Dynamics of arbuscule development and degeneration in mycorrhizas of Triticum aestivum L. and Avena sativa L. with reference to Zea mays L.

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1 New Phytol. (1988), 110, Dynamics of arbuscule development and degeneration in mycorrhizas of Triticum aestivum L. and Avena sativa L. with reference to Zea mays L. BY TOM ALEXANDER!, ROSE MEIER^ RONALD TOTH^ AND HANS CHRISTIAN ^ Fachbereich Biologie, Philipps Universitdt, 3550 Marburg, West Germany ^Department of Botany, U?tiversity of Minnesota, St Paul, Minnesota 55108, USA ^ Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115, USA {Received 12 February 1988; accepted 23 June 1988) SUMMARY A quantitative light and electron microscope study of developing and degenerating arbuscules of the mycorrhizal fungus Glomus fasciculatum (Thax. setisu Gerd.) Gerd. & Trappe in Triticum aestivum L. (wheat) and Avena sativa L. (oats) was carried out in order to estimate three parameters during the colonization cycle and to compare these parameters to those in Zea mays L. The parameters are: (1) V^ (f,c), the fraction of the host cell volume (c) occupied by a volume of fungus (f); (2) K,,(cy,c), the fraction of the host cell volume occupied by host cytoplasm (cy); (3) Sj,(p,c) the surface area-to-volume ratio of the host protoplast (p) to the whole host cell. Uninfected cortical cells of wheat had an S,,(p,c) of 011 //m" //m ^ and those of oats 0-10 //m^/«m"^. As the fungus penetrates the cell wall, the protoplast invaginates, causing a decrease in protoplast volume and an increase in ^i.. In wheat this increase reached 117/<m"//m"^ and in oats, 104//m^/^m"^. When the arbuscule is mature, the fungus occupies 35% of the cell in wheat with 20% as branches and 15% as trunk. In oats, the fungus occupies 36% of the cell, with 23 % as branches and 13 % as trunk. In wheat, host cell cytoplasm was initially 3-9 % and increased to 21-6% and in oats, from 3-6 to 28-8%. These values are similar to those observed in Zea mavs. The rates at which the parameters F,,(f, c) and 5,(p,c) changed during arbuscule development and degeneration were similar. The amount of encasement and host cell cytoplasm showed the greatest variation among host species. Key words: Arbuscule, vesicular-arbuscular, mycorrhiza, rnorphometry, cereals. appears that the amount of fungal material in INTRODUCTION,,,,,, ^ ru collapsed arbuscules does not account tor the In a vesicular-arbuscular (VA) mycorrhizal as- observed phosphorus obtained by the host (Cox & sociation, a highly branched haustorium known as the Tinker, 1976; Toth & Miller, 1984). arbuscule develops in cells (usually roots) of land The changes which occur in cortical cells during plants (Gerdemann, 1968). This development can be arbuscule development and degeneration follow a dramatic, with a large volume of host cell occupied definite cycle (Toth & Miller, 1984). Arbuscules in by fungal branches (Toth & Miller, 1984). The rapid general follow this cyclic pattern (Mosse, 1973; breakdown of the arbuscule, and the possible 're- Brown & King, 1982a,6; Scannerini & Bonfanteinfection' of a cortical cell by a new arbuscule are Fasolo, 1983; Bonfante-Fasolo, 1984) although only processes which have attracted attention. It has been the arbuscular cycle in Zea inays (maize) has been postulated that nutrients are absorbed by the host quantified (Toth & Miller, 1984). Preliminary results during 'digestion' of the old arbuscule (Burgefl, from wheat (Meier, 1983) and oats (Alexander, Toth 1938, cited in Cox & Tinker, 1976) although it is not & Weber, 1985) indicated that some aspects of the known if the fungus reabsorbs the arbuscule or if it cycle were similar but that differences did occur, is truly digested by the cortical cells. In any case it Considering that over 90",, of land plant families are

2 364 T. Alexander, R. Meier, R. Toth and H. C. Weber mycorrhizal and VA mycorrhizas are the most prevalent (Gerdemann, 1968) one might expect to find variations in arbuscule structure and patterns of development. The purpose of this study is to determine if variability exists between arbuscular cycles and if so how much and in which parameters. Since Zea mays L. (maize) has already been quantified in some detail, it was decided to investigate more fully the grass species, Triticum aestiiium L. (wheat), and Avena sativa L. (oats), and compare them to maize, keeping the fungal component, Glomus fasciculatum (Thax. sensu Gerd.) Gerd. & Trappe as the constant. In the previous study on maize, several parameters were quantified (Toth & Miller, 1984). These were: (1) [^,,(cy,c), the fraction of the host cell (c) volume occupied by cytoplasm (cy); (2) F,,(t,c) the fraction of the host cell occupied by the arbuscular trunk (t); (3) F,,(b,c) the fraction of the host cell occupied by the arbuscular branches (b); and (4) S,,(p,c), the ratio of the surface area of the host protoplast (p) to the volume of the host cell. The study of maize was incomplete for several reasons: (1) the curves were presented on a relative rather than an absolute scale; (2) the sum of the branch and trunk curves did not add up to the arbuscule curve due to the choice of size classes; (3) the volume of the host cell occupied by 'clumps' (collapsed branches surrounded by encasement) was not quantified; (4) a cycle time was only estimated and not determined accurately. For these reasons we decided to quantify not only F,,(t,c), F,,(b,c), V,, (cy,c), and S.Xp.c) but also F,,(cl,c), the volume of host cortical cells occupied by clumps (cl), to present the cycle curves on an absolute rather than a relative scale, to use different size classes, and to obtain a more accurate estimation of cycle time not only for wheat and oats, but also for maize and to compare the data from this study wivh those previously obtained for maize (Toth & Miller, 1984). MATERIALS AND METHODS Seeds of Triticum aestivum L. (wheat) (cv. Arthur 71, obtained from Carolina Biological Supply Co., Burlington, North Carolina, USA), Avena sativa L. (oats) (cv. Holden, source unknown) and Zea mays L. (maize) (cv. W64A, DeKalb-Pfizer Genetics, DeKalb, Illinois, USA) were grown in the same soil employed by Toth & Miller (1984), containing spores of the VA mycorrhizal fungus Glomus fasciculatum which had been stored at 4 C. Two sets of plants were grown. One set included wheat, oats and maize and were used for cycle time estimations. Seeds were selected for uniformity of size and shape and placed in Petri dishes on wet filter paper. Only seeds which imbibed water were planted in the previously mentioned inoculum in 2 in. pots, three per pot, and sampled every 24 h from the time of planting for 30 d. Roots were washed free of soil, fixed in 4% formaldehyde for several weeks, washed in water, processed in hot KOH and stained with trypan blue (Phillips & Hayman, 1970). Roots were squashed on microscope slides and assayed for the time of initial infection, arbuscule initiation, maturation, and degeneration. For morphometric studies, seeds of wheat and oats were planted in 8 in. pots containing the same soil (containing spores of VA mycorrhizal fungi) used in the cycle time studies. Roots were harvested 8 weeks after seed germination, washed free of soil, and 1 mm sections were processed for electron microscopy using standard techniques (Toth & Miller, 1984). Micrographs were overlaid with a grid of horizontal and vertical lines drawn on a clear acetate sheet. Line intersects were used for point counts for volume (F,,) determinations and horizontal lines were used for line intersections of host plasmalemma for surface area (SJ determinations (Toth, 1982). R E.S U L T S 1. Descriptive cytology Control (uncolonized) cortical cells of wheat averaged 3-9 % cytoplasm (protoplast minus vacuoles and nucleus), 93-1% vacuole, and 2-6% wall by volume. Cells averaged 44/<m in diameter and 140/^m in length. The surface area-to-volume ratio (o,,) of an uncolonized cortical cell was measured as 0-11 //,m^//m"^. Control cells of oats averaged 3-6 % cytoplasm, 93-5% vacuole, and 2-4% wall by volume. Cells averaged 50 /im in diameter and 113 /im in length. The surface area-to-volume ratio (SJ of uncolonized cortical i cells was measured as 0-\0 jiim~'-'. The cortical cells of both wheat and oats were approximately cylindrical in shape. The descriptive cytology of arbuscule development and degeneration for both wheat and oats were similar to that observed in maize (Toth & Miller, 1984) and need not be described in detail. 2. Cycle times Cortical cells from cleared and stained roots of wheat, oats and maize all contained arbuscules in very early stages of development 2 d after infection. After a further 2 d all species contained mature arbuscules. At 3 d, numerous arbuscules were observed in early stages of degeneration. From these observations we assume that the development phase of the arbuscule is approximately 25 d and is definitely less than 3 d. 3. Arbuscular cycles Each parameter (V,,(f,c), etc.) of the arbuscular cycle was treated in an analogous way to a nucleus going

3 XI E Dynamics of arbusciile dez^elopment in mycorrhizal cereals 365 Development Degeneration I II I I I Size classes for percent trunk volume Figure 1. Histogram con.structcd from wheat trunk data. The volume of the wheat cell occupied by trunk, K,,(t,c) is divided into ten size classes. The height of each bar indicates the number of cells containing K^Xt.c) fractions within each size class. through mitosis. In such a case the number of cells observed in each 'phase' is proportional to the time that phase occupies in the total cycle. However, arbuscule development and degeneration cannot be divided into discrete phases as can mitosis. In order to carry out a cycle analysis, all cells were first quantified for all parameters and the values for a mature arbuscule were calculated. Five of the most mature arbuscules, from cells with the highest total fungus, cytoplasm and largest S^., were averaged to obtain an estimation of the characteristics of a mature arbuscule. The values obtained for each parameter of a mature arbuscule were then divided into ten size classes (five for development and Hve for degeneration). Histograms were constructed for each of the parameters, showing the number of cells in each size class (Fig. 1). From each histogram, a curve was constructed to show the velocity at which each parameter changed during the arbuscular cycle. By plotting cycle time (number of cells) on the x axis and percentage of the way through the cycle (size classes) on the y axis, a velocity curve for each parameter was constructed (Figs 2-9). (A) Wheat and oats. The developmental phase of the arbuscular cycle occupied 35 " of the total time in wheat, and 31 % in oats (Figs 2-9). During this time the volume of the trunk I^,,(t,c) increased at a linear rate in both wheat and oats, and finally reached a maximum volume of 15 " in wheat [F,,(t,c) = 0-15]* * As a matter of convenience in constructing.sentences {V^) fractions will, in most cases, be jjiven as percent of host cell volume. For example, V,. (t, c) = 0-1.S means that 15 % of the host cell was occupied by iun^us as arbuscular trunk. and 12-7",, in oats (Fig. 2). The rate of trunk degeneration was slower than in development in both species (Fig. 2). In wheat, arbuscular branches developed rapidly at first, then more gradually, whereas branches in oats developed more slowly at first and then more rapidly (10-25 "o of the cycle time) (Fig. 3). Branches occupied a maximum of 20% of the host cell in wheat and 23-3 " in oats, at the end of the development phase (Fig. 3). Several processes occurred simultaneously during the degeneration of the branches. In both species, branch collapse was very rapid (Fig. 3). Large numbers of collapsed branches were surrounded by 'encasement' material resulting in the formation of large 'clumps' of material (Fig. 4). Within these clumps are the collapsed branches (Fig. 5). Therefore, the difference between the curves in Figure 4 (dotted lines) and Figure 5 (dotted lines) represents the volume of the host cell occupied by the 'encasement' material. The total fungus (arbuscule) is essentially a summation of the curves for trunk and branch volume (Figs 6, 7) and occupied 35 " of the host cell in wheat and 36% in oats. The curves for wheat and oats in Fig 6 represent the total volume of the cells occupied by the fungal material (arbuscule): branches, collapsed branches and trunk. The curves in Figure 7 represent the total volume occupied by the arbuscule plus the encasement. Host cell cytoplasm increased from 3-9 "o to a maximum of 21-6 % in wheat and from 3-6 to 28-8 % in oats (Fig. 8). In wheat this increase was rapid at first but levelled off by 15 (, of the cycle time, whereas in oats, cytoplasm increased slowly at first (0-10 % of the cycle) but increased during the rest of the developmental phase (F'ig. 8). Degeneration of the arbuscule in both wheat and oats was marked by an initial rapid decrease in host cell cytoplasm followed by a more gradual decline in the amount (F,,) of cytoplasm until pre-arbuscule levels were reached at the end of the cycle (Fig. 8). The ratio of surface area of the host protoplast to the volume of the cell 5',.(p,c) also increased during colonization by the mycorrhizal fungus. In both species, the protoplast is initially tightly pressed to the wall so that the 5,, of the protoplast is approximately equal to the 5,, of the cell S,.(c,c). This value was calculated to be 0-11//m"//m"'* in wheat and 0-10//m'^/tm"^ in oats from light microscopic observations. As the fungus entered the host cells, the protoplast shrank to accommodate the fungus and invaginated around the trunk and branches so that an increase in protoplast surface area occurred. These changes resulted in the typical increase in 5,,(p,c) in host cells. In wheat this increase was almost linear, whereas in oats it was slower at first (0-10 % of the cycle) but increased at the end of development (Fig. 9). The

4 366 T. Alexander, R. Meier, R. Toth and H. C. Weber Arbuscule 6 M Trunk W, M Branches 3 M Arbuscule 7 plus encasement Branches 4 and clumps M Branches and collapsed branches Cycle (%) Cycle (%) Figure 2-9. Composites of arbuscular cycles in Triticum aestivum (wheat) (w), Avena sativa (oats) (O), and Zea mays (maize) (M) for the various parameters. In all cases, the percentage of the cycle (time through one arbuscular cycle) is plotted on the x axis and the volume fractions (F,,) or surface area-to-volume ratios (S,) are plotted on the y axis. For matters of convenience, volume fractions are plotted as percent of host cell occupied by each parameter. For example, a F,,(t,c) fraction of 0-10 means that 10% of the host cell is occupied by fungus in the form of arbuscular trunk. Figures 2-5. Composites of cycles for trunk and branch development and degeneration. In Figure 3, branch refers to functional branches. In Figure 4, clumps refer to collapsed branches surrounded by encasement, and in Figure.S, collapsed branches respresent the clumps minus the encasement. Figures 6-9. Composites of cycles for arbuscule (total fungus), host cytoplasm and surface area-to-volume ratio of the host protoplast to the host cell, S,(p,c), changes during one cycle of arbuscule development and degeneration. In Figure 6, arbuscule refers to the total fungus (branches, collapsed branches and trunk) minus the encasement material. maximum 5',,(p,c) for the host protoplast in wheat was 117//m'''//m * and 1-04/^//m"'' in oats. Degeneration of the arbuscule brought about a rapid decrease in protoplast S,, as branches collapsed and clumps were formed (Fig. 9). At this stage the host protoplast membrane surrounded clumps of many branches rather than individual branches, resulting in the observed rapid decrease in 5,,(p,c). In both species this decrease levelled off at about 50 % of the way through the total cycle time (Fig. 9). Although all 'functional' branches were gone by 35% of the cylce time in both species (Fig. 3), collapsed branches in clumps persisted as did significant amounts of trunk (Fig. 2), so that the original.s,,(p,c) was not reached until the end of the cycle. The increase in S,,{p,c) from 0-11 to M7 //m'^ //m"'' in wheat and 0-10 to l-04/im'-//m ^ in oats does not represent a 10-fold increase in the surface of the cells, but just the protoplasts, since, as in maize (Toth & Miller, 1984) the cell wall, and therefore cell size and volume were unaffected. Also, since the fungus occupied 35% of the cell volume in wbeat and 36 % in oats and the interfacial matrix* was 5 o in wheat and 4% in oats, the protoplasts shrank to occupy only 59-4 % [97-4 % - (33 % -f- 5 %)] of the cell volume in wheat and 57-6% [97-6 %-(36 %- -4 %)] in oats. In order to calculate the S^XPiP) of the protoplast to its own volume rather than to the volume of the cell, the S,.(p,c) ratio must be * The interlacial matrix is that zone between the fungal cell wall and the host cell membrane. This parameter was calculated for cells containing mature arbuscules.

5 Dynamics of arbuscule developinent in tnycorrhizal cereals 367 divided by the fraction of the cell occupied by the protoplast F,,(p,c). In wheat this equals 59 4% (F,,(p,c) = 0-594) and in oats 57-6% (F.,(p,c) = 0-576). Therefore, the ratio of the surface area of the protoplast to its own volume in wheat is 1-72 /im'^ fim^'' (M7//m''//m~70-594) and \-81/im'fim"-' {\-04- /im'' /U.m~^/0-576) in oats. In both species, this represents approximately an 18-fold increase in the surface area-to-volume ratio for the living contents of wheat and oat cells with mature arbuscules. (b) Maize. A description of the changes in parameters during the maize'cycle has previously been reported (Toth & Miller, 1984) and should not be repeated here. However, during this study on wheat and oats, numerous cells were discovered which contained fungal structures ranging from 2-8 //m diameter which were initially thought to be developing trunks. Further analysis revealed that these were intracellular hyphae and not assoicated with arbuscule development. This caused a re-evaluation of the maize micrographs with the result that several cells previously assumed to be in early stages of arbuscule development were removed from the cycle analysis data. Because of this, the development phase is now estimated to occupy 37 % of the total cycle (Figs 2-9) and not 42 % as was previously thought. For maize, the size classes were constructed using maximum values for each parameter. This resulted in an error because the cycle curves for branch and trunk development did not add up to the arbuscule curve. In the present study the cycle curves for niaize were reconstructed using new size classes based on averages of mature arbuscules (Figs 2-9). This results in the branch and trunk curves adding up to the arbuscule curve. The maize data were also recalculated using the new^ size classes and plotted on an absolute rather than a relative scale. This allows for a more meaningful comparison between arbuscules in the different host species (Figs 2-9). 4. Absolute arbuscular volume and host protoplast surface area The 5,. and F,, fractions generated by morphometric analysis from electron micrographs can be converted into absolute amounts if the cell volume is known. Cortical cells of all three species are approximately cylindrical in shape. In longitudinal section they are rectangular but in cross-section they are not truly circular. However, the slight indentation caused by contact with neighbouring cells do not increase surface area very much. For this reason they can be considered cylindrical in shape for the purpose of calculating cell volume. Using the 5^ and V,. ratios calculated from micrographs, absolute values for arbuscule volume and host protoplast surface area in uncolonized host cells (initial) and cells with mature arbuscules (final) were calculated (Table 1). Also, initial and final volumes of host cytoplasm as well as initial and final surface area-to-volume ratios of host protoplast to host cytoplasm S,.(p, cy) were calculated (Table 2). All three species had similar sized arbuscules (/<m^) and similar initial and final protoplast surface areas (Table 1). They also had similar initial cytoplasmic fractions F,,(cy,c) and absolute amounts of cytoplasm (//m'') (Table 2). In the average cortical cell, just inside of the cell membrane there is a thin layer of cytoplasm bounded by the plasmalemma itself and the tonoplast (vacuolar membrane). The surface area-to-volume ratio of the protoplast (amount of membrane) to the cytoplasmic volume, 5',(p,cy), is an indicator of the relative thickness (amount) of this layer. Host cells initially had similar F,,(cy,c) fractions, 5'.(p,c) ratios and absolute amounts of cytoplasm and protoplast surface area (Tables 1 and 2). Therefore, their 5',(p,cy) ratios were also similar (Table 2). This would indicate that the thickness of the layer of cytoplasm was uniform from cell to cell. However, in cells with mature arbuscules, the final 5',,(p,cy) ratios Table 1. Summary of arbuscide (a) volume fractions of host cells (c) F,,(a,c), surface area-to-voltwte fractions of host protoplasts (p) to host cells (c) volume 5',,(p,c), in uncolonized cortical cells (initial) as well as cells zvtth mature arbuscules (final). Also, calculations of cell volumes V(c), arbuscule volumes, V(a), and inittal and final surface areas of host protoplast, S(p) Host species Wheat Oats Maize Cell size (/im) 44&120 50x X 125* Arbusctile Cell volume volume fraction V(c) (jum' ) ^'.(a.c) Arbuscule volume V(a) (//m^) S^,(p,c) (/im^ fim '' protopl ast surface a rea S(p) (// m^) ) [F(c)x ^.(P.c)] I'inal i >,(P.'^) (. Dm' /im-') Final protoplast surface area S(p) (//m-) [F(c)x5,,(p,c) * In Toth & Miller (1984), maize cells were listed as being 34/;,m diam and 120//m long. These figures were not corrected for shrinkage.

6 368 T. Alexander, R. Meier, R. Toth and H. C. Weber Table 2. Stimmary of cell volume V {c), volume fraction of host cytoplasm F,. (cy,c), in uncolonized cortical cells (c) {initial), and in cortical cells with mature arbuscules (final), as well as calculations of initial and final cytoplasmic z'olume V (cy) and initial and final surface area-to-volume fractions of host protoplast S{p) to cytoplasmic volume K(cy), S,,(p,cy) Host species Cell volume* V(c) (//m^) l^,,(cy,c) volume of host cytoplasm F(cy) (/tm') S,,(p,cy) (//m-/<m"'') [S(p)*/K(cy)] Final ^'..(cy.c) Final Host cytoplasm Klcy) (/im') Final S,,(P, cy) (/'m- /tm'") */ I'Xcy)] Wheat Oats Maize * From Table 1. varied more than before arbuscule development (Table 2). This would indicate that in addition to changes in S,,(p,c), the thickness of the layer of host cytoplasm also changed between host species. DISCUSSION In general, the dynamics of arbuscule developnrient and degeneration were similar in the three host species. Trunk development and degeneration was gradual in all hosts with maize having a slightly faster rate. Branch development was also similar from host to host as was branch collapse. In all cases, branch collapse was very rapid so that in wheat no cells contained functional branches once the degeneration phase began. This collapse seems to be a synchronous event in which all of the branches collapse in a short time. The timing of this event appears to be under the control of the fungus and is the major event separating the development phase from the degeneration phase. The length of the development phase was estimated to be 2-5 d for all three species in this study which correlates well with the time of arbuscule development in leek (Brundett, Piche & Peterson, 1985). In their study arbuscule development began four days after leek seedlings were transplanted into pots containing an establish leek mycorrhiza (their Fig. 4) and in 6-d-old samples (their Fig. 22) almost mature arbuscules were formed. Therefore, within 2 d, well-developed arbuscules were formed. AK though they did not specifically report on the timing of arbuscule degeneration, the rates of arbuscule development in leek colonized by G. versiforma were similar to the present study. From the electron microscopic observations, the developmental phase occupied 35 % of the cycle time in wheat, 31 "' in oats and 37 "4 in maize, giving an average of 34%. If 34% of the cycle represent 2-5 d then the total cycle is estimated to be 7 d. This range (31-37 "o) would not be detectable from the light microscopic determinations of cycle time but can clearly be observed from electron micrographs of cortical cells containing either developing or degenerating arbuscules. This variation may be due to the methods of sampling and not represent real differences in cycle time across host species. During the early stages of root colonization by a VA mycorrhizal fungus (first few days) a sample would produce a large percentage of developing arbuscules. This would lead to the conclusion that the development phase occupied most of the cycle time. Conversely, at the end of the growing season, arbuscule formation would cease. During the time old arbuscules were degenerating, a sample would yield mostly degenerating arbuscules. This would lead to the conclusion that degeneration occupied almost all of the cycle time. Obviously somewhere in between these two extremes a balance occurs. When the colonization process has reached a steady state. then arbuscule formation is in equilibrium with degeneration. Sutton (1973) refers to this as the 'phase of constancy'. Cells sampled at this time would reflect the true number of cells in development and degeneration, and therefore the percent of the cycle occupied by each phase. Maize, oat and wheat plants were grown under greenhouse conditions using the same inoculum. However, they were grown in three successive springs. Also, maize and wheat plants were harvested 8 weeks after planting the seeds, whereas oats were harvested 8 weeks after seed germination. Preliminary results from several non-grass species grown along with oats indicate that the development phase occupies approximately 33 "o of the cycle time and is uniform from host to host. Due to the previously listed factors which could account for the variation, we feel that the duration of the development phase of cycle is constant across host species. The variability can be accounted for by difterent growing conditions and time of sampling roots. It appears that the time at which the arbuscule begins to degenerate is under the control of the fungus rather than the host. Further studies should help to confirm this hypothesis. The production of the encasement material ^'aries

7 Dynamics of arbtiscule development in mycorrhizal cereals 369 across host species. In the early stages of branch underlying the plasmalemma changed between host collapse in wheat, 10 /,, of the cell volume was species a the same time the 5,,(p,c) changed. This occupied by encasement whereas in oats and maize, resulted in difference in F,,(cy,c) and 5';,(p,cy) ratios. encasement was produced slowly and only occupied We therefore believe that the changes in host 4 a n d 7"b of the cell respectively. This range cytoplasm are both under the control of the fungus (4 10%) represents the largest relative difference and the host cell. between species for any of the parameters in this The surface area-to-volume ratio S,, of the host study. It has always been assumed that the en- protoplast in cells with mature arbuscules was similar casement material is produced hy the host across hosts. Since 5',,(p,c) correlates closely with (Scannerini & Bonfante-Fasolo, 1983; Bonfante- branch development (Toth & Miller, 1984) and Fasolo, 1984) and might therefore show \ariation hranch development was similar across hosts, the from host to host. It is assumed that the dynamics of similarities in 5,.(p,c) across hosts would be exthis parameter are under the control of the host pected. Because of the uniformity of this parameter cell. we assume that this process (although a property of T'he parameter which showed the next highest the host cell) is under the control of fungus. degree of variability was host cytoplasm. ly, An unexpected finding was the great similarity in cortical cells contained a thin layer of cytoplasm ahsolute volume of the arhuscules which developed surrounding the large central vacuole(s). As the in different host cells. Although maize cells had the fungus entered the cell, the protoplast invaginated smallest volume, they had the largest volume ot and surrounded each trunk and branch. As the fungus and therefore contained arbuscules similar in S,,{p, c) of the protoplast increased, so proportionately size to those in wheat and oats. Because of the should have the amount of host cytoplasm since a uniformity between hosts we assume that this thin layer of cytoplasm is always found just inside characteristic is primarily under the control of the the plasmalemma. It was shown that there was a fungus. As was explained above, 5',(p,c) was similar close correlation between branch volume (F,,(b,c) across hosts. Although this is a parameter of host and cytoplasm F,,(cy,c) in maize (r = 0-676, P< cells, it is a direct response to ahsolute arbuscular 0-001) (Toth & Miller, 1984). This was due to the size which is similar in different host cells. Therefact that a certain amount of branch development fore, it seems reasonable to expect that the absolute causes a proportional increase in membrane surface increase in host protoplast surface area S(p) would which is underlied hy a layer of cytoplasm of a also be uniform across hosts. Indeed this is what was certain thickness. One would expect the thickness of ohserved (Tahle 1). the cytoplasmic layer to be under th control of the In conclusion, it appears that arbuscule dehost and the 5',,(p, c) increase under the control of the velopment and degeneration in the VA mycorrhizal fungus since plasmalemma invagination is a reaction fungus Glomus fascicidattim is similar in three species to branch development, a fungal characteristic. of grass plants and that encasement production and Indeed in maize the correlation hetween branch K,, host cell cytoplasm show the most variation. (b,c) and 5'^(p,c) was so exact as to be considered a cause and effect phenomenon (r = 0928, P < 0001) ( T o t h & Miller, 1984). ACKNOWLEDGIVIENT.S If the thickness of the layer of cytoplasm remained The authors wish to thank Ms Carol Garner and Ms uniform during the S,,(p,c) increase, then one would Freyja Kerwin for technical assistance. expect the proportional changes m F,,(cy,c) across host species to be related to those for K,,(b,c) and 5^,(p,c). This would result in the ratio of the REFERENCES protoplast (p) surface area to a volume (thickness) of ALEXANDER, T., T O T H, R. & WEBER, H. C. (1985) Ultrastrukurelle cytoplasm (cy), 5,(p,cy) to also remain uniform. If untersuchungen zur entvvicklung und degeration der arbuskeln the different host species reacted differently to fungal von VAM..pilzcn. fith Symposium Morphotogie, Anatomie Und Systematik, Hamburg (no pages listed) ingress in response to cytoplasm production, there BONFANTE-FASOLO, P. (1984). Anatomy and morphology of VA would be differences in 5',,(p,cy) ratios. ly, all mycorrhizae. In: VA Mycorrhiza (Ed. by C. L. Powell & D, J. three grasses had similar 5',,(p,cy) ratios (2-75Bagyaraj), pp CRC Press, Boca Racon, Florida /xra^ /<m~') (Table 2). Regardless of the absolute BROWN, M. F. & KING, E. J. (1982a). Morphology and histology of vesicular-arbuscular mycorrhizae. A..Anatomy and Cysurface area increases in the host protoplast 5(p), if tology. In : Methods and Principtes of Mycorrhizal Research (Ed. the amount of cytoplasm per unit membrane reby N. C. Schcnck), pp American Phytopathological Society Press, St Paul. mained uniform in thickness between host species, BROWN, M. F. & KING, E. J. (1982A). Electron microscopy ot the S^(p,cy) ratios would remain constant. This was m y c o r r h i z a e. I n : Methods and Principtes of Mycorrhizal Renot the case. In host cells with mature arhuscules the search (Ed. by N. C. Schenck), pp American Phytopathological Society Pre.ss, St Paul. 'S',j(p,cy) ratios varied considerably (from 3-61BRUNDRETT, M. C, PICHE, Y. & PETER.SON, R. L. (1985). A 5-71 /^mvm-^) (Tahle 2). developmental study of the early stages of vesicular arbuscular mycorrhiza formation. Canadian jfourtial of Botany 63, Clearly, the thickness of the layer of cytoplasm

8 370 T. Alexander, R. Meier, R. Toth and H. C. Weber Cox, G. & TINKER, P. B. (1976). Translocation and transfer of SCANNERINI, S. & BONFANTE-FASOLO, P. (1983). Comparative nutrients in vesicular-arbuscular mycorrhizae. I. The arbuscule ultrastructural analysis of mycorrhizal associations. Canadian and phosphorus transfer; a quantitative ultrastructural study. Journal of Botany. 61, New Pliytologi.<:t 77, SUTTON, J. C. (1973). Development of vesicular arbuscular GERDEMANN, J. W. (1968). Vesicular-arbuscular mycorrhizae and mycorrhizae in crop plants. Canadian Journal of Botanv 51, plant growth. Annual Review of Phytopathology 6, MEIER, R. A. (1983). Dynamics of arbuscule development and TOTH, R. (1982). An introduction to morphometric cytology and degeneration in roots of Triticum aestivum L., MSc Thesis. its application to botanical research. American Journal of Northern Illinois University, DeKalb, Illinois 60115, U.S.A. Botany 69, MossE, B. (1973). Advances in the study of vesicular-arbuscular TOTH, R. & MILLER, R. M. (1984). Dynamics of arbuscule mycorrhiza. Annual Rezjiew of Phytopathology 11, development and degeneration in a Zea mays mycorrhiza. PHILLIPS, J. M. & IIAYMAN, D. S. (1970). Improved procedure American Journal of Botany 71, 449^60. for clearing roots and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Myeological Society 55,

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