Transgenic Betula pendula expressing sugar beet chitinase IV forms normal ectomycorrhizae with Paxillus involutus in vitro

Abstract

Mycorrhizal fungi contain chitin in their cell walls and may be influenced by transgenic chitinases. This study examined the ability of a transgenic tree, silver birch (Betula pendula Roth), constitutively expressing the sugar beet chitinase IV gene, to form ectomycorrhizae with Paxillus involutus (Batsch) Fr. Eight transgenic lines showing varying levels of sugar beet chitinase IV expression and the non-transgenic control plants were inoculated by P. involutus in vitro, and the morphology of the mycorrhizae, mycorrhization efficiency and shoot and root fresh weights were studied. All the transgenic birch lines were able to form normal ectomycorrhizae containing distinctive mantles and Hartig nets. The level of sugar beet chitinase IV expression had no significant influence on mycorrhizal colonization. The only significant differences between transgenic and control plants were detected in weight parameters. According to these results, the expression of sugar beet chitinase IV in silver birch is not deleterious to formation of ectomycorrhizae between birch and P. involutus.     

外生菌根真菌纯培养的生态学研究

本文研究了Boletus griseus Frost.(灰盖牛肝菌),Boletus sp.和Suillus grevillei (Klotz.) Sing. (厚环乳牛肝菌)3株菌根菌在纯培养条件下对温度、pH、盐浓度和水势等4个因子的生态适应性以及同油松和湿地松纯培养合成菌根的能力。结果表明,pH和盐浓度对菌根菌生长的影响比较小,而温度和水势则对真菌的生长具有很大的影响。其中B.sp.与S.grevillei具有很强的生态适应能力,而B.griseus则稍差。此外,结果还表明PEG4000可能会促进菌根菌在液体培养中的生长。在菌根纯培养合成研究中,共形成5种菌根,其中油松-B.griseus属新发现的外生菌根。5种菌根中,以油松-S.grevillei和湿地松-B.griseus较为理想。     

Dynamics of symbiotic establishment between an IAA-overproducing mutant of the ectomycorrhizal fungus Hebeloma cylindrosporum and Pinus pinaster

To clarify the early steps of symbiotic establishment, we studied the dynamics of Pinus pinaster (Ait.) Sol. tap root colonization and mycorrhiza formation by an IAA-overproducing mutant of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi and by the corresponding wild type strain. Differences between wild type and mutant strains were quantitative rather than qualitative and were detected two days after inoculation. Both fungal strains established a typical Hartig net when they colonized the tap roots. Consequently, colonized tap roots exhibited features of a true mycorrhiza and fungal colonization enhanced plant growth. Fungal colonization and Hartig net formation were more rapid with the mutant than with the wild type. Colonization, especially with the mutant strain, increased rhizogenesis and the production of mycorrhizas. The mutant formed a hypertrophic Hartig net in tap roots and mycorrhizal short roots and we obtained evidence that the process of short root transformation into mycorrhiza started before their emergence from the tap root. Hyphae of the Hartig net in the tap root penetrated the cortex of young lateral roots at the beginning of their elongation, after the endodermis layer broke under the pressure of the elongating lateral root. Colonization was inhibited when triiodobenzoic acid was added to the culture medium, providing circumstantial evidence that auxin is involved in mycorrhiza formation.     

毛白杨VA菌根与外生菌根关系的研究

定期从毛白杨扦插育苗地挖取根样，并从中原地区的毛白杨人工林地采集细根（直径小于２ｍｍ）根样，观察统计ＶＡ菌根、外生菌根、混合菌根以及未受菌根菌侵染的１ｃｍ长根段的百分比。结果表明，在苗木的菌根侵染初期，ＶＡ菌根菌的侵染势较强，但优势很快被外生菌根菌取代，两种菌根菌间的负交互作用随苗龄的增加而不断增强。造林地种类和土壤的ｐＨ值对毛白杨两种菌根间的负交互作用关系有制约作用。在强碱性砂土地上，ＶＡ菌根的     

Light sources with different spectra affect root and mycorrhiza formation in Scots pine in vitro

We studied the effects of broad-spectrum light quality on the interaction between the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch and Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings cultured in vitro. The light sources were cool white (CW), warm white (WW) and red-rich daylight (RD) fluorescent lamps. Inoculation with P. tinctorius enhanced adventitious root formation of the cuttings in all light treatments. Rooting of the inoculated cuttings was highest in WW light (89%), followed by CW (73%) and RD light (66%). During 6 weeks of in vitro culture, rooted cuttings formed only a few lateral roots. The fungus grew over lateral roots, but the Hartig net was absent in all light treatments. In non-inoculated cuttings, neither root formation nor subsequent root growth was affected by light quality. In the seedling experiment, inoculation in the WW treatment resulted in a significantly (P < 0.05) greater number of lateral roots than inoculation in the RD treatment. The percentage of lateral roots covered with fungal hyphae was also highest in WW light (62%), followed by CW (50%) and RD (27%) light. A similar pattern was observed in the intensity of Hartig net formation. We conclude that effects of broad-spectrum light quality on the ectomycorrhizal fungus-root interaction are dependent on the developmental stage of the root.     

Laser microprobe mass analysis (LAMMA) of aluminum and lead in fine roots and their ectomycorrhizal mantles of Norway spruce (Picea abies (L.) Karst.)

Fine roots and ectomycorrhizal root tips were sampled in a Norway spruce (Picea abies (L.) Karst.) stand in the eastern part of the Belgian Ardennes. The cellular and partly subcellular localizations of aluminum and lead were identified by the micro-analytical laser microprobe mass analysis (LAMMA) technique. In fine roots with secondary structure, localization of aluminum was limited to the peripheral cell layers. Lead was found in the outer layers, and also in the primary phloem. Aluminum penetrated the mycorrhizal mantle, but lead was seldom detected in ectomycorrhizae.     

毛白杨优良外生菌根菌的选择*

在毛白杨外生菌根菌生态学特性和共生关系研究的基础上，对与毛白杨有良好共生关系的外个菌根菌的研究表明，硬皮马勃能耐温，在碱笥条件下仍能生长，在中性基质中，均能与毛白杨形成菌根，并大幅度地提高毛白杨的生长量。但在碱性土壤中，仅有硬皮马勃能与毛白杨形成一定数量的菌根，并能明显地提高毛白杨的生长量、地上部分生物量，改善菌木Ｎ、Ｐ的营养状况，接种效果随接种量的变化而差异明显。     

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.     

彩色豆马勃菌剂的应用研究

外生菌根真菌——彩色豆马勃(豆包菌)Pisolithus tinctorius(Pers.)Coker and Couch(简称P_t)。应用彩色豆马勃商品化菌剂对我国一些重要用材树种进行菌根化的研究,评价这些树种对P_t菌剂的反应,各种菌剂的效果及其应用前景,为在我国林业生产和环境保护中推广和应用P_t技术及其菌剂提供基础。     

Only two weeks are required forTricholoma matsutake to differentiate ectomycorrhizal Hartig net structures in roots ofPinus densiflora seedlings cultivated on artificial substrate

There has been conflicting debate over many years regarding the trophic status ofTricholoma matsutake (Ito et Imai) Sing., and further investigations are necessary to better understandT. matsutake physiology, particularly carbon nutrition, during ectomycorrhizal symbiosis. For this purpose, we developed a technique to rapidly synthesizein vitro ectomycorrhizas betweenPinus densiflora Sieb. et Zucc. andT. matsutake on artificial substrate (vermiculite: perlite: peat: beech sawdust; 5:5:1:1.), without added sugar in the nutrient solution. Only 1 week was required before the first rudimentary Hartig net ‘palmetti’ could be observed in roots. Well-developed Hartig net structures appeared in taproots after 2 weeks and in lateral roots after 3 weeks. Such rapid root infection may be attributed to the quality of the substrate and the inoculum used. This research was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN).     

Response of juvenile Norway​ spruce (Picea abies [L.] Karst.) to ectomycorrhizal inoculation of perlite-peat substrates in a nursery

Nursery growth substrate and ectomycorrhizal symbiosis are important circumstances influencing seedling development. In this study, the effect of pre-sowing inoculation of pure peat and peat+perlite mixtures (2:1 and 1:1, v:v) with an ectomycorrhizal mycelium-bead inoculum prepared in the laboratory and commercial inoculum Ectovit on ectomycorrhiza formation, growth, and foliar nutrients concentration of 1-year-old bare-root Norway spruce seedlings was estimated. Seedlings grown in the substrates inoculated with bead inoculum had significantly larger shoot dry weight and slightly higher other growth parameters than those grown in non-inoculated and Ectovit-inoculated substrates. However, the use of inocula formed neither treatment-specific ectomycorrhizal morphotypes nor higher mycorrhization of roots compared to control. Ectomycorrhizae were most probably formed by naturally occurring fungi; nevertheless, based on the abundance of the morphotypes, any participation of the applied fungi in ectomycorrhiza formation and/or their non-nutritional effects is not excluded. Seedlings grown in peat+perlite (2:1) mixture were slightly higher-developed than those in the two other substrates, but significant effect of the substrates neither on seedling growth nor on ectomycorrhiza formation was detected. In all treatments, sufficient macroelements concentration in needles was found. The results suggest difficulties to reach a complex positive effect of ectomycorrhizal inoculation in operational conditions and its dependence on various circumstances.     

Boric acid-phenolic relationships within the Pinus echinata-Pisolithus tinctorius ectomycorrhizal association

At germination, container-grown shortleaf pine seedlings were inoculated with Pisolithus tinctorius (Pers.) Coker & Couch or left uninoculated, and both groups were fertilized semiweekly with a modified Hoagland's solution supplemented with 0 or 0.4 mM boric acid. After 12, 16 and 24 weeks, seedling root tissue was analyzed for ectomycorrhizal colonization, phenolic concentration and phenoloxidase activity. In addition, phenoloxidase activity was assayed in P. tinctorius that had been cultured in a liquid medium containing boric acid. Inoculation with P. tinctorius increased the root phenolic concentration of 16- and 24-week-old seedlings, and increased root phenoloxidase activity in 12-, 16- and 24-week-old seedlings. Fertilization with boric acid reduced the phenolic concentration of P. tinctorius ectomycorrhizae after 24 weeks. Although boric acid fertilization did not affect the phenoloxidase activity of 12-, 16- and 24-week-old inoculated roots, or that of 16- and 24-week-old uninoculated roots, it increased the phenoloxidase activity of P. tinctorius grown in vitro and 12-week-old uninoculated roots. We conclude that boric acid fertilization influences the phenolic relations of the shortleaf pine-P. tinctorius ectomycorrhizal association, possibly through a boric acid-induced increase in phenoloxidase activity.     

Distribution of Frankia, ectomycorrhizal fungi, and bacteria in soil after the eruption of Miyake-Jima (Izu Islands, Japan) in 2000

Miyake-Jima, a round island of about 8 km in diameter, is located about 200 km south of Tokyo, Japan (34°08′ N, 139°53′ E). In July 2000, the central cone of this island collapsed and a new eruption started with the falling of volcanic ash and SO₂ emission. In October 2001, we measured the distribution of bacteria, and Frankia, a symbiotic nitrogen-fixing actinomycete that forms root nodules, and ectomycorrhizal fungi associated with alder in newly deposited ash and its underlying soil by cultivation of Alnus sieboldiana seedlings in these soils. There were fewer bacteria growing on the nutrient broth agar and fewer bacteria growing on 100-fold diluted nutrient broth agar in newly deposited volcanic ash than there were growing in the buried old soil. In four out of five sites, little or no nodulation was observed in newly deposited ash; abundant root nodules were formed in the underlying soil. Ectomycorrhizae formed in the seedlings cultivated in the underlying soil. In May 2003, the distribution of Frankia and ectomycorrhizal fungi in soil at different depths (up to 160 cm) was also investigated. Frankia and ectomycorrhizal fungi were relatively abundant in surface soil layers.     

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.     

Binucleate Rhizoctonia (Ceratorhiza spp.) induce adventitious root formation in hypocotyl cuttings of Pinus sylvestris L.

Adventitious rooting of Scots pine (Pinus sylvestris L.) hypocotyl cuttings was promoted by binucleate Rhizoctonia (BnR) and to a lesser extent by ectomycorrhizal fungi. Four BnR isolates (251, 266, 268 and 269) differentiated root meristems and significantly induced adventitious rooting in young derooted seedlings. Rooting rates were significantly higher in BnR treatments than in either indol-3-butyric acid (IBA) pretreatment (200 µm) or co-cultivation with the ectomycorrhizal fungi Suillus bovinus or Laccaria bicolor. In pre-IBA-treated cuttings, adventitious root numbers were higher in the treatments with isolates 251 and particularly 268, while more similar in other BnR treatments. In the 251, roots emerged from distal positions along the hypocotyl and not from the cut base as in the other three BnR isolate treatments. Loss of turgor and hypocotyl wilt was commonly detected up to the point of root initiation. Lateral roots and dichotomous short roots subsequently appeared at high frequency from primary adventitious roots. Mechanisms involved in root meristem differentiation, e.g. auxin production, wound response and oligosaccharide signals, are discussed with respect to host-fungal signalling mechanisms. These beneficial BnR and ectomycorrhizal fungi could provide promising new tools in the development of efficient clonal propagation methodology for this highly recalcitrant Pinus species.     

西澳州桉树林地土壤菌根菌剂接种潜力研究(英文)

对澳大利亚西澳州桉树人工林菌根类型及其形成情况进行了调查 ,并采用生物测定法 (Bioas saymeasurement)研究了桉树林地土壤菌根菌剂的接种潜力 .根系菌根检查和生物测定法试验均表明 ,在自然条件下桉树可以和不同真菌共生而形成三种类型的菌根 ,即外生菌根、VA菌根和混合菌根 ;林分成熟程度与菌根形成有一定的相关性 ,与共生体的类型也有一定影响 .在幼林中 ,桉树根系主要与内囊霉菌共生形成VA菌根 ,而成熟林主要与担子菌共生形成外生菌根 ,混合菌根表现为一种中间类型 .在收集的林地土壤中移植的菌根诱饵植物 (三叶草和蓝桉 )分别检测出土壤中存在有一定量的VA菌根菌和外生菌根菌 ,但菌根菌繁殖体数量及接种潜力相对较小 ,并且也揭示了按树年龄对土壤菌剂的相对接种潜力影响较大 .本文对桉树人工林土壤菌剂接种潜力进行了评价 ,并就引进优良菌根菌对桉树人工林生产的重要性进行讨论     

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.     

Ectomycorrhizal colonisation of roots and ash granules in a spruce forest treated with granulated wood ash

Granulated wood ash has been proposed as a slow release fertiliser suitable for forest soils. In this study ectomycorrhizal colonisation of roots and ash granules was studied in a 40-year-old spruce forest treated with 0, 3 or 6 t ha⁻¹ granulated wood ash. We used PCR-RFLP methods for ITS-typing and identification of ectomycorrhizal fungi. In total 20 different ITS-types were recognised on roots in the organic soil horizon. Five of these were identified to species and two to genus. Six species, Tylospora fibrillosa Donk, Cortinarius sp. 3, Piloderma sp. 1, and three unidentified ITS-types (Ve-95-1, Tö-95-3 and Ve-95-9) each occurred on over 5% of the total root tips analysed. Together these comprised 55% of the ectomycorrhizal community on the screened roots.

Ash granules collected from the fertilised plots were normally colonised by fungal mycelia. PCR-RFLP analysis of these mycelia revealed the presence of four ITS-types. Three of these (Piloderma sp. 1, Ha-96-3 and Tor-97-1) were also present on the mycorrhizal roots. Piloderma sp. 1 was the most abundant species colonising roots. A possible role of ectomycorrhizal mycelia in the direct mobilisation of nutrients from ash granules is discussed.     

Ectomycorrhizal fungal community of naturally regenerated Pinus thunbergii seedlings in a coastal pine forest

To add to knowledge of ectomycorrhizal (ECM) community structure on the roots of Pinus thunbergii seedlings in a Japanese coastal forest, we sampled naturally regenerated current-year and 1- to 5-year-old seedlings. We classified the 667 root tips on current-year seedlings and the 1,927 root tips on older seedlings into 13 phylotypes based on morphological and genetic analyses. Cenococcum geophilum, members of the families Clavulinaceae, Russulaceae, or Thelephoraceae or the genus Trichophaea, were indicated to be fungi forming P. thunbergii ectomycorrhizas. Among them, C. geophilum and Clavulinaceae sp. 1 were the most or second most dominant species. A species accumulation curve based on the number of samples nearly reached a plateau, with observed species richness equal to 11 species and the Jackknife2 and Chao2 richness estimators indicating 14 and 12 species, respectively. In addition, Simpson’s 1/D was 3.89 and Shannon–Wiener’s H′ was 1.71, indicating a relatively low taxonomic diversity. There was no significant difference in the ectomycorrhizal formation rate, or the occurrence frequency and the number of ECM phylotypes between current-year and older seedlings. These results indicated that less diverse fungi were involved in ectomycorrhizal formation on coastal pine seedlings compared with those in comparable inland forests in the study area.     

Mycorrhizal status of an ozone-sensitive poplar clone treated with the antiozonant ethylene diurea

The antiozonant ethylene diurea is proven to prevent growth reductions in forest trees induced by ozone. The community of mycorrhizal fungi could be useful indicator of environmental stress. In this study, response of mycorrhizal fungi and fine roots to a 4-year exposure to ambient ozone and treatment with antiozonant was investigated in ozone-sensitive poplar clone under field conditions. The community of ectomycorrhizal fungi and root length colonization with ectomycorrhizal, arbuscular mycorrhizal fungi, and root endophytic fungi was analyzed in antiozonant-treated poplar plants and in poplar plants irrigated with water. In general, plants protected by antiozonant showed higher total number of fine roots, number of ectomycorrhizal types, Shannon–Weaver diversity index, and Species richness index compared to the plants treated with water. The ectomycorrhizal community shifted from contact exploration type in the trees irrigated with water to short-distance exploration type in ethylene diurea-treated trees. Ozone protectant may beneficially affect the belowground community of mycorrhizal fungi colonizing roots of ozone-sensitive poplar clone.