom. An increased frequency of frost injury symptoms indicated that the winter hardening process was disturbed in the needles of ozone-treated seedlings.     

Influence of laser light on mycelial growth of Hebeloma mesophaeum and ectomycorrhizal development on Scots pine

 The effects of exposure to helium-neon (He-Ne) or Argon (Ar) laser light (λ=632.8 nm and 514 nm, respectively) on the growth of Hebeloma mesophaeum mycelium in pure culture were studied. Growth rates were highest after exposure to the He-Ne laser (1×60 s)+Ar laser (2×60 s) and to the He-Ne laser for 3×30. Container-grown Pinus sylvestris (pine) seedlings were inoculated with a water suspension of H. mesophaeum mycelium previously exposed to different kinds of laser light. After 3 months, the percentage of mycorrhizal associations on pine roots was 34.3% higher after He-Ne laser treatment and 47.1% higher after Ar laser treatment than in the controls with untreated fungus. However, seedlings infected with treated fungus were smaller than the control. Overall, laser light stimulated growth of H. mesophaeum mycelia in pure culture and enhanced mycorrhizal development on Scots pine seedlings.     

An ericoid shrub plays a dual role in recruiting both pines and their fungal symbionts along primary succession gradients

Relative importance of positive and negative interactions between plant species may change along disturbance and resource gradients. Positive interactions are suggested to prevail in low resource, low productivity (high stress) conditions and negative interactions in high resource availability. A dwarf shrub, mountain crowberry Empetrum nigrum ssp. hermaphroditum, is known to have allelopathic impacts on both Scots pine Pinus sylvestris and its ectomycorrhizal symbionts. We aimed to study if the outcome of Empetrum impacts on Scots pine changes along primary succession gradients on the dune shores of Bothnian Bay, in Finland, where abiotic stress gradually changes to biotic stress along the succession. We found that Empetrum may act as a facilitator despite its allelopathic effects, since the proportion of Scots pine seedlings established in Empetrum patches was higher than in patches without the shrub in early and mid succession stages, whereas patches without Empetrum were preferred in late succession. The amount of mycelial fungal biomass (ergosterol) in the soil in the vicinity of the seedling roots was higher in Empetrum patches than in patches without Empetrum and it increased along the succession gradient. Proportion of pine root tips colonised by suilloid morphotypes with abundant external mycelia and the diversity of ectomycorrhizal morphotypes were higher in mid successional stage in Empetrum patches compared to patches without Empetrum. Our results suggest that in the harsh physical conditions of the dune shore Empetrum facilitates pine seedling establishment in the early and mid stages of succession by providing mechanical and physical shelter whereas in late succession negative interactions (competition and allelopathy) between the shrub and the pine are dominating. To our knowledge we present the first finding that an ericoid mycorrhizal shrub could enhance both the performance of the ectomycorrhizal host tree and the tree's fungal symbionts.     

Elevated atmospheric CO2 increases fine root production, respiration, rhizosphere respiration and soil CO2 efflux in Scots pine seedlings

In this study, we investigated the impact of elevated atmospheric CO₂ (ambient + 350 μmol mol^–1) on fine root production and respiration in Scots pine (Pinus sylvestris L.) seedlings. After six months exposure to elevated CO₂, root production measured by root in-growth bags, showed significant increases in mean total root length and biomass, which were more than 100% greater compared to the ambient treatment. This increased root length may have lead to a more intensive soil exploration. Chemical analysis of the roots showed that the roots in the elevated treatment accumulated more starch and had a lower C/N-ratio. Specific root respiration rates were significantly higher in the elevated treatment and this was probably attributed to increased nitrogen concentrations in the roots. Rhizospheric respiration and soil CO₂ efflux were also enhanced in the elevated treatment. These results clearly indicate that under elevated atmospheric CO₂ root production and development in Scots pine seedlings is altered and respiratory carbon losses through the root system are increased.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Altered root growth and plant chemistry ofPinus 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.     

The co-occurrence of ectomycorrhizal,arbuscular mycorrhizal,and dark septate fungi in seedlings of four members of the Pinaceae

Although roots of species in the Pinaceae are usually colonized by ectomycorrhizal (EM) fungi, there are increasing reports of the presence of arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi in these species. The objective of this study was to determine the colonization patterns in seedlings of three Pinus (pine) species (Pinus banksiana, Pinus strobus, Pinus contorta) and Picea glauca x Picea engelmannii (hybrid spruce) grown in soil collected from a disturbed forest site. Seedlings of all three pine species and hybrid spruce became colonized by EM, AM, and DSE fungi. The dominant EM morphotype belonged to the E-strain category; limited colonization by a Tuber sp. was found on roots of Pinus strobus and an unknown morphotype (cf. Suillus–Rhizopogon group) with thick, cottony white mycelium was present on short roots of all species. The three fungal categories tended to occupy different niches in a single root system. No correlation was found between the percent root colonized by EM and percent colonization by either AM or DSE, although there was a positive correlation between percent root length colonized by AM and DSE. Hyphae and vesicles were the only AM intracellular structures found in roots of all species; arbuscules were not observed in any roots.     

Effect of mycorrhiza and nitrate nutrition on nitrate reductase activity in Scots pine seedlings

In vivo nitrate reductase (EC 1.6.6.1) activity was measured in seedlings of Scots pine ( Pinus sylvestris L.) inoculated with Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutus (Batsch:Fr) Fr, Piloderma croceum Erikss, & Hjortst, and Suillus variegatus (Fr.) O. Kuntze. The activity was higher in the mycorrhizal pine roots than was previously found in the fungus symbiont alone, but lower than in the roots of nonmycorrhizal pine seedlings. The differences observed in a previous study between the fungal species under pure culture conditions were not found in the present work for mycorrhiza synthezised with the same fungal species. An increase in the nitrate concentration of the nutrient solution increased the proportion of the nitrate reductase activity in the needles. The mycorrhizal root tips had higher nitrate reductase activity than nonmycorrhizal root tips in the same root system.     

Distribution of Cren- and Euryarchaeota in Scots Pine Mycorrhizospheres and Boreal Forest Humus

Archaeal 16S rRNA gene sequences have been found in a variety of moderate-temperature habitats including soil and rhizospheres. In this study, the differences of archaeal communities associated with Scots pine (Pinus sylvestris L.) short roots, different types of mycorrhizospheric compartments, and uncolonized boreal forest humus were tested by direct DNA extraction, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE), and sequencing. The results indicated that mycorrhizal colonization of Scots pine roots substantially influence the archaeal community of pine rhizospheres. Colonization of short roots by most mycorrhizal fungi tested increased both archaeal frequency and diversity. Most of the archaeal sequences encountered in mycorrhizas belonged to the phylum Euryarchaeota, order of Halobacteriales. The difference in archaeal diversity between the mycorrhizospheric compartments and humus was profound. Most compartments with fungal components contained euryarchaeotal 16S rRNA gene sequences, whereas a high diversity of crenarchaeotal sequences and no euryarchaeotal sequences were found in forest humus outside mycorrhizospheres.     

Growth and reproduction of aphids and levels of free amino acids in Scots pine and Norway spruce in an open-air fumigation with ozone

To estimate the susceptibility of conifer seedlings to aphids under future tropospheric ozone levels, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings were exposed to ambient and elevated ozone levels in an open-air exposure system. Growth and reproduction of the aphids Schizolachnus pineti and Cinara pinea on Scots pine and Cinara pilicornis on Norway spruce were monitored. Levels of free amino acids in foliage and young shoots were used as indicators of host plant quality. In elevated treatment plots the ozone doses were between 1.2 and 1.7 times the dose in ambient plots in 1990–93. Half of the seedling material in 1992–93 was subjected to nitrogen fertilization treatment to evaluate the effects of increased N deposition. In 1990, population density of S. pineti on pine did not differ between ambient and elevated ozone treatments during growing season, but remained higher in the elevated ozone plot than in the ambient plot at the end of the growing season. This was associated with elevated levels of glutamic acid in foliage. In August 1992, the numbers of S. pineti were consistent between the two ambient ozone plots, but deviated highly between the two ozone-fumigated plots. Glycine concentration in pine foliage was elevated by ozone, but free amino acid concentrations were not related to aphid performance. In 1993, ozone and nitrogen did not significantly affect the relative growth rate (RGR) of S. pineti or C. pinea nymphs on Scots pine, but glutamic acid concentration in foliage was increased by nitrogen fertilization. On Norway spruce, fecundity of C. pilicornis females was higher in elevated ozone treatment, but RGR of nymphs was not affected in 1992. In 1993, RGR of C. pilicornis nymphs was increased by nitrogen fertilization in June, but not affected by ozone. Nitrogen fertilization increased the levels of total free amino acids, aspartic acid, glutamic acid and proline in elongating shoots of Norway spruce, and ozone reduced the concentrations of valine and γ-butyric acid. Our results suggest that availability of nitrogen from soil has a stronger impact on the concentrations of free amino acids in conifer seedlings than ozone. Some episodes of high ozone concentration may increase free amino acids in foliage. Aphid response to ozone was extremely variable, in agreement with previous laboratory experiments. The expected 20–70% increase in ambient concentrations of tropospheric ozone may in some occasions enhance aphid performance on Scots pine and Norway spruce seedlings, but in most cases the ozone effect on the susceptibility of conifer seedlings to sucking insect pests is not important.     

The fungicides Terrazole and Terraclor and the nematicide Fenamiphos have little effect on root colonisation by Glomus mosseae and growth of cotton seedlings

 The effect of three pesticides on the initiation and early development of arbuscular mycorrhiza in cotton was examined in experiments under controlled conditions. The fungicides Terrazole and Terraclor initially inhibited mycorrhizal infection of roots of cotton. The inhibition disappeared after 4 weeks, and neither fungicide had a lasting effect. The nematicide Fenamiphos slightly increased shoot dry weight at 6–10 weeks from planting and had no effect on mycorrhizal infection. We conclude that these pesticides have no sustained, detrimental effect on mycorrhizal infection or growth of cotton seedlings when applied at recommended rates. Accepted: 30 May 1997     

Evaluation in the greenhouse of the effects of fungicides on the development of mycorrhiza on leek and wheat

Summary The effect of 11 fungicides, viz Aliette, Bayleton 5, Benlate, Dithane M45, Euparene, Karathane, Orthocide 83RP, Pelt 44, Tilt C, Tilt 125 and Tripomol 80, on vesicular-arbuscular mycorrhizal symbiosis in leek and wheat growing in a calcined clay was studied in the greenhouse at four concentrations applied to the foliage. Fungicides had minimal effects when applied to leek or wheat plants with well-established mycorrhiza. Mycorrhizal infection was not drastically affected by any fungicide at any application level. However, mycorrhizal infection of leek plants was reduced by systemic fungicides, and that of wheat by non-systemic fungicides. Aliette was the only fungicide that stimulated mycorrhizal development.