The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.     

Contrasting responses to ectomycorrhizal inoculation in seedlings of six tropical African tree species

Five caesalpinioid legumes, Afzelia africana, Afzelia bella, Anthonotha macrophylla, Cryptosepalum tetraphylum and Paramacrolobium coeruleum, and one Euphorbiaceae species, Uapaca somon, with a considerable range in seed sizes, exhibited different responses to inoculation by four species of ectomycorrhizal (ECM) fungi, Scleroderma dictyosporum, S. verrucosum, Pisolithus sp. and one thelephoroid sp. in greenhouse conditions. Thelephoroid sp. efficiently colonized seedlings of all of the five caesalpinioid legumes except U. somon, but provided no more growth benefit than the other fungi. Thelephoroid sp. and S. dictyosporum colonized seedlings of U. somon poorly, but stimulated plant growth more than the other fungi. The relative mycorrhizal dependency (RMD) values of the caesalpinioid legumes were never higher than 50%, whilst U. somon had RMD values ranging from 84.6 to 88.6%, irrespective of the fungal species. The RMD values were negatively related to seed mass for all plant species. Potassium concentrations in leaves were more closely related than phosphorus to the stimulation of seedling biomass production by the ECM fungi. Our data support the hypothesis that African caesalpinioid legumes and euphorbe tree species with smaller seeds show higher RMD values than those with the larger seeds.     

Morphological and molecular analyses of ectomycorrhizal diversity in <Emphasis Type="Italic">Pinus densiflora</Emphasis> seedlings

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification) and molecular analysis (internal transcribed spacer region amplification and sequencing), we assessed diversity and composition of the ectomycorrhizal fungal community colonizing P. densiflora seedlings (from 1 to 5 years ) in a naturally regenerating area of a mature forest, in Dongning County of Hei Longjiang Province, China. A total of 22 ectomycorrhizal taxa were determined on the roots of 100 seedlings. The most frequent species was Cenococcum geophilum, which colonized 31.2% of the root tips and occurred in 93.8% of the all samples. It was followed by Clavulina cinerea, Thelephora sp.2, and Russula albidula. They colonized in 22.7%, 17.5%, 13.4% of the root tips and occurred in 63.4%, 42.6%, 29.3% of the samples respectively. These results suggested that ectomycorrhizal diversity increased in P. densiflora seedlings with increasing age.     

Distribution of ectomycorrhizal and pathogenic fungi in soil along a vegetational change from Japanese black pine (Pinus thunbergii) to black locust (Robinia pseudoacacia)

The nitrogen-fixing tree black locust (Robinia pseudoacacia L.) seems to affect ectomycorrhizal (ECM) colonization and disease severity of Japanese black pine (Pinus thunbergii Parl.) seedlings. We examined the effect of black locust on the distribution of ECM and pathogenic fungi in soil. DNA was extracted from soil at depths of 0–5 and 5–10 cm, collected from the border between a Japanese black pine- and a black locust-dominated forest, and the distribution of these fungi was investigated by denaturing gradient gel electrophoresis. The effect of soil nutrition and pH on fungal distribution was also examined. Tomentella sp. 1 and Tomentella sp. 2 were not detected from some subplots in the Japanese black pine-dominated forest. Ectomycorrhizas formed by Tomentella spp. were dominant in black locust-dominated subplots and very little in the Japanese black pine-dominated forest. Therefore, the distribution may be influenced by the distribution of inoculum potential, although we could not detect significant relationships between the distribution of Tomentella spp. on pine seedlings and in soils. The other ECM fungi were detected in soils in subplots where the ECM fungi was not detected on pine seedlings, and there was no significant correlation between the distribution of the ECM fungi on pine seedlings and in soils. Therefore, inoculum potential seemed to not always influence the ECM community on roots. The distribution of Lactarius quieticolor and Tomentella sp. 2 in soil at a depth of 0–5 cm positively correlated with soil phosphate (soil P) and that of Tomentella sp. 2 also positively correlated with soil nitrogen (soil N). These results suggest the possibility that the distribution of inoculum potential of the ECM fungi was affected by soil N and soil P. Although the mortality of the pine seedlings was higher in the black locust-dominated area than in the Japanese black pine-dominated area, a pathogenic fungus of pine seedlings, Cylindrocladium pacificum, was detected in soil at depths of 0–5 and 5–10 cm from both these areas. This indicates that the disease severity of pine seedlings in this study was influenced by environmental conditions rather than the distribution of inoculum potential.     

Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.     

Soil transfers from valley oak (Quercus lobata Nee) stands increase ectomycorrhizal diversity and alter root and shoot growth on valley oak seedlings

 Soils from valley oak (Quercus lobata Nee) riparian areas of the Cosumnes River Nature Conservancy Preserve near Sacramento, California were added to growth medium of valley oak seedlings grown in a greenhouse or in agricultural fields at Cosumnes which probably once supported valley oak trees and are now replanted with native riparian vegetation or allowed to revegetate naturally. Agricultural field soil from the Cosumnes River Preserve was presumed to be low or lacking in ectomycorrhizal inoculum. The study was designed to (1) determine whether valley oak stand soil transfer could cause mycorrhizal infection on valley oak seedlings in an agricultural field and in a greenhouse, (2) describe ectomycorrhizal morphological types formed on valley oak seedlings, and (3) determine whether seedling growth is enhanced more by transfer of natural valley oak stand soil than agricultural field soil. In the field study, transfer of forest soil increased average ectomycorrhizal diversity (2.4 types) more than transfer of agricultural field soil (1.2 types). Valley oak seedlings were responsive to ectomycorrhizal infection in the field study. With increase in mycorrhizal infection there was an increase in shoot growth at the expense of root growth. In the greenhouse study, both percent mycorrhizal infection and mycorrhizal diversity were increased more by transfer of oak forest and woodland soils than agricultural field soil. Eight morphotypes occurred on seedlings in forest and woodland soils but only three morphotypes in agricultural soil. This result strongly suggests that the agricultural field also harbors ectomycorrhizal propagules but forest and woodland soils support a more abundant and diverse ectomycorrhizal flora. Accepted: 17 August 1997     

连续三年夜间增温和施氮对云杉外生菌根及菌根真菌多样性的影响

以西南亚高山针叶林建群种粗枝云杉(Picea asperata)为研究对象,采用红外加热模拟增温结合外施氮肥(NH₄NO₃ 25 g N m^-2 a^-1)的方法,研究连续3a夜间增温和施肥对云杉幼苗外生菌根侵染率、土壤外生菌根真菌生物量及其群落多样性的影响。结果表明:夜间增温对云杉外生菌根侵染率的影响具有季节性及根级差异。夜间增温对春季(2011年5月)云杉1级根,夏季(2011年7月)和秋季(2010年10月)云杉2级根侵染率影响显著。除2011年7月1级根外,施氮对云杉1、2级根侵染率无显著影响。夜间增温对土壤中外生菌根真菌的生物量和群落多样性无显著影响,施氮及增温与施氮联合处理使土壤中外生菌根真菌生物量显著降低,但却提高了外生菌根真菌群落的多样性。这说明云杉幼苗外生菌根侵染率对温度较敏感,土壤外生菌根真菌生物量及其群落多样性对施氮较敏感。这为进一步研究该区域亚高山针叶林地下过程对全球气候变化的响应机制提供了科学依据。     

Growth response of Afzelia africana Sm. seedlings to ectomycorrhizal inoculation in a nutrient-deficient soil

 The growth and mineral nutrition responses of seedlings of two provenances of Afzelia africana Sm. from Senegal and Burkina Faso, inoculated with four ectomycorrhizal (ECM) fungi (Scleroderma spp. and an unidentified isolate) from the same regions were assessed in a pot experiment in a savanna ECM-propagule-free soil deficient in NPK. There was little variation in the ability of the different fungal species to colonize roots of either provenance of A. africana or to produce external hyphal in soil. Root colonization by ECM fungi and their hyphal development were not related to mineral nutrition or ECM dependency. Differences in P, N, Mg and Ca concentrations in the leaves of inoculated and non-inoculated Afzelia seedlings were not always associated with production of biomass. Only leaf K concentration increased in both provenances after ECM inoculation. However, the Burkina Faso provenance responded better to inoculation with the two fungal isolates than the Senegal provenance in terms of biomass production. This was due to stimulation of root dry weight of the Burkina Faso provenance. Therefore, the hypothesis arises that non-nutritional rather than nutritional effects explain the contribution of ECM inoculation to the growth of A. africana seedlings. Accepted: 27 April 1999     

Ectomycorrhizal fungi promote growth of <Emphasis Type="Italic">Shorea balangeran</Emphasis> in degraded peat swamp forests

Shorea balangeran is an important component of peat swamp forests in Southeast Asia and is an important source of timber. However, S. balangeran has been decreasing in number due to overexploitation. The objective of this study was to investigate the effect of inoculation of native ectomycorrhizal (ECM) fungi on growth of S. balangeran in degraded peat swamp forest. Spores of Boletus sp., Scleroderma sp., and Strobilomyces sp. were collected from natural peat swamp forest in Indonesia. Seedlings of S. balangeran were inoculated with or without (control) spores and grown in sterilized peat soil under nursery conditions for 6 months. Then, the seedlings were transplanted into a degraded peat swamp forest and grown for 40 months. ECM colonization was 59–67% under nursery conditions and increased shoot height and weight. Shoot height, stem diameter, and survival rates were higher in inoculated seedlings than in control 40 months after transplantation. The results suggest that inoculation of native ECM fungi onto native tree species is useful for reforestation of degraded peat swamp forests.     

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.     

Interaction between a dark septate endophytic isolate from Dendrobium sp. and roots of D. nobile seedlings

Interactions between an isolate of dark septate endophytas (DSE) and roots of Dendroblum nobile Lindl.seedlings are reported in this paper.The isolate was obtained from orchid mycorrhizas on Dendrobium sp.in subtropical forest.The fungus formed typical orchid mycorrhiza in aseptic co-culture with D.nobile seedlings on modified Murashige-Skoog (MMS)medium.Anatomic observations of the infected roots showed that the DSE hyphae invaded the velamen layer,passed through passage cells in exodermis,entered the cortex cells,and then formed fungal pelotons of orchid mycorrhiza.D.nobile seedlings' plant height,stem diameter,new roots number and biomass were greatly enhanced by inoculating the fungus to seedlings.The fungus was identified as Leptodontidium by sequencing the polymerase chain reaction-amplified rDNA ITSt-5.8S-ITS2 (internal transcribed spacer (ITS)) regions and comparison with similar taxa.     

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.     

Ectomycorrhizal fungal biomass in roots and uptake of P from apatite by Pinus sylvestris seedlings growing in forest soil with and without wood ash amendment

Forest soil from an experimental Norway spruce forest with four levels of wood ash addition (0, 1, 3 and 6 tonnes ha^–1) was used to inoculate pine (Pinus sylvestris) seedlings with indigenous ectomycorrhizal (EM) fungi. Uptake of ³²P and ⁸⁶Rb in a root bioassay was used to estimate the demand for P and K by seedlings grown in the different soils. Utilisation of P from apatite was tested in a laboratory system where uptake by the ectomycorrhizal mycelium was separated from uptake by roots. The demand for P and K in the seedlings was similar regardless of the ash treatment. Variation in EM levels, estimated as fungal biomass (ergosterol) in roots, was large in the different soils, but not related to ash addition. Uptake of P from apatite was, on average, 23% of total seedling P and was not related to EM levels. It was concluded that the improved P uptake from apatite by EM fungi found in earlier studies is probably not a general phenomenon among EM fungi. The small effect of ash addition on EM levels and P uptake suggests that addition of granulated wood ash is a forest management treatment that will have only minor influence on ectomycorrhizal symbiosis.     

Growth,nutrient acquisition and ectomycorrhizae of <Emphasis Type="Italic">Eucalyptus regnans</Emphasis> F. Muell. seedlings in fertilized or diluted air-dried and undried forest soil

Seedlings of Eucalyptus regnans (mountain ash) grow poorly in undried forest soil, where they develop purple coloration in the foliage, but their growth is markedly improved when forest soil has been air dried. Whether this growth promotion is purely due to improved nutrient status of the soil, as a result of air drying, was investigated. In several pot experiments, E. regnans seedlings were grown (i) in air-dried and undried forest soil with addition of different levels of complete fertiliser, (ii) in air-dried or undried soil diluted to different extents with sand, or (iii) in undried soil mixed with different amounts of air-dried soil. Seedling dry weight, P content and incidence of ectomycorrhizal root tips were determined.In all experiments, the dry weights of seedlings were 3–6 times greater in 100% air-dried soil than in 100% undried soil. Fertiliser application resulted in a significant increase in dry weight of seedlings in both air-dried and undried soil, but the dry weights in air-dried soil were always significantly greater than those in undried soil at the same level of fertiliser application. Even at the highest level of fertiliser application, the growth difference between seedlings in air-dried and undried soil remained. When air-dried soil was diluted with sand, there was a significant reduction in seedling dry weight only when soil was diluted to 20% or less (air-dried soil:total mix). Conversly, when air-dried soil was mixed with undried soil, there was a proportional decrease in seedling dry weight with increasing amounts of undried soil. In all experiments, the dominant ectomycorrhizal morphotypes in 100% air-dried soil were different from those in undried soil. Fertilisation and dilution of air-dried and undried soil did not result in a reduction in the overall incidence of ectomycorrhizal root tips, although the frequency of occurrence of different ectomycorrhizal morphotypes was affected.It is concluded that the growth difference between seedlings in air-dried and undried forest soils is not due solely to differences in the direct availability of nutrients in the soils, and different ectomycorrhizae may indirectly affect nutrient availability to the plant.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

The effects of insect pathogenic soil fungi and ectomycorrhizal inoculation of birch seedlings on the survival of Otiorhynchus larvae

• 1 Weevil larvae of the genus Otiorhynchus are a serious problem in agriculture and forestry, causing damage to a wide range of plant species, primarily by larval feeding on roots. Otiorhynchus larvae are a serious pest in forest plantations in Iceland, causing 10–20% mortality of newly‐planted seedlings.
• 2 We studied the effects of soil fungi on the survival of Otiorhynchus sulcatus larvae. The larvae were introduced into pots with birch seedlings grown in: (i) nursery peat; (ii) nursery peat inoculated with three different species of ectomycorrhizal fungi; (iii) nursery peat inoculated with insect pathogenic fungi; (iv) nursery peat inoculated with ectomycorrhizal fungi and insect pathogenic fungi; and (v) nursery peat inoculated with natural forest soil from Icelandic birch woodland.
• 3 Larval survival was negatively affected by inoculation of: (i) the ectomycorrhizal fungus Laccaria laccata; (ii) the ectomycorrhizal fungus Cenococcum geophylum; (iii) the insect pathogenic fungus Metarhizium anisopliae; and (iv) forest soil. Inoculation with the ectomycorrhizal fungus Phialophora finlandia did not have any significant effect on larval survival. No significant synergistic effect was found between insect pathogenic and ectomycorrhizal fungi.
• 4 It is concluded that ectomycorrhizal and insect pathogenic fungi have a significant potential in biological control of Otiorhynchus larvae in afforestation areas in Iceland. Further studies are needed to establish the effect of these fungi in the field and to analyse how mycorrhizal fungi affect root‐feeding larvae.

     

<Emphasis Type="Italic">Aurifilum,</Emphasis> a new fungal genus in the Cryphonectriaceae from <Emphasis Type="Italic">Terminalia</Emphasis> species in Cameroon

Native Terminalia spp. in West Africa provide a popular source of construction timber as well as medical, spiritual and social benefits to rural populations. Very little is, however, known regarding the diseases that affect these trees. During an investigation into possible diseases of Terminalia spp. in Cameroon, orange to yellow fungal fruiting structures, resembling those of fungi in the Cryphonectriaceae, were commonly observed on the bark of native Terminalia ivorensis, and on dead branches of non-native Terminalia mantaly. In this study the fungus was identified based on morphological features as well as DNA sequence data (ITS and β-tubulin) and its pathogenicity was tested on T. mantaly seedlings. Our results showed that isolates of this fungus represent a previously undescribed genus in the Cryphonectriaceae, which we describe as Aurifilum marmelostoma gen. et sp. nov. Pathogenicity tests revealed that A. marmelostoma is pathogenic on T. mantaly. These tests, and the association of A. marmelostoma with disease symptoms on T. ivorensis, suggest that the fungus is a pathogen of this important tree.     

Mycorrhiza status of Gnetum spp. in Cameroon: evaluating diversity with a view to ameliorating domestication efforts

A field survey was carried out to investigate the diversity of mycorrhizal fungi associated with Gnetum spp. in Cameroon. The extent and variation of ectomycorrhizal colonisation as well as the degree of host specificity were evaluated. Gnetum spp. were found to be almost always ectomycorrhizal in all sites visited. There were just two ectomycorrhizal morphotypes (‘yellow’ and ‘white’) associated with this plant. Such low diversity is unusual for an ectomycorrhizal plant. The yellow morphotype was the most widespread and prevalent and was identified by morphological and molecular methods to have been formed with Scleroderma sinnamariense. Propagules of this fungus were present in soil collected from farm lands, cocoa plantations, Chromolaena and bush fallows, as well as in a relatively undisturbed forest harbouring ectomycorrhizal legumes. The fungus responsible for the white morphotype was identified as also belonging to the genus Scleroderma by ITS sequence similarity. Arbuscular mycorrhizal structures were absent in cleared and stained portions of the roots.