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A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. I. Morphological and cellular changes in mycorrhizas formed by Tylospora fibrillosa (Burt.) Donk and Paxillus involutus (Batsch. ex Fr.) Fr

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A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. I. Morphological and cellular changes in mycorrhizas formed by Tylospora fibrillosa (Burt.) Donk and Paxillus involutus (Batsch. ex Fr.) Fr
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   w  Phytol.  (1992), 122, 141^152 A study  of  ageing  of  spruce  [Picea sitchensis (Bong.) Carr.] ectomycorrhizas.  I a Morphological  and  cellular changes  in niycorrhizas formed  by  Tylospora fibrillosa (Burta) Donk and  Paxillus involutus (Batscha  ex Fr.) BY  G. M.  DOWNES\  I. J. ALEXANDERS  AND  J. W. G. CAIRNEY^ Department of Plant and Soil Science, University of Aberdeen, Meston Building,Aberdeen  AB9  2UE,  UK  Received 22 July 1991; accepted 10 April 1992) SUMMARY An age sequence  of  mycorrhizas formed by  Paxillus involutus  (Batsch. ex Fr.) Fr. and  Tylospora fibrillosa  (Burt.)Donk  was  sampled from 6-9-month-old seedlings  of  Picea sitchensis  (Bong.) Carr. grown  on  peat  in  rootobservation chambers. Information about  the  ageing process  was  obtained from co-ordinated studies  of mycorrhizal morphology, anatomy, ultrastructure and cell vitality (indicated  by  FDA-staining).Newly produced mycorrhizas were pale  and  turgid over most  of  their length with obvious extramatricalmycelium. Most mycorrhizas over 50 d old bad  a  light turgid apical portion and  a  darkened wrinkled proximalportion: extramatrical mycelium was less obvious. Many remained in this state until the sampling stopped  at  day142 but some were completely darkened before day 50. Tbe morphological appearance  of  a mycorrhiza was nota good indicator  of its  chronological age. Most mycorrbizas displayed periodic busts  of  growth which addedportions  of  young turgid cortex  to an  ageing axis.The percentage  of  cortical cells showing nuclei  in  1  fim  sections of tbe apical 1 mm declined from 40-50   in mycorrhizas  <  25 d old to 5-15 % in mycorrhizas 110-140 d old. Cortical cells became more vacuolate as they agedand the number  of  organelles appeared to decline. Senescence proceeded from the outer to tbe inner cortex andfrom proximal to distal regions. Degenerate cortical cells were present in the Hartig net zone of mycorrbizas over25 days old Cortical cell degeneration preceded, but was closely followed by, degeneration of adjacent Hartig net.Tbe FDA study supported tbe general pattern and timing of cell deatb interpreted from tbe morpbological andultrastructural study. Cortical/Hartig net Huorescence declined markedly  in  mycorrbizas over 70-85  d  old. Tbestele was the last tissue  in  the mycorrhiza  to  degenerate.  . ,  TT  • It is suggested that a mycorrbiza ceases to function in nutrient and water uptake when no living cortical/Hartignet interface remains. In'this study  a  few mycorrbizas became non-functional  by  day 31. For most mycorrbizastbe major decline  in  function took place after 85 days. This estimate  is in  broad agreement with tbe estimates ofmycorrbizal life span obtained from biomass studies  in  tbe field. Key words: Ectomycorrhiza,  Ptcea sttchensis, Paxillus involutus, Tylospora fibrillosa,  ageing. been estimated either from direct observation ofINTRODUCTION parts of the fine root system of trees or from Ectomvcorrhizas are the organs of water and nutrient measured seasonal fluctuations  in  fine ^oot biomassuptake'  in  most coniferous forests  and are an  Using  the  former approach Orlov (1957, I960)important sink for carbohydrate. Their longevity has recorded changes  m  small (12x12 mn.) portions  of the absorbing root system  of  100-yr-old  Picea abies ' Present address: CSIRO, Division  of  Forest Products,  ^ ^  the Russian taiga Over periods  of  Up tO 5 yr.  He ' TTest't  l^lTDe^rZZ  of1;.t  Sc n e.  Un.vers.ty  of  concluded that while  10 % of the  mycorrhizas died  in Adelaide, Waite Agricultural Research Institute, Glen Osmond,  ^^^  y^^j. ^hey were produced, 40  %  lasted  for  more,  g y y p SA 5064, Australia.  ^y^^^  3  yj.. Many  of the  estimates  of  mycorrhizal life^ To whom correspondence should be addressed.  142  G. M. Downes I.  J.  Alexander and J. W. G. Cairney span obtained using tbe biomass approacb (i.e. meanfine root longevity  =  mean annual fine root biomassdivided by annual mortality) are considerably lowertban tbose obtained by Orlov, e.g. 2-5 to 6-2 montbs(roots  <  1 mm diam.,  Pinus radiata  D. Don, Santantonio   Santantonio, 1987);  2-5  months(roots  <  2 mm diam.,  Pinus sylvestris  L.,  Persson,1979);  3-8  montbs (roots  <  1 mm diam.,  Picea sitchensis  (Bong.) Carr., Fairley, 1983; Alexander Fairley, 1983). Even tbe greatest estimates tbat canbe derived by tbe biomass metbod from tbe data setof Vogt, Grier Vogt (1986) are between 10 and 12montbs.Tbe reasons  for  tbe discrepancy between directand indirect approaches are not clear. It may be thatthe technical difficulties  in  obtaining data  on  fineroot biomass  and the  conceptual difficulties  in developing  a  reliable model  to  estimate fine rootmortality (Santantonio Grace, 1987) have led  to overestimates  of  root mortality, and thus to under-estimates  of  longevity. Further uncertainty arisesbecause  of the use of  morphological criteria  to distinguish living from dead mycorrhizas. Biomassstudies recognize  a  transition from young, light-coloured, turgid mycorrhizas  to  older, darker,wrinkled tips where only the apex remains light-coloured (Harvey, Larsen Jurgensen, 1976; Vogt,Edmonds Grier, 1981), and  it  is the existence ofthis light-coloured apex which is taken as the primaryindication  of a  living mycorrhiza. Orlov alsorecognized this progression and his diagrams (Orlov,1957, 1960) do show some mycorrhizas with light-coloured apices persisting  for  3 yr; however,  the longevities quoted appear rather to have been basedon  the  condition  of  the stele  as  viewed  in  cross-section.From a functional point of view the critical aspectof a living mycorrhiza  is  the existence  of  a physio-logically active interface between the symbionts, andit is not clear how this relates to the morphologicalcriteria outlined above. Ultrastructural studies  of VA and ericoid mycorrhizas have shown that  in those systems  the  interface  (i.e. an  individualarbuscule  or  coil)  is  maintained for  a  few weeks  at most (Harley, 1984). Ultrastructural studies  of ectomycorrhizas usually record living cortical  or epidermal cells  in  contact with  the  Hartig  net (Scannerini   Bonfante-Fasolo, 1983)  but  somerecord dead cells (Scannerini, 1968), and in a growthpouch study  of the  Pinus strobus L ./Pisolithustinctorius  (Pers.) Coker Couch association (Piche et  al 1983) cortical cells became necrotic as early as9 d after inoculation.There can clearly be several developmental stageswithin  a  single ectomycorrhiza. Atkinson (1975)described three zones  of  contact between host andfungus behind the root meristem of  Eagus sylvatica L.;  a  pre-Hartig net zone,  a  Hartig net zone, and  a late Hartig net zone in which epidermal and corticalcells become penetrated by fungal hyphae and thesheath begins  to  senesce. The pre-Hartig net zoneappears  to be a  consistent developmental stage  in ectomycorrhizas  but  the extent  to  which  the  lateHartig  net  stage  is  only  a  feature  of  certainhost-fungus combinations  is not  clear. Atkinsonhimself found no late Hartig net zone in  Betula  sp.or indeed in the ultimate order laterals of  Eagus  sp.where  a  more general senescence occurred. As wellas these developmental differences along the axis ofthe mycorrhiza there  are  obviously also develop-ments in a radial direction with the passage of time.Orlov considered that the death  of  the cortex andfungal sheath preceded that of the stele (see above).StruUu (1976) presented ultrastructural evidencethat ectomycorrhizal senescence begins in the outerlayers of the fungal sheath and proceeds inwards. Aninvestigation  of  cell vitality  in  spruce mycorrhizasusing fluorescein diacetate (Ritter, Kottke   Oberwinkler, 1986) supported this view. Someauthors (e.g. Fogel, 1985) consider  it  possible thatthe host tissue remains physiologically active afterthe death of the fungal symbiont, while others (e.g.Heikurainen, 1957) have maintained that the fungalmantle can remain 'active' after the death  of  thehost. Whether or not the mycobiont outlives its hosthas considerable implications  not  only  for the movement  of  carbon  and  nutrients within  the mycorrhizal system  but  also  for  decompositionprocesses and nutrient cycling in forest communities.In view  of  the uncertainties concerning the life-span and senescence of ectomycorrhizas, and to givea basis for further physiological work on carbon andnutrient metabolism  in  mycorrhizas  of  differentages,  we  have carried  out a  light  and  electronmicroscope study  of  ageing, using mycorrhizas  of known ages. Our main aims were to relate morpho-logical changes  to  cellular events,  to  see  at  whatstage host cells  in  contact with the Hartig net losetheir integrity, and to examine the relative longevityof host and fungal tissues. We examined mycorrhizasformed by two contrasting fungi,  Paxillus involutus (Batsch. ex Fr.) Fr. and  Tylospora  fibrillosa  (Burt.)Donk (Taylor   Alexander, 1991),  on  Picea sitchensis. MATERIALS AND METHODS Plant  and  fungal material Seedlings of  icea sitchensis  were established duringJan 1988  in  20 x 20 cm Perspex root observationchambers (Finlay Read, 1986) containing  a  non-sterile peat:vermiculite (1:7) mixture. The lowerPerspex plate  of  each chamber was etched with  a 1 cm grid  and  each grid square was referenced.Seedlings were inoculated with either  Tylosporafibrillosa  (isolate obtained from surface-sterilized P.  sitchensis  roots)  or  Paxillus involutus  (isolateobtained from a basidiome growing in a  P.  sitchensis  Ageing of spruce ectomycorrhizas. I 143 plantation) from agar cultures. Inoculated seedlingswere periodically watered with tap water and main-tained in a glasshouse with photoperiod extended to16 h by supplementary incandescent light and withroot temperature controlled to c. 15 °C using acooled water bath. During the following six monthsseedlings were periodically transferred to freshpeat: vermiculite medium, each transfer resulting ina brief flush of production of short lateral roots.Finally they were transferred to peat withoutvermiculite.An age sequence of mycorrhizas was sampled from6-9-month-old seedlings in the chambers. To avoidvariation between mycorrhizas owing to root en-vironment, host genotype and host carbon balance,the sampling sequence of each mycorrhizal type wastaken from a single seedling.Sampling was initiated at the end of June 1988, 3weeks following the final transfer, when a minimumof 130 new mycorrhizas had formed on each seedling.At the time of first sampling mycorrhizas werebetween 1 and 24 d old (T.  fibrillosa or 1 and 22 dold  {P. involutus .  Immediately prior to this sam-pling, each of the grid squares on the observationchambers containing one or more of the newlyproduced mycorrhizas was sketched so that eachmycorrhiza could be identified in later samplings.  T. fibrillosa  mycorrhizas were sampled at day 24, 31, 51, 71,  85, 105, 122, 135 and 142 and  P. involutus mycorrhizasatday22, 31,51,71,85,92, 116 and 132after the seedlings had been transferred to the peat.At each sampling, 10 mycorrhizas were selected atrandom and a morphological description of each wasprepared, supplemented by a photograph takenthrough a dissecting microscope. Five mycorrhizaswere subsequently processed for transmission elec-tron microscopy and five for vitality assessmentusing fluorescein diacetate. Assessment of mycorrhizal vitality by fluorescencemicroscopy Fluorescein diacetate (FDA) is a fluorogenic sub-strate which is non-fluorescent until enzymaticallyhydrolysed. It has been used to assess the vitality offungi in pure culture and in soil (e.g. Soderstrom,1977) and ectomycorrhizas (Ritter  et al.,  1986).Mycorrhizas were carefully excised from the rootsystem and rinsed in deionized water. Radial longi-tudinal sections were cut by hand, stained in anaqueous solution of FDA (0-05 mg ml ^) for 15 min,mounted in the same solution, examined and photo-graphed using epi-illumination with a 450^90 nmband pass excitation filter and a 520-560 nm bandpass barrier filter. Fluorescence of the meristem, stele,  fungal sheath, and Hartig net/cortical regionwere recorded on a scale of 0-3 (0 = no fluorescence,1 = < 33  o ^  of cells in tissue fluorescing or very faintfluorescence, 2 = 33-66% of cells in tissuefluorescing, 3 = > 66 % of cells in tissue fluorescing). Light microscopy and transmission electronmicroscopy Mycorrhizas were divided into two segments, anapical segment  {c.   mm), and the region immediatelyproximal to the apex (mature cortex). Mycorrhizalsegments were fixed in 2-5 % glutaraldehyde in0-03  M  PIPES buffer (pH 8 0) for 2 h at roomtemperature with continuous mixing. Followingthree washes in buffer, the tissue was post-fixed in1 % OsO, in 0-015  M  PIPES buffer (pH 7-4) for 1 hat 4 °C and dehydrated through an ethanol series.The segments were infiltrated with LR white resin(London Resin Company) over several days, using aregime combining continuous mixing at room tem-perature and vacuum-assisted infiltration at 10 °C,and embedded in gelatin capsules at 60 °C for 24 h.The low-viscosity LR white resin was used as itwas found to give the most reliable tissue infiltration.Preservation of membranes and cellular detail wasnot as good as with, for example, Epon 812 but sinceonly five mycorrhizas were available for analysis ateach harvest, reliable infiltration was regarded asmore important. Even using LR white, mycorrhizaswere increasingly difficult to infiltrate as they aged:where the cortical tissue had collapsed specimenstended to float on the resin surface and could not beprocessed further.Sections (1  /im  for light microscopy and80-120 nm for TEM) were prepared on an LKBUltramicrotome. For light microscopy sections werestained with 0-1 %Toluidine blue in O in acetatebuffer (pH 4-4) for 4 min. Thin sections for TEMwere mounted on formvar-coated grids, stained in asaturated aqueous solution of uranyl acetate for40 min followed by 15 min in Reynolds lead citrate(Reynolds, 1963) and viewed on a Philips 301 TEMusing an accelerating voltage of 80 kV.The number of cortical cells in the   /J,m  section ofthe apical 1 mm of each mycorrhizas was countedand the proportion of cells in which the sectionpassed through a nucleus was calculated. R SU TS General morphology T. fibrillosa  mycorrhizas.  Mycorrhizas in the firsttwo samples were pale and turgid over most of theirlength (Fig. la), with obvious extramatrical my-celium. By the third sample (day 51) the proximalcortex of some mycorrhizas in the sample haddarkened and collapsed. In subsequent samples themajority of the mycorrhizas were dark and wrinkledexcept for the light-coloured turgid apex (Figs 2a, 3a).  Extramatrical mycelium appeared to be lessabundant around these mycorrhizas than in earlier  144  G. M. Downes I. J. Alexander and J. W. G. Cairney Figure 1 A single  Tylospora jibrillosa Picea sitchensis  mycorrhiza, 24 days old. (a) Morphology: note light-coloured turgid appearance over entire length, (b) Freehand radial longitudinal section of apical  1  mm stainedwith FDA. Figure 2 As above but between 47 and 71 days old. (a) Morphology: note light-coloured, turgid apical portionand collapsed proximal portion, (b) FDA-stained section of the same mycorrhiza. Figure 3 . \s above but between 98 and 12 days old. (a) Morphology: note small light-coloured apex, (b) FDA-stained section of the same mycorrhiza: note fluorescence only in stele and pericycle, brightest in the proximalportion. The outline of the mycorrhiza, viewed in bright field, is shown. Figure 4 A single  Paxillus involutus/Picea  sitchensis  mycorrhiza, between 63 and 85 days old. Freehand radiallongitudinal section viewed with (a) bright-Reid and (b) fiuorescence microscopy. Note brightly fluorescingmeristem delineated by metacutin (m) and patchy cortical fluorescence (C). Figure 5 Tylospora fibrillosa/Picea  sitchensis  mycorrhiza between 27 and  51  days old. Semi-thin  1  //m) sectionof apical  1  mm stained with Toluidine blue O. Sheath (S), Hartig net (H), metacutin (m). Figure 6 Paxillus involutus/Picea  sitchensis  mycorrhizas photographed on (a) day 22 and (b) day 132. Note thechange in appearance of the mycorrhizas and the disappearance of mycelium and rhizomorphs. Figures 1-5,bar =  c.  500 //m; Figure 6, bar  = c. 2  mm.  Ageing of spruce ectomycorrhizas. I 145 50  u  o c o o TJ  t  o D Z 40302010 3060 90Time days)120150 Figure 7 The relationship between age and tbe meanpercentage of nucleated cortical cells in a   //m thick radiallongitudinal section of the apical   mm of mycorrbizas of Picea sitchensis  formed by  Paxillus involutus  (O) and Tylospora fibrillosa  (•). Each point is tbe mean of 2-5samples. , y = 49-2-0-20x,  r =  0 69;  ,  3;  =41-8-0-24X,  r^  = 0-68.samples. Some mycorrhizas appeared to have formedby a continuous period of extension, with progressivedarkening of the proximal cortex, and to have thenpersisted in a quiescent state with a light turgidapical portion of variable length. Others showed thebeaded appearance associated with discontinuousextension growth. Mycorrhizas with a completelydarkened apex were first encountered in the sampleon day 85. At the final sample (day 142), six of theten mycorrhizas still had a light, turgid apex. T. fibrillosa  mycorrhizas had a compact pros-enchymatous sheath 3-7 hyphal elements in depth.Sheath hyphae were embedded in an interhyphalmatrix which stained blue-purple with Toluidineblue O. The Hartig net penetrated to the en-dodermis. In the samples on days 24 and 31 mostmycorrhizas showed a clear pre-Hartig net zoneextending 3-6 cortical cells behind the apical tissue.Seventy-five per cent of the mycorrhizas in thesesamples had ceased apical growth and metacutin hadformed. One mycorrhizas on day 31 showed evidenceof renewed growth (cortical cells distal to metacutin).At later samplings in those mycorrhizas wheremetacutin had formed behind the apex, and norenewed extension had occurred, Hartig net waspresent immediately proximal to the metacutin, i.e.there was no pre-Hartig net zone.In most mycorrhizas sampled after day 31 therewas evidence of more than one period of extension(Fig. 5). Hartig net formed around the new corticalcells by the ingrowth of hyphae from the sheath. Apre-Hartig net zone was present in the most recentlyformed cohort of cortical cells in some cases but notin others. It seems reasonable to assume that a pre-Hartig net zone was a feature of actively extendingmycorrhizas or mycorrhizas which had recentlycompleted a period of extension, and that in non-extending mycorrhizas the Hartig net progressively'caught up' with the metacutin behind the apex.Although one or more layers of metacutin were a 10 feature of most mycorrhizas in all samples, a singlemycorrhiza sampled on day 134 had no metacutinand appeared to have extended continuouslythroughout the sampling period. This mycorrhizahad a pre-Hartig net zone.From 25 to 50 % of the cortical cells in a section ofthe apical 1 mm of the mycorrhizas at day 24contained nuclei. At subsequent sampling dates theproportion of nucleated cortical cells was variable,being greater in those mycorrhizas which showedevidence of extension growth, but in general theproportion declined with time and was 10-20% atday 134 (Fig. 7). P.  involutus  mycorrhizas.  Mycorrhizas in the firsttwo samples (day 22, 31) were pale grey-buff andturgid over most of their length, with obviousextramatrical mycelium and rhizomorphs (Fig. 6a).More than half of the mycorrhizas were beaded, i.e.displayed morphological evidence of intermittentextension. In a number of mycorrhizas the root apexwas not covered by the fungal sheath. By the thirdsample (day 51) the mycorrhizas had darkened andthe proximal cortex in some had started to collapseleaving a lighter turgid apical portion of variableextent. The extramatrical mycelium had dis-appeared. One tip was completely brown with nolighter apex. At the final sample (day 132, Fig. 6b)six of eight mycorrhizas still had a light turgid apex. P.  involutus  mycorrhizas had a loose patchyprosenchymatous sheath. In mycorrhizas from day85 onwards the sheath appeared more compactexcept where new extension growth had beencolonized by a loose sheath. Throughout the sam-pling period the Hartig net was never extensive,often patchy, and in many mycorrhizas little or noHartig net was observed. In the first sample (day 22)six of the ten mycorrhizas had a layer of metacutinand four of these had renewed extension (i.e. corticalcells distal to metacutin). After day 92 all themycorrhizas sampled had at least one layer ofmetacutin.About 25-50% of the cortical cells in a section ofthe apical 1 mm of mycorrhizas at day 22 containednuclei. As with  T. fibrillosa  mycorrhizas this pro-portion declined over time, but counts of < 15%were more common in  P. involutus  mycorrhizas(Eig. 7). Ultrastructure T. fibrillosa  mycorrhizas.  At the second sampling(day 31), hyphae of the fungal sheath in the Hartignet zone were embedded in an electron-lucentextracellular material. Hyphae were cytoplasmic,though many, particularly in the Hartig net andinner part of the sheath, contained large vacuoles(Fig. 8). Outer and inner cortical cells of the hostwere generally highly vacuolated, often with only a ANP 122
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