Interactions between Tricholomopsis rutilans and ectomycorrhizal fungi in paired culture and in association with seedlings of lodgepole pine and Sitka‐spruce

Interactions between two isolates (Avondhu and CBS) of Tricholomopsis rutilans and ectomycorrhizal fungi (Hebeloma crustuliniforme, Laccaria laccata and Paxillus involutus) were examined on agar medium in the presence or absence of woodchips. The CBS isolate showed more competitiveness than the Avondhu isolate, when paired with ectomycorrhizal fungi. There was an inhibition of the growth of mycelium of the ectomycorrhizal fungi ranging from overgrowth to avoidance. The ectomycorrhizal fungi exhibited hyphal abnormalities such as discoloration, excessive vacuolation and curling. Formation of mycorrhizas by H. crustuliniforme in Pinus contorta and Picea abies was unaffected by the presence of the CBS isolate, whereas a reduction occurred in the presence of the Avondhu isolate. In non‐mycorrhizal seedlings of both conifers, the Avondhu isolate reduced root and shoot dry mass and number of root laterals and caused a lower number of short roots in lodgepole pine. The significance of these interactions between T. rutilans and ectomycorrhizal fungi in paired culture and during symbiosis is discussed.     

Diversity of ectomycorrhizal fungus assemblages on nursery grown European beech seedlings

? Context

An important feature of forest nursery management is the production of first-rate planting stock, which is closely connected with an adequate development of ectomycorrhizas on seedling roots.

? Aims

The aim of this study was to provide a detailed assessment of the ectomycorrhizal (ECM) fungal diversity of European beech grown in bare-root forest nursery conditions.

? Methods

Morphological and molecular analyzes were used to identify ECM fungi, quantify fungal richness, and record differences in the relative abundance of individual taxa.

? Results

Twenty-seven different mycorrhizal fungal taxa were found in four tested nurseries. Of these 27, only 6 were present in more than one nursery. Taxa richness in individual nurseries ranged from 6 to 13, indicating that ECM colonization potential is nursery-dependent. The most frequent taxa were: Tuber sp. 1, Cadophora sp., Cenococcum geophilum agg., Hebeloma sacchariolens, and Laccaria tortilis. According to the analysis of similarity, ECM fungal communities were significantly different between nurseries (R?=?0.7741; p?<?0.0001).

? Conclusion

Beech seedlings grown in bare-root nurseries are colonized by a highly diverse suite of ECM fungi; thus, artificial inoculation of seedlings is generally not necessary in nursery practice.     

Effect of three species of bacteria on damping-off,root rot development,and ectomycorrhizal colonization of lodgepole pine and white spruce seedlings

Interactions between three species of bacteria (Burkholderia cepacia, Pseudomonas chlororaphis and Pseudomonas fluorescens), an ectomycorrhizal fungus (Paxillus involutus), and three root pathogenic fungi (Fusarium moniliforme, Fusarium oxysporum and Rhizoctonia solani) were studied. Burkholderia cepacia significantly reduced the in vitro mycelial growth of P. involutus, whereas, B. cepacia, Ps. chlororaphis, Ps. fluorescens and P. involutus reduced the mycelial growth of F. moniliforme, F. oxysporum and R. solani. Culture filtrates of B. cepacia, Ps. chlororaphis, Ps. fluorescens and P. involutus reduced conidial germination and increased the formation of chlamydospores of F. moniliforme and F. oxysporum. Burkholderia cepacia also reduced the formation of ectomycorrhizal short roots by P. involutus on lodgepole pine (Pinus contorta) and white spruce (Picea glauca) seedlings 2 months after inoculation. However, no significance difference in mycorrhizal short roots was observed after 4 months. The fungicide Anchor (a mixture of carboxine and thiram) significantly reduced root rot severity and increased the survival of lodgepole pine seedlings grown in a growth mix infested with F. moniliforme, F. oxysporum and R. solani. Control of the diseases of white spruce caused by these pathogens was not as successful. Treatment of seeds with either B. cepacia or P. involutus alone significantly increased the survival of seedlings grown in a mix that was inoculated with F. moniliforme and reduced the root rot severity caused by F. moniliforme and F. oxysporum, but not R. solani. Higher seedling survival and lower root rot severity were observed when conifer seeds were concomitantly inoculated with one of the bacterial species, P. involutus and Anchor.     

Effects of ectomycorrhizal fungi on damping‐off and induction of pathogenesis‐related proteins in Pinus tabulaeformis seedlings inoculated with Amanita vaginata

The formation of mycorrhiza on Pinus tabulaeformis (Pt) seedlings by ectomycorrhizal fungi (ECMF), the protective effect of the ECMF on damping‐off caused by Rhizoctonia solani and the expression of pathogenesis‐related proteins (PR‐proteins) induced by Amanita vaginata and/or R. solani in Pt seedlings were investigated. The results indicated that: (i) among the 10 ECMF tested, five species (Gomphidius viscidus, Suillus laricinus, Suillus tomentosus, A. vaginata and Basidiomycetes no. 058) formed mycorrhiza with Pt seedlings. There was a positive correlation between mycorrhizal colonization levels and protective effects (r = 0.9207). Among the five species, A. vaginata had the highest colonization level and the best protective effect against damping‐off; however, G. viscidus had no protective effect even though it had a high mycorrhization level. (ii) Chitinases, β‐1,3‐glucanases and PR‐proteins in the seedlings were not induced by A. vaginata colonization, but were induced when A. vaginata‐mycorrhizal seedlings were attacked by R. solani. It is possible that chitinases, β‐1,3‐glucanases and 108, 97, 78, 44, 37 and 14‐kDa PR‐proteins induced both by A. vaginata and R. solani protect Pt seedlings from R. solani infection.     

Water relation patterns of bare-root and container jack pine and black spruce seedlings planted on boreal cut-over sites

Water relation patterns and subsequent growth were studied on bare-root and container jack pine (Pinus banksiana Lamb.) and black spruce (Picea mariana (Mill.) B.S.P.) seedlings during the first growing season on boreal cut-over sites.Containerized seedlings of both species had greater needle conductance compared to bare-root seedlings over a range of absolute humidity deficits. Needle conductance of containerized seedlings in both species remained high during periods of high absolute humidity deficits and increasing plant moisture stress. Bare-root seedlings of both species had a greater early season resistance to water-flow through the soil-plant-atmosphere continuum (SPAC) than container seedlings. Resistance to water flow through the SPAC decreased in bare-noot seedlings of both species as the growing season progressed, and was comparable to container seedlings 9 through 14 weeks after planting. Four weeks after field planting jack pine container seedlings had greater new root development compared to bare-root seedlings, while at the end of the summer both stock types had similar new root development. Black spruce bare-root seedlings had greater new root development compared with container seedlings throughout the growing season.     

Growth stimulation of young oak plantations inoculated with the ectomycorrhizal fungus Paxillus involutus with special reference to summer drought

Nursery-grown Quercus petraea and Q. robur seedlings were inoculated with the ectomycorrhizal fungi Paxillus involutus, Hebeloma crustuliniforme or Laccaria laccata. At the end of the first growing season, the seedlings with roots colonized with the inoculated fungi were outplanted at two sites in northeastern France in 1985 or 1988. P. involutus was found to be the most competitive and efficient of the three fungi tested; its mycorrhizas were present 7 yrs after outplanting and it markedly improved the growth of both oak species. In the case of Q. robur, the growth stimulation was more marked on years with a dry summer. These results are discussed in terms of water relations and of competitiveness of the introduced versus resident symbiotic fungi.     

Root-associated fungi of healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries

The aim of this study was to assess fungal communities in roots of healthy-looking Pinus sylvestris L. and Picea abies (L.) Karst. seedlings in nine forest nurseries in Sweden using a combination of traditional culturing and direct sequencing of internal transcribed spacer of fungal ribosomal RNA (ITS rRNA) from the roots. Culturing from 1800 surface-sterilised root segments resulted in 2387 fungal cultures representing 42 different taxa. Direct sequencing from 180 root segments resulted in 119 ITS rRNA sequences representing 25 different taxa. In total, 55 different fungal taxa were detected using both methods. Although direct sequencing was more efficient than culturing in detecting different fungal taxa, both methods provided complementary information about fungal communities in roots since each detected rather different groups of fungi. The most dominant taxa detected by culturing were Trichoderma viride Pers. (19.5%), Phoma mucivora Davey & Currah (19.1%), Phialocephala fortinii Wang & Wilcox (17.4%) and Meliniomyces variabilis Hambl. & Sigler (10.2%), while Thelephora terrestris Ehrh. (26.1%), Unidentified sp. NS126 (25.2%) and Heliotales sp. C20 (10.1%) were most commonly detected by direct sequencing. In conclusion, results showed that forest nurseries in Sweden harbour diverse communities of fungi associated with the roots of healthy-looking P. sylvestris and P. abies seedlings. Although fungal communities were often dominated by saprotrophs and endophytes, several facultative pathogens were also detected indicating that under suitable conditions they may be a potential threat to the plants.     

Selection of efficient VA mycorrhizal fungi for Casuarina equisetifolia—second screening

An investigation was carried out to screen and select efficient vesicular arbuscular mycorrhizal (VAM) fungi for inoculating the forest tree species, Casuarina equisetifolia. The seedlings were inoculated with 10 different VAM fungi, obtained from various sources. Inoculated seedlings generally had greater plant height, stem girth, biomass and P content than uninoculated plants. They also had more mycorrhizal root colonization and spore numbers in root zone soil. C. equisetifolia seedlings responded best (in biomass) to inoculation with Glomus mosseae (Nicolson and Gerdemann) Gerdemann and Trappe, closely followed by Acaulospora laevis Gerdemann and Trappe and G. fasciculatum (Thaxter Sensu Gerdemann) Gerdemann and Trappe; all the three being statistically on par with each other.     

Seed spacing and seedling biomass: Effect on root growth potential of loblolly pine (Pinus taeda)

Results from three studies conducted in bare-root nurseries indicate a positive linear relationship between seed spacing and root growth potential of Pinus taeda L. seedlings. However, root growth potential was also correlated with several morphological variables, including seedling weight, root collar diameter, root weight, root volume, foliage weight and stem weight. Because seedlings grown at wider spacings were larger in diameter and biomass, covariate analyses (using either root collar diameter or foliage ovendry weight) were also conducted. These analyses indicate that differences in root growth potential between seed spacings can be accounted for by differences in seedling size.     

Prior contact of <Emphasis Type="Italic">Pinus tabulaeformis</Emphasis> with ectomycorrhizal fungi increases plant growth and survival from damping-off

Pinus tabulaeformis (Chinese pine) is a widely planted conifer species in northern China and is used for soil and water conservation on the Loess Plateau. Due to its strong reliance on ectomycorrhizae and low survival rate from damping-off during seedling cultivation and in nurseries, we explored the early influence of three ectomycorrhizal fungi (Handkea utriformis, Suillus lactifluus, and Suillus tomentosus) on the growth, root morphological characters, root vitality, and survival of P. tabulaeformis seedlings from subsequent damping-off in a pot experiment. In addition, the in vitro suppression of three ectomycorrhizal fungi on the damping-off pathogen (Fusarium solani) was evaluated and observed using a scanning electron microscope. We found that all three ectomycorrhizal fungi could colonize more than 40% of the roots of P. tabulaeformis, promote plant shoot and root growth, increase the proportion of large-diameter roots, improve root vitality, and increase survival rates 3 months after inoculation. All three ectomycorrhizal fungi suppressed the growth of F. solani to a different extent in vitro. Ectomycorrhizal fungal hyphae proliferated and wrapped around the hyphae of F. solani when the hyphae encountered each other. The colonization rate of P. tabulaeformis roots was highly correlated with root vitality and root growth parameters, while the survival rate of P. tabulaeformis seedlings was highly correlated with the colonization rate of P. tabulaeformis roots, root vitality and growth. Our results suggest that early contact between P. tabulaeformis and ectomycorrhizal fungi could improve plant growth and resistance against damping-off.     

Procedure for establishing sheathing mycorrhizas on tree seedlings

A simple procedure is described where seedlings emerging from surface sterilised seeds (of Betula pendula, B. pubescens, Pinus contorta and Picea sitchensis) were grown axenically in a peat/vermiculite mixture which had been previously inoculated, and incubated, with sheating mycorrhizal fungi (including Hebeloma sacchariolens, Paxillus involutus and Amanita muscaria). Mostly, seedlings were grown in split polystyrene tubes (50 mm tall x 13 mm internal diameter) which were arranged $\text{140}\text{150}$ per tray, the trays being covered with transparent propagator lids so forming a unit that could be sealed and sterilised before use with gamma radiation. With the formation of sheating mycorrhizas after about 7 weeks the sterility of the container was broken to enable seedlings to be hardened off prior to transplanting.Roots of Betula pendula seedlings grown in tubes were smaller, as judged by numbers of ‘fragments’, than those of plants which were not ‘restricted’. ‘Tubing’ increased: (a) the proportion of root fragments with mycorrhizas; and (b) the sizes of individual mycorrhizas. The sizes of the ‘tubing’ effects differed with different isolates of A. muscaria, so suggesting, because of the sensitivity of the system, that environmental and genotypic factors must be carefully controlled.     

Ectomycorrhizal inoculation of Douglas-fir transplanted container seedlings with commercially produced inoculum

Commercially produced vegetative inocula of Laccaria laccata and Hebeloma crustuliniforme successfully formed ectomycorrhizae with Douglas-fir transplanted container (plug+1) seedlings. After 4.5 months in containers, 83% and 90%, respectively, of short roots were mycorrhizal. L. laccata- or H. crustuliniforme-inoculated seedlings had significantly more mycorrhizal and total short roots than Pisolithus tinctorius-inoculated (4% mycorrhizal root tips) or uninoculated control seedlings. No significant differences were detected in seedling growth at the end of the container phase.After transplantation and growth in nursery beds for 17 months, mean new short root colonization of all seedlings was 80%. H. crustuliniforme persisted as a dominant mycorrhizal fungus on seedlings initially inoculated with this fungus. L. laccata-inoculated seedlings had 40% of their short roots colonized by L. laccata and another 40% by native fungi Rhizopogon and Thelephora spp. All mycorrhizae of control seedlings and those inoculated with P. tinctorius were formed by fungi native to the nusery beds. A significant fungal treatment effect was detected for shoot height only. Control seedlings were significantly taller than L. laccata-inoculated seedlings after transplanting.This article is part of senior author's thesis in partial fulfillment of the Ph.D. degree in the Department of Forest Science at Oregon State University, Corvallis.     

First report of Calonectria montana causing damping‐off disease on pine and spruce seedlings in Europe

In 2015–2016, damping‐off symptoms were observed on Pinus sylvestris and Picea abies seedlings in four forest nurseries in Poland. Some Calonectria isolates were obtained from symptomatic seedlings by culturing pieces of necrotic tissue on malt extract agar.Morphological and molecular analyses revealed that isolates represent Calonectria montana, a recently described species from natural forests in China. The isolates represent the first detection of this species outside of Asia. Pathogenicity tests demonstrated that C. montana is a severe pathogen of P. sylvestris seedlings.     

Effect of selected forestry herbicides on ectomycorrhizal development and seedling growth of lodgepole pine and white spruce under controlled and field environment

In tests under aseptic conditions, the herbicides hexazinone (Pronone^TM5G® and Velpar L.R.®), glyphosate, and triclopyr reduced seedling growth and mycorrhizal development of Pinus contorta var. Litifolia and Picea glanca. Triclopyr was most toxic of the tour herbicide formulations. Under greenhouse condition, only 2 formulations of hexazinone (Pronone ^TM5G® and Velpar L.®) were tested. At high concentrations (2 and 4 kg/ha) it reduced growth and mycorrhizal infections significantly but showed recovery with time. No adverse effects were observed at low concentration (1 kg/ha). In general, seedlings inoculated with mycorrhizal fungus (Suillus tomentosus), were more sensitive to herbicide than the ones without mycorrhizal inoculation. Under field conditions overall effects of herbicide application were less intense. Only 4 kg/ha rates of hexazinone resulted in reductions in seedling growth and mycorrhizal infections. The nonmycorrhizal seedings planted in the field developed mycorrhizae in over 40% of short roots within 2 months after planting in all herbicide treatments.     

Mosaic stunting in bareroot Pinus banksiana seedlings is unrelated to colonization by mycorrhizal fungi

Mosaic stunting, the occurrence of random patches of chlorotic seedlings with reduced shoot and diameter growth amidst more robust cohorts within bareroot nurseries, is classically associated with poor colonization by mycorrhizal fungi. We examined possible relationships among soil fertility, mycorrhizas, and random patches of mosaic stunting in bareroot Pinus banksiana Lamb. and suggest this paradigm is not universal. Stunted seedlings were distributed among healthy seedlings, occupied field space for 2–3 years, and used nursery resources (i.e. irrigation, fertilization); consequently high rates of culling at harvest resulted in an economic stress for the nursery. Thus, an understanding of the cause(s) of stunting was necessary. Stunted 1 + 0 seedlings had significantly lower levels of nitrogen, phosphorus, potassium, and zinc than their healthy cohorts, despite similar soil nutrient levels. The numbers of mycorrhizal root tips on stunted and healthy seedlings were similar, and the taxa of mycorrhizal fungi, determined by isolations and DNA sequencing, were not consistently associated with stunted or healthy seedlings. We conclude that differences in Pinus banksiana mycorrhizas are not responsible for mosaic stunting, but may be caused by localized low soil N availability due to uneven distribution of added organic matter amendments.     

Field survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi

Red pine and jack pine seedlings growing in styroblocks were inoculated 8 wk after sowing with mycelium/agar slurries of 3 mycorrhizal fungi (Laccaria bicolor, Scleroderma citrinum, and an unidentified basidiomycete), and one suspected mycorrhizal fungus (Cantharellula umbonata). Seedlings inoculated with L. bicolor developed mycorrhizae earlier and in greater numbers than the other inoculation treatments, with red pine out-performing jack pine in both respects. At 34 wk following sowing, seedlings were outplanted on a cleared xeric site in Baraga Co., in Michigan's Upper Peninsula. Seedlings inoculated with C. umbonata failed to form mycorrhizae and were not outplanted. Inoculation treatments did not affect shoot or root weight at outplanting. Red pine inoculated with L. bicolor averaged 21% and 19% greater survival compared with control seedlings after one and two years in the field, respectively. Other inoculation treatments failed to increase seedling survival for either tree species. Jack pine demonstrated higher overall survival than did red pine for both years in all corresponding inoculation treatments.     

In vitro ectomycorrhizal formation of Pinus patula, P. pseudostrobus, P. oocarpa and P. elliotii grown in southern India

In vitro syntheses of ectomycorrhizas of Pinus patula, P. pseudostrobus, P. oocarpa and P. elliotii with Amanita muscaria, Laccaria fraterna, L. laccata, Pisolithus tinctorius, Rhizopogon luteolus, Scleroderma citrinum and Thelephora terrestris were attempted. Most of the fungi initiated mycorrhizal formation within 2 weeks after inoculation. Laccaria fraterna and S. citrinum formed mycorrhizas after 6 weeks of inoculation. Amanita muscaria failed to form mycorrhizas during the 12 week period of observation. The degree of colonization of different mycorrhizal fungi varied with different species of Pinus.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Ectomycorrhizas and cadmium toxicity in Norway spruce seedlings

We studied the effects of ectomycorrhizal colonization by Laccaria bicolor (Maire) Orton S238 and Paxillus involutus (Batsch) Fr. 533 on cadmium (Cd) toxicity in Norway spruce seedlings (Picea abies (L.) Karst.). Both mycorrhizal and nonmycorrhizal seedlings were exposed to 0 (control), 0.5 or 5 &mgr;M CdSO(4) for 9 weeks in a sand culture system with frequent addition of nutrient solutions. In pure culture, P. involutus and L. bicolor showed similar Cd tolerance. However, in symbiosis, the Cd treatments decreased colonization by L. bicolor but not by P. involutus. Paxillus involutus ameliorated the negative effects of 0.5 &mgr;M Cd on shoot and root growth and chlorophyll content of old needles, whereas L. bicolor did not. Mycorrhizal colonization did not affect Cd concentrations of old needles and roots of seedlings. Despite differences between the ectomycorrhizal fungi in colonization and ability to alleviate Cd toxicity of seedlings, both species reduced Cd concentrations of young needles to a similar degree compared with nonmycorrhizal seedlings. However, in the 0.5 &mgr;M Cd treatment, the Cd content of needles of seedlings colonized by P. involutus was increased, whereas the Cd content of needles of seedlings colonized by L. bicolor was similar to that of needles of nonmycorrhizal seedings. When the amount of Cd translocated to needles was expressed on a root length basis to account for differences in the size of the root systems, the amount of Cd translocated to the needles was similar in seedlings mycorrhizal with P. involutus and in nonmycorrhizal seedlings. All mycorrhizal seedlings were similarly affected by 5 &mgr;M Cd, indicating that the amelioration efficiency of ectomycorrhizal fungi is dependent on the metal concentration to which the roots are exposed. Concentrations of P, K, Ca, Mg and Mn were decreased by 5 &mgr;M Cd to a similar extent in both nonmycorrhizal and mycorrhizal seedlings. In contrast to L. bicolor, P. involutus increased P uptake and altered patterns of root branching. We conclude that mycorrhizas alleviate Cd-induced reductions in growth of Picea abies seedlings. Although the two mycorrhizal fungi examined differed in their ability to alleviate Cd toxicity, these differences were not related to differences in Cd uptake or translocation to the shoot of the mycorrhizal seedlings. We suggest that amelioration of Cd toxicity by P. involutus may be a result of improved P nutrition or changes in root morphology, or both.     

Rock phosphate and vesicular-arbuscular mycorrhiza effects on growth and nutrient uptake of Faidherbia albida (Del.) seedlings in an alkaline sandy soil

Response of Faidherbia albida (Del.) to five levels of Kodjari rock phosphate (KRP) application (0, 310, 620, 1240 and 2480 ppm P, equivalent to 0, 775, 1550, 3100 and 6200 kg P/ha) and vesicular-arbuscular mycorrhizal fungi (VAMF) Glomus manihotis Howeler, Sieverding & Schenck or Glomus aggregatum Schenck & Smith emend. Koske was evaluated in an alkaline sandy soil. The F. albida seedlings grew poorly without mycorrhizal colonization and without KRP applications. For non-VAM Faidherbia, the maximum growth response and both P and N uptake in shoots was achieved with the 620 ppm P. However, even without KRP application, VAM plants achieved better results in terms of biomass. VAM plants with G. manihotis and G. aggregatum improved plant growth and increased nutrient contents at any KRP application rate. Although mycorrhizal colonization was comparable at all levels of KRP application, the impact of nutrient content of the shoot varied. Finally, VAM plants did not accumulate more biomass than non-VAM plants at 620 ppm P and above. Growth response and mycorrhizal dependency decreased as KRP applied levels increased. These results suggest that VAM Faidherbia seedlings take up more P from soil and KRP than non-VAM.