Báo cáo lâm nghiệp: "Physiology and metabolism of ectomycorrhizae"

Chia sẻ: Nguyễn Minh Thắng | Ngày: | Loại File: PDF | Số trang:9

Thêm vào BST

Báo xấu

70
lượt xem 3
download

Download Vui lòng tải xuống để xem tài liệu đầy đủ

Tuyển tập các báo cáo nghiên cứu về lâm nghiệp được đăng trên tạp chí lâm nghiệp Original article đề tài: Physiology and metabolism of ectomycorrhizae...

Chủ đề:

Bình luận(0) Đăng nhập để gửi bình luận!

Lưu

Nội dung Text: Báo cáo lâm nghiệp: "Physiology and metabolism of ectomycorrhizae"

metabolism of Physiology and ectomycorrhizae C. Bledsoe D. Brown M. Coleman W. Littke 2 1 3 Rygiewicz P. 1 5 and J. Ammirati J. A mn ati 5 5 U. SSanqwar , S. Rogers U. 4 S. oge angwanit 1 WA 98 t 95 U.S.A. University of Washington, Seattle, College of Forest Resources AR-f 0, 2 Weyerhaeuser Corp., Centralia WA, U.S.A., 3 US EPA, Corvaltis, OR, U.S.A., 4 Forest Bangkok, Thailand, and Biology, Kasetsart University, 5 Washington, Seattle, WA, U.S.A. of Botany Dept., University mycorrhizal group in forestry at the Uni- Introduction versity of Washington. Our research pro- gram has focused on two central ques- the forests of today. forests tions: How do ectomycorrhizal fungi affect Managed are In these forests, growth and yield are processes of nutrient uptake by forest tree improved by forest fertilization. Application species? And, do fungal species differ in of fertilizers, often nitrogen, has created a their abilities to affect physiological pro- need for more understanding of how min- cesses in general? eral nutrients, roots and soils interact. This need has produced new partnerships among forest soil scientists, root physiol- ogists, soil microbiologists, tree nutri- uptake and metabolism Nutrient tionists and mycorrhizal research workers. The study of mycorrhizae is a critical inter- Inorganic nitrogen uptake face in understanding the processes by which nutrients are transferred from the soil through fungal hyphae into roots, then Inorganic ammonium and nitrate are as- metabolized and distributed throughout sumed to be the major forms in which the tree. This interface between root and nitrogen is taken up by tree roots. Forest fungus is illustrated in Fig. 1. soils generally contain more ammonium than nitrate, although levels of either ion The following is a discussion of ectomy- are relatively low. Although organic nitro- corrhizal fungal physiology and its effects gen is also an important form of nitrogen, on coniferous trees, particularly effects on more attention has been directed to inor- nutrient uptake, tree nutrition and water ganic forms. Soil pH is both affected by stress. This discussion focuses on 10 and affects uptake of ammonium and years of research conducted by our
red to solutions for short uptake periods. nitrate. With ammonium uptake, hydrogen ions are released into the rhizosphere, Uptake rates decreased with increasing while uptake of nitrate results in hydroxyl acidity, so that rates at pH 3 were only ion release. These exchanges balance 50-70% of rates at pH 7. Mycorrhizal charge in the roots and substantially alter plants generally had higher uptake rates pH around the roots. This affects availabi- over the entire pH range, particularly Douglas fir. Mycorrhizal effects were much lity and uptake of many ions (particularly more noticeable for ammonium uptake phosphorus). than for nitrate uptake. Unlike many crop In our lab, we measured uptake of species, uptake rates for ammonium were ammonium and nitrate by 3 conifer spe- higher, about 10-fold, than nitrate uptake cies (Douglas fir, western hemlock and rates. Since ammonium levels in forest Sitka spruce) which were either non- soils are gene-rally higher than nitrate, mycorrhizal or mycorrhizal with Hebelo- higher uptake rates might be expected. ma crustuliniforme (Bull. ex. Fr.) Que!. Mycorrhizal roots did not release as (Rygiewicz al., 1984a, b). Seedlings et grown in solid media, then transfer- much H+ per ammonium taken up as did were
mycorrhizal root cells. Half-lives of of non-mycorrhizal roots. This finding sug- potassium in all 3 cellular compartments gested that mycorrhizal fungi may buffer were increased by mycorrhizal fungi. For ammonium uptake, allowing uptake to example, in the vacuolar compartment, the continue at faster rates by reducing acidifi- half-life was 25 h for mycorrhizal roots, but cation in the rhizosphere. Another interest- only 6.6 h for non-mycorrhizal roots. ing observation was that ions were not These data suggest that mycorrhizal fungi only being taken up by roots, but were also being released - sometimes in sub- can alter ion fluxes through roots, reducing efflux and resulting in increased retention stantial amounts. Potassium efflux was in the roots. Higher fungal metabolic rates noted. Clearly, loss or efflux of ions must may increase energy for active uptake and be a temporary phenomenon, since plants increase in size and nutrient content over retention of ions. time. Our results simply indicated that influx or efflux of a particular ion may Cation-anion balance change from time to time, depending upon conditions. We have shown cation efflux during a period of rapid ammonium uptake These mycorrhizal effects on ionic fluxes by Douglas fir roots (Cole and Bledsoe, led us to ask whether mycorrhizal fungi 1976). When all the ammonium in the can change total ionic flux into cells. A solution was depleted, cations were reab- cation-anion balance sheet was deter- sorbed. Although it may seem inefficient, mined for mycorrhizal and non-mycorrhizal plant roots both take up and release ions Douglas fir seedling roots during a short at rapid rates. Some ions are certainly uptake period (Bledsoe and Rygiewicz, retained, but this may be a small percent- 1986). Influx and efflux of cations (ammo- age of the total flux. nium, potassium, calcium, H and anions ) + (phosphate, sulfate, chloride and bicarbo- nate) were measured using stable and Potassium fluxes in roots radioisotopes and chemical analyses. In this experiment, mycorrhizae had little Our interest in ionic fluxes into and out of effect on total fluxes, but they did increase roots led to a study of mycorrhizal effects anion uptake and bicarbonate release. For on these fluxes. Using a compartmental all treatments, cation fluxes were much analysis technique, we labeled Douglas fir more rapid than were anion fluxes; 25 roots for 24 h with radioactive rubidium times more cations enter and leave root (potassium tracer) (Rygiewicz and Bled- cells than anions. This massive cation 1984). After labeling, rubidium efflux influx was not balanced by parallel anion soe, tracked for 10 h. Mathematical anal- influx, but by efflux of H and potassium. + was yses of efflux data allowed data to be The very small amount of anion influx was separated into fluxes and pool sizes for 3 balanced by bicarbonate efflux. Most compartments: cell wall/free space, cyto- cations were presumably stored in plasm and vacuole. vacuoles as salts of organic acids. Our calculations suggest that both mycorrhizal There was rapid influx and efflux of and non-mycorrhizal coniferous roots syn- potassium. About 95% of all potassium thesize large amounts of organic acids. entering roots was subsequently released; net accumulation was only 5% of total flux. Using data from the literature, we com- Mycorrhizal fungi did alter fluxes, with pared our data on coniferous roots to more storage of potassium in the vacuoles those on several major crop species and
found a major difference. Coniferous roots mycorrhizal with Cenococcum geophilum take up cations at about twice the rate of Fr., H. crustuliniforme, or Suillus granula- herbaceous crop species -27 vs 14 tus (L.: Fr) Kuntze. Net charge of these microequivalents per gram dry wt. of roots amino acids was either neutral (alanine), per hour. Since hydrogen ions are the pri- plus (aspartic acid) or minus (arginine) at mary ion released to balance cation up- pH 5.5, the uptake solution pH. take, coniferous roots acidify the external Uptake was measured by appearance in medium (or soil) to a much greater extent [14C)arnino acids. The use of axe- roots of than do roots of crop species. Conifer nic seedlings precluded microbial degra- roots also synthesize greater quantities of dation of the amino acids, which would organic acids than do crop species. Table have separated 14 label from the amino C I shows these fluxes. acid. Ionic charge had little effect on rates, since they were similar - about 50 nmol per mg of root per hour x 10- (Bledsoe 2 and Sangwanit, submitted). The single Organic nitrogen exception was lower rates (25 nmol) for arginine uptake by hemlock. Similarly, As indicated earlier, little attention has choice of host species had little effect on been paid to organic nitrogen uptake by uptake rate, with the exception noted plants or to fluxes and pool sizes of above for hemlock and arginine. Fungal soluble organic nitrogen in forest soils. effects were significant. Compared to non- Early work by Melin in the 1950’s demon- mycorrhizal seedlings, rates for seedlings strated amino acid uptake by mycorrhizal mycorrhizal with Hebeloma and Cenococ- roots. There have been few reports since cum were 25 and 33% higher, while rates then. We investigated uptake and utiliza- for Suillus were lower - only 75% of the tion of organic nitrogen, since this path- control rates. Thus choice of the fungal way may be important for carbon and partner did affect amino acid uptake rates. nitrogen assimilation by roots. Amino acid metabolism Amino acid uptake Metabolism of these 3 amino acids and a 3 different amino acids, uptake 4th amino acid, glycine, was affected both Using rates by roots of Douglas fir and western by type of amino acid and by mycorrhizae. measured in solution cul- After a 4 h uptake period, little glycine had hemlock were and Bledsoe, submitted; been metabolized (90% unaltered gly- (Sangwanit ture and Bledsoe cine). In contrast, about 50% of the ala- Sangwanit, submitted). either nine was converted into non-amino carbon Seedlings non-mycorrhizal or were
Using microautoradiography, the loca- compounds; less than 30% alanine re- glycine in root tissues was deter- mained. About 70 and 50% of arginine tion of and aspartate, respectively, remained. mined (Sangwanit and Bledsoe, submit- When root extracts were chromatograph- ted). In a time series uptake experiment, ed on thin-layer chromatograms, many mycorrhizal and non-mycorrhizal Douglas C_labeled 1a compounds were found. fir roots were exposed to glycine for 1, 4, Mycorrhizal roots often contained 14 C-la- 12 and 24 h. Then root tips were frozen in bel not found in non-mycorrhizal roots - liquid N freeze-dried at -70°C, and , 2 such as ’C in Fig. 2. This unknown com- vacuum-embedded with a low viscosity, ’ 7 pound was produced in 5 of the 6 mycor- non-water soluble resin (to prevent move- rhizal treatments, but not in ment of water-soluble glycine). After cut- non-mycorrhi- zal(NM) ones. These mycorrhizal-specific ting ultramicrotome sections, root sections compounds were not identified. covered with a film emulsion and were stored. After photographic development, black dots on the film indicated [ C]gly- 14 Amino acid transfer and storage cine in root tissues. Glycine appeared in the stele of non- mycorrhizal roots at 1 h; transport con- might be expected to Mycorrhizal roots tinued throughout the 24 h experiment store amino acids in fungal tissues and to (Sangwanit and Bledsoe, submitted). For transfer less to the stele, in contrast to mycorrhizal roots, however, much of the non-mycorrhizal roots. Using [C]glycine 14 glycine was stored in the fungal mantle. (Sangwanit and Bledsoe, submitted), we Gradually, glycine was transferred to the found that non-mycorrhizal roots did trans- stele over the 24 h experiment. These fer much of their glycine directly to the results indicate that mycorrhizal roots can shoot, whereas mycorrhizal roots stored storage organ for organic nitro- more glycine in the roots. serve as a
gen in roots. Perhaps in a forest soil, or- very difficult to characterize root-associ- ganic nitrogen may be taken up directly by ated fungi based solely on color and cul- the fungi and stored in the mantle. At later ture characteristics (Bledsoe, 1987) and times, these amino acids could be used many mycorrhizal fungi have not been as grown in culture. of both carbon and nitrogen for a source growth as well as for root or tree fungal We are developing an identification pro- growth. cedure based on rDNA patterns (Rogers et al., 1988). Using about 1-100 mg from fruit bodies (fresh or dried), fresh-cultured fungal mycelia or mycorrhizal roots, rDNA Fungal physiological diversity was extracted using a CTAB microprepa- ration method (Fig. 3). After extraction and purification, the DNA was restricted with Our previous discussion has documented EcoRl, run on an agarose gel and South- the beneficial effects of mycorrhizae on ern blots were made with a yeast pBD4 nutrient uptake and metabolism. These probe. results led to the following question. Do fungal species differ in their abilities to Fig. 4 shows an autoradiograph of rDNA affect physiological processes in general? blot-hybridizatio!n patterns from mycorrhi- For example, there are a large number of of Rhizopogron vinicolorA.H. Sm: lane zae fungal species - more than 1000 - that mycelial culture only; lane 2 Douglas 1 = = may form mycorrhizae with Douglas fir fir mycorrhizal roots; lane 3 uninfected = (Trappe, personal communication). Why roots. The position of fungal bands was there so many different fungi? Do they are separate from those of the conifer roots. have different ecological niches? Do they Thus, the fungus infecting the root could carry out different functions in association be identified by comparison to patterns with tree roots? This puzzling fungal di- from a ’library’ of mycorrhizal fungal pat- versity is the focus of our current work. terns. Since fungal and root patterns did not overlap, it is not necessary to separate and root tissues before DNA extrac- fungal Identification of fungi on roots considerable advantage. tion - a Before studying fungal diversity, it is Fungal physiological diversity necessary to know whether diversity of fungal fruit bodies is related to mycorrhizal diversity. Are fungi which form fruit bodies Although many fungi fruit in association also functioning mycorrhizae? We are stu- withDouglas fir, little is known about which dying fruiting patterns and mycorrhizal pat- fungus is appropriate for any set of envi- terns on roots in the same field plots ronmental conditions. We studied one (Rogers, personal communication). If aspect of physiological diversity - the abil- there are correlations between fruiting pat- of to tol,erate water stress ity fungi (Cole- terns and root-associated mycorrhizal aL, 1988). Over 50 isolates were et man fungi, then we can assume that taxonomic tested in pure culture, using polyethylene diversity is related to mycorrhizal diversity. glycol to adjust medium water potential. In order to know which fungi are present In response to stress, 3 different growth on roots, it is necessary to identify root patterns were observed (Fig. 5). For type fungi. However, fungal taxonomy is based I, fungi were intolerant of stress and grew on characteristics of the fruit body. It is
only at the lowest level of stress (-0.02 MPa). For type II, fungi did tolerate some Growth rates decreased with stress. maximum growth occur- increasing stress; red in the lowest stress level. For type III, fungi were much more tolerant of stress and even grew faster at a moderate stress level. Laccaria spp. were type I. Most of the isolates (80+%) were type II. Only 7 isolates were type III, including C. geophi- lum and H. crustuliniforme (Coleman et al., 1988). These results indicate that fungi do differ in their abilities to grow under imposed water stress in pure culture. We have synthesized mycorrhizal seedlings with some of these isolates and are stu- dying their effects on the water relations of Douglas fir seedlings (Coleman, personal communication).
ed by Blasius et al. These research results Summary and Conclusions illustrate the intense interest in under- standing how mycorrhizae affect host nutrition and physiology. In addition to ourwork, other papers pre- sented at this symposium report on With the of and techniques use new mycorrhizal physiology. For example, the methods, we are now able to understand soils work in Germany by Ritter and not only how fungi affect nutrient uptake coworkers shows effects of liming soils on and tree nutrition but also to study more species diversity of fungi. In nutritional stu- specific effects of individual fungal spe- dies, Rousseau and Reid from Florida, cies. In the future, we expect that we will USA, have focused on phosphorus uptake understand fungal physiological diversity and translocation, while Vezina et al., from sufficiently to be ;able to select certain fun- Laval, Quebec, evaluated metabolism of gal partners for specific field and environ- nitrogen supplied in different forms. More mental conditions. New areas of research detailed metabolic studies at the Univers- will probably include: host-fungus recog- ity of Nancy by Chalot and coworkers nition, genetic engineering of mycorrhizal showed more efficient uptake of am- fungi, studies of :>patial patterns of mycor- monium by mycorrhizal plants. Not only rhizal roots in forest soils and microbial nutrients but also carbon biochemistry is interactions in the rhizosphere. affected by mycorrhizae as reported by Namysl et al., also at the University of Nancy. Several papers discussed inter- Acknowledgments actions in the rhizosphere, such as El- Badaqui et al.’s report on mycorrhizal pro- duction of extracellular phosphatases. We appreciate the technical assistance provid- Succession of different mycorrhizal types ed by Suzanne Bagshaw, Faridah Dahlan, Kelly on seedlings and young trees was report- Leslie, Kim Do and HCathy Parker.
References Hebeloma crustuliniforme and other Pacific NW mycorrhizal fungi. Can. J. Bot. 62, 647-652 Rogers S.O., Rehner S., Bledsoe C., Mueller Bledsoe C.S. (1987) Ecophysiological diversity G.J. & Ammirati J.F. (1989) Extraction of DNA of ectomycorrhizae. In: Current Topics in from fresh, dried & lyophilized fungus tissue for Forest Research, U.S. Forest Service S.E. Exp. ribosomal DNA hybridizations. Can. J. Bot. 67, Stn. Tech. Report No. SE-46, pp. 14-19 9 1235-1243 Bledsoe C. & Rygiewicz P.T. (1986) Ectomycor- P.T. & Bledsoe C.S. (1984) Mycorrhi- Rygiewicz rhizae affect ionic balance during ammonium zal effects on potassium fluxes by Northwest uptake by Douglas fir roots. New Phytol. 102, coniferous seedlings. Plant Physiol. 76, 918- 271-283 923 Cole D.W. & Bledsoe C.S. (1976) Nutrient Bledsoe C.S. & Rygiewicz P.T., Zasoski R.J. dynamic of Douglas fir. IVI IUFRO World (1984a) Effects of ectomycorrhizae & solution Congress Proceedings, Div. II, pp. 53-64 pH on 15N ammonium uptake by coniferous Coleman M.D., Bledsoe C.S. & Lopushinsky W. seedlings. Can. J. For. Res. 14, 885-892 (1989) Pure culture response of ectomyc. fungi Rygiewicz P.T, Bledsoe C.S. & Zasoski R.J. to imposed water stress. Can. J. Bot. 67, 29-39 (1984b) Effects of ectomycorrhizae and solution pH on 15 nitrate uptake by coniferous seed- N Littke W.R., Bledsoe C.S. & Edmonds R.L. lings. Can. J. For. Res. 14, 893-899 & in vitro by (1984) Nitrogen uptake growth