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Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. IV. Host-endophyte interactions during
arbuscular deterioration192
DARRELL A. KINDEN A N D MERTON F. BROWN Drpnrt~nent ofPIont Pathology, University ofMissouri, Colurnhin, Missorrri 65201
Accepted September l I , I975
K I N D E N , D. A., and M. F. BROWN. 1976. Electron microscopy of vesicular-arbuscular mycor- rhizae of yellow poplar. IV. Host-endophyte interactions during arbuscular deterioration. Can. J . Microbiol. 22: 64-75.
Scanning electron stereoscopy and transmission electron microscopy were used to correlate morphological alterations and cytological phenomena associated with deterioration of arbuscules in yellow poplar mycorrhizae. Arbuscular degradation was initiated at the tips of the finest branches and progressed basipetally. Cytoplasm in arbuscular hyphae progressively deteriorated and was followed by collapse of the fungal walls. Degraded portions of thearbuscules aggregated into clumps comprised of host wall material and the distorted fungal walls. Host nuclei, abundant mitochondria, and proplastids were closely associated with arbuscular branches undergoing cytoplasmic deterioration and with clumped portions of the arbuscule which contained degraded hyphal branches. Most of the arbuscules observed had deteriorated to the clumped stage. Some cortical cells contained several clumped arbuscules and nearly mature, intact arbuscules which indicated that reinfection occurs even as degradative phenomena are in progress. It is suggested that substantial quantities of mineral nutrients may be made available to the host via degradation of fungal cytoplasm in the arbuscular hyphae preceding aggregation of degraded hyphae into discrete clumps.
K I N D E N . D. A,, et M. F. BROWN. 1976. Electron microscopy of vesicular-arbuscular mycor- rhizae of yellow poplar. IV. Host-endophyte interactions during arbuscular deterioration. Can. J . Microbiol. 22: 64-75.
La stereoscopie par balayage electronique et la microscopie par transmission electronique furent utilisees pour relier les alterations rnorphologiques, et les phenomenes cytologiques associes, avec la deterioration des arbuscules des mycorrhizes du tulipier. La deterioration arbusculaire debute a la pointe des plus fines ramifications et progresse basipetalement. Le cytoplasme des hyphes arbusculaires degenere progressivement avec affaissement successif des parois fongiques. Les portions degenerees des arbuscules sont rkunies en touffes lesquelles sont constituees de materiel parietal de I'hbte et de parois d'hyphes rabougris. Les noyaux de I'hbte, des mitochondries nornbreuses et des proplastes sont etroiternent associes aux ramifications arbusculaires qui subissent la deterioration cytoplasmique et aux portions de touffes de I'arbuscule qui contient des ramifications d'hyphes degenkres. La plupart des arbuscules observes prisentent une deterioration avancee au stade de touffe. Certaines cellules corticales contiennent plusieurs arbuscules touffus et des arbuscules intacts, presque parvenus B rnaturite, ce qui indique qu'une re-infection prend place rnOme en presence de la progression des phenomenes de deterioration. Ceci suggere que des quantites substantielles de nutriments mineraux peuvent devenir disponibles pour I'hbte par suite de la degradation du cytoplasme des hyphes arbusculaires, avant que survienne I'aggregation en touffes discretes des hyphes degeneris.
[Traduit par le journal]
Introduction uptake and plant growth in low-fertility soils is
Vesicular-arbuscular (VA) mycorrhizae con- well established and has been reviewed recently
stitute the most common type of mycorrhizal by Gerdemann (6) and Mosse (14). Excellent
association formed by higher plants (15). The light-microscopic descriptions of VA infections
role of VA lnycorrhizae in increasing nutrient (3, 12, 13) suggest that the arbuscular cornpo- nents of these endophytes possess the greatest potential for nutrien; tiansf& to the host. HOW-
'Received June 10, 1975. ZContribution from the Missouri Agriculture Experi- ever, autoradiographic studies, conducted to
rnent Station. Approved by the Director as Journal series determine Sites of "P localization in the endo- paper 7324. phyte and, subsequently, in the host, yielded
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KINDEN AND BROWN: HOST-ENDOPHYTE INTERACTIONS 65
inconsistent results (1). Ultrastructural studies have been directed toward descriptive aspects of VA associations on various hosts (2, 9, 16). Little effort has been made to relate these ultra- structural observations t o the functional aspects of these mutualistic associations. The present study was conducted to determine the magnitude of arbuscular deterioration in naturally infected roots and the potential for nutrient transfer as a consequence of this process.
Materials and Methods Natural mycorrhizal infections of 1- and 2-year-old
yellow poplar (Lirioa'endron trclipi/era L.) seedlings were used in the study. The predominant endophyte involved in this association is considered to be Glon7us mosseae (Nicol. & Gerd.) Gerd. & Trappe. Spores of this fungus were consistently recovered by wet-sieving (4) soil from pots in which the seedlings were grown. Samples were collected and prepared for transmission (TEM) and scanning electron microscopy (SEM) as described before (11). Scanning electron stereopairs were taken with a 12' separation angle between individuals of each pair and have been mounted for viewing with a pocket stereo viewer. Figures 2, 3, 4, and 5 were enlarged photo- graphically, with some loss of resolution, to illustrate those structures at comparable magnifications. Polarizing microscopy and the IKI-H2S04 histochemical reaction were used to detect cellulose (8).
Results
Scanning Electron Microscopy Scanning microscopy of longitudinally sec-
tioned and chemically cleaned root segments permitted direct examination of several thousand arbuscules in various stages of degeneration. The use of stereoscopic techniques permitted an accurate determination of morphological altera- tions which were particularly valuable in corre- lation with TEM observations of the deteriora- tion process. An estimated 90% or more of all arbuscules examined by SEM showed some evidence of degeneration which resulted in dis- tinct morphological alterations of the arbuscular system.
Deterioration of arbuscules was initially mani- fested by collapse of the terminal hyphal branches and progressed toward the main trunk. However, the extent of degradation often varied markedly within individual portions of a single arbuscule. Frequently, arbuscules exhibited collapse of some bifurcate terminal branches, indicating initial stages of degeneration, while other por- tions of the same arbuscule were aggregated into compact clumps typical of later stages of this process (Fig. 1).
As deterioration progressed, substantial por- tions of the arbuscule were aggregated into loose, irregular clumps composed of degenerated fine branches (Fig. 2). Individual branches within the clumps were barely visible and appeared to be covered by loose, flocculent substances. At this stage of deterioration, the major branches, main trunk, and subtending hyphae in adjoining cells generally retained their morphological integrity. As the major branches subsequently collapsed, they were incorporated into existing clumps which remained attached to the main trunk of the arbuscule (Fig. 3). Throughout the deteriora- tion process, the arbuscules diminished in size and the hyphal components comprising the aggregations became less distinct. In the latter stages of deterioration, the arbuscules occupied only a small portion of the total cortical cell volume and were condensed about the residual trunk a t the original point of penetration through the host cell wall (Figs. 4, 5). At this stage, the clumps possessed an irregular but relatively smooth surface texture and individual arbuscular branches were completely obscured. Since col- lapsed hyphal components of degenerating ar- buscules could not be resolved by SEM in the late stages of deterioration, and since the surface texture of the clumps became progressively smoother, the deterioration process appeared to be accompanied by abundant deposition of sub- stances on the fungal surfaces. Deposition of such substances was most clearly defined as an encrusting layer on the trunk of the arbuscule (Fig. 5). Many cortical cells contained several degraded arbuscules and, frequently, they also contained mature functional arbuscules. This strongly suggested that reinfection and develop- ment of new arbuscules occurred while degrada- tive processes were in progress (Fig. 6). The occurrence of clumped arbuscules was wide- spread in root samples examined by both SEM and TEM. About 80-90% of the arbuscules observed were in the late stages of deterioration and exhibited a clumped morphology (Fig. 7) .
Transmission Electron Microscopy In agreement with SEM observations, nearly
all arbuscules observed by TEM exhibited some evidence of deterioration and most showed both collapse of fine branches and aggregation of degraded branches into clumps (Fig. 8). In such host cells, the major arbuscular branches ap- peared cytologically functional and contained nu- clei, numerous small vacuoles, and mitochondria.
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66 CAN. J . MICROBIOL. VOL. 22, 1976
The major branches were surrounded by a thin, but well-defined, layer of host wall material as previously reported on intracellular hyphae in this association (10). All components of the arbuscular system were enclosed by the host plasmalemma and cytoplasm which contained abundant mitochondria, proplastids, endoplas- mic reticulum, and ribosomes (Figs. 8 ,9 , 11). An electron-translucent interspace separated the host plasmalemma from the endophyte. Since host cytoplasm conformed to the branched structure of the arbuscules, vacuoles in the host cell were much smaller, more numerous, and more irregu- lar in outline than those in uninfected cortical cells (Fig. 8).
The first evidence of arbuscular deterioration observed by T E M was disorganization of cyto- plasmic structure in the smallest branches. Loss of membrane integrity was followed by a pro- gressive degradation and disintegration of the cytoplasm in these branches. Ultimately, little cytoplasmic residue remained and the fungal walls became compressed (Fig. 9). Viable small branches containing dense cytoplasm and small, distinct vacuoles were often interspersed among deteriorating branches. Often, smaller branches, which lacked cytoplasm and exhibited wall collapse, were attached to larger branches which appeared to be cytologically functional. This suggested a progressive deterioration from the
FIG. 1 . Scanning electron stereopair of a cortical cell containing arbuscules exhibiting initial collapse of terminal parts of arbuscular branches (arrows). One portion of the arbuscule has aggregated into an amorphous clump (C) indicative of a late stage of digestion while the globose arbuscule (left) shows no evidence of deterioration. x 1500. FIG. 2. Stereopair showing terminal arbuscular branches aggregated into irregular lobate clumps. Components of the clumps are poorly defined. Major arbuscular branches and the hypha from which the arbuscule was produced in a contiguous host cell remain intact (arrow). x 1300. FIG. 3 . Stereopair of an arbuscule showing condensation of individual clumps into a central aggregation surrounding the main trunk. One portion of the trunk (arrow) is collapsed but the subtending hypha in an adjoining cell is intact. x 1200.
FIG. 4. Stereopair of an arbuscule composed of several compacted clumps. The surface of the clumps appears relatively smooth and arbuscular branches cannot be defined. The surface texture of the trunk (T) and clumps is comparable. x 2000. FIG. 5, Stereopair showing a nearly completely deteriorated arbuscule composed of a single small but irregular clump. The trunk appears encrusted with amorphous material similar in appearance to the surface of the clump. x 2000. FIG. 6. Stereopair of a cortical cell containing several clumped arbuscules in a late stage of digestion and two mature arbuscules (arrows). x 1500.
FIG. 7. Scanning micrograph of a tangential plane through the inner cortex demonstrating the widespread occurrence of arbuscular digestion. Nearly every cell contains one or more arbuscules in late stages of digestion (see Figs. 4, 5). x 500.
FIG. 8. Ultrathin section showing portions of two cortical cells containing arbuscules. Major branches and some smaller branches contain nuclei (N) and appear cytologically functional while many individual branches are collapsed (arrows). A clump (C) of degraded branches is present. All fungal structures are enclosed by host cytoplasm and larger branches are surrounded by a distinct layer of host wall material (HW). Cortical cell wall (CW); vacuole (V); vesicular bodies (VB). x 5200.
FIG. 9. Portion of an arbuscule showing progressive degradation of fungal cytoplasm in the fine branches (D,-D2) and ultimate collapse of the fungal walls (FW). Collapsed walls remain attached to functional portions of the branch system but septa (arrows) separate viable and degraded portions of the hyphae. Host cytoplasm containing small proplastids (Pr) and numerous mitochondria surround all components of the endophyte. x 9000. FIG. 10. Portion of a collapsed arbuscular branch con- taining condensed cytoplasm and transverse septa (S). Accumulations of flocculent material are present between the fungal wall (FW) and host plasmalemma (P). x 19 300.
FIG. I I . Peripheral portion of a deteriorating arbuscule demonstrating the intimate association of the host nucleus (N) and degenerating branches. Numerous mitochondria (arrows) and proplastids (PI) are present in the host cytoplasm. x 6800. FIG. 12. Portion of an arbuscule with functional and deteriorated branches illustrating clump formation. Individual collapsed branches are progressively distorted (see Fig. 9). Groups of individual branches, surrounded by accumulations of flocculent material (arrows), aggregate into discrete clumps (C). Functional hyphae (H). x 5500.
FIG. 13. Oblique section through a portion of an arbuscular clump containing residual osmiophilic fungal walls (FW) and membrane-bound vesicular bodies (arrows). The fibrillar composition of the clump matrix is evident. x 49 500. FIG. 14. Portion of a host cell containing an arbuscule in a late stage of deterioration. Accumulations of the fibrillar clump matrix completely surround the arbuscular remnants which are aggregated into several large clumps. The host nucleus (N), enlarged proplastids (Pr) containing numerous osmiophilic plastoglobuli, and mitochondria (arrows) are associated with such arbuscular clumps. x 8400.
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74 CAN. J. MICROBIOL. VOL. 22, 1976
branch tips basipetally toward the arbuscular trunk such as observed by SEM (Fig. 1). Occa- sionally, collapse of some branches appeared to occur before total degradation of fungal cytoplasm. In such instances, the cytoplasm con- tained no discernible organelles or vacuoles and appeared as an amorphous osmiophilic mass (Fig. 10). Such branches often contained trans- verse septa separating deteriorating and func- tional portions of the arbuscular system. Varying amounts of flocculent material, similar in appear- ance to the host wall material surrounding intra- cellular hyphae and larger arbuscular branches, were present in the interspace between the host plasmalemma and the walls of both functional and deteriorating branches (Figs. 9, 10).
The host nucleus was often observed in close association with arbuscular branches undergoing deterioration (Fig. 11). The nuclei and nucleoli were markedly larger than in uninfected cells and nuclei were irregularly lobed since they were located among the branches of the arbuscule. Host cytoplasm surrounding the nucleus in such cells invariably contained large numbers of mitochondria and small proplastids.
After individual branches had collapsed, they were progressively distorted and aggregated into separate clumps (Figs. 2, 12). The relative size of the clumps appeared to be dependent upon the number of branches involved. There was no
shown in Fig. 3. Concurrently, the host vacuoles increased in size to nearly the volume observed in uninfected cells. The host nucleus was frequently located among the larger clumps and its shape conformed to the available space between them. A thin band of host cytoplasm surrounded the large clumps and was most abundant between closely associated clumps. While host mito- chondria were observed along the periphery of the clumps, they were more numerous adjacent to host nuclei. Proplastids in such cells were larger and contained more numerous plasto- globuli than proplastids in cells containing arbuscules which were predominantly in early stages of deterioration.
Examination of fresh root samples and paraffin sections of roots with polarized light revealed no birefringence in clumped arbuscules. Similarly, histochemical tests for cellulose yielded incon- clusive results. However, comparable tests made on root segments used for SEM produced definite birefringence and a positive reaction for cellulose in the clumps. Materials such as pectic and hemicellulosic substances, which may have masked the cellulose in untreated clumps, were apparently extracted by the alkaline hydrolysis treatment used with these samples.
Discussion Scanning electron microscopy, combined with
evidence of fragmentation of collapsed fungal stereoscopic techniques and removal of host walls. Concomitant with clumping, there was an cytoplasm to expose the endophyte, provided increase in the quantity of flocculent material in significant advantages over previous techniques the interspace between the fungal walls and the used to study deterioration of arbuscules within host plasmalemma. This material was noticeably root cortical cells. These procedures permitted condensed in well-defined clumps and at high direct observation of large numbers of arbus- magnification a fibrillar composition was appar- cules and identification of subtle, morphological ent (Fig. 13). Numerous membrane-bound vesi- alterations which often occurred initially in only cular bodies were present within the fibrillar a small portion of the arbuscular structure. matrix and were interpreted as remnants of host Progressive collapse of terminal branches basi- cytoplasm and ( o r ) organelles incorporated during aggregation of degraded hyphae and smaller clumps. Some of the compressed osmio- philic fungal walls within the clumps appeared less intensely stained and somewhat amorphous. Such variations in fungal wall structure ap- peared to result from their orientation within the clump and the plane at which they were sectioned rather than the result of degradation of fungal wall components. In later stages of deterioration (Fig. 14), larger clumps were formed by coales- cence of individual degraded branches and aggre- gation of smaller adjacent clumps such as those
petally toward the trunk and subsequent aggre- gation of degraded branches into discrete clumps, erroneously called 'sporangioles' by Janse (7), were clearly defined and were in agreement with descriptions of the deterioration process reported in VA-mycorrhizal associations at the light microscope level (3, 5, 12, 13). The encrusting material on the surface of the clumps, which obscured collapsed arbuscular branches in S E M preparations, undoubtedly correlates with the layered and flocculent host wall material around functional branches and that which .further ac- cumulated around branches undergoing deterio-
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KINDEN AND BROWN: HOST-ENDOPHYTE INTERACTIONS 75
ration and aggregation as observed by TEM (Fig. 14). In our samples, deterioration of arbuscular branches and subsequent aggregation of col- lapsed branches into clumps was always accom- panied by accumulation of host wall material which formed the matrix of the clumps. While accumulation of a similar material was reported in onion mycorrhizae by Cox and Sanders (2), this process was inconsistent in its occurrence. These workers reported that, in many instances, arbuscules underwent complete deterioration without becoming encased by such materials.
Host-metabolic activity, indicated by the abun- dance of mitochondria, endoplasmic reticulum, and ribosomes, was greatly increased in associ- ation with degenerating branches. Similar indi- cations of increased host metabolism continued throughout the development of compacted clumps. Since the clump matrix was clearly of host origin, these activities may be attributed to processes of host wall synthesis and deposition.
The presence of relatively few immature or mature, intact arbuscules, even in areas near the root apex where infections would be newly established, indicated that these structures are ephemeral. It appeared that degradative pro- cesses were initiated immediately after matura- tion of the arbuscule or simultaneously with maturation of portions of the structure. SEM observations clearlv revealed that reinfection of cortical cells was a common occurrence and that individual cells often contained several arbus- cules. Consequently, because of the complexity of host-arbuscular interactions, one cannot be certain that all fungal components observed by TEM within an individual host cell re~resent components or phenomena related to a single arbuscule. However, TEM observations clearly indicated that deterioration and disintegration of cytoplasm in arbuscular hyphae preceded collapse of the fungal walls and aggregation of wall residues into c lum~s . These observations are in agreement with data obtained in ultra- structural studies of tobacco (9) and onion (2) mycorrhizae.
I t is, therefore, most probable that nutrient transfer to the host occurs before clump forma- tion and as a consequence of solubilization of fungal cytoplasm in the arbuscule. With con- sideration given to the total volume of cytoplasm contained within active arbuscules throughout an entire mycorrhizal system, the extensive and rapid deterioration of arbuscules, and the poten-
tial for reinfection, it is apparent that substantial quantities of nutrients could be made available to the host in this manner. We do not regard processes involved in clump formation to be significant with respect to nutrient transfer. Rather, this phenomenon is interpreted as a belated host response to infection which, through host wall deposition, confines degenerated por- tions of the endophyte.
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