Mycorrhiza (1995) 5:405-408 �9 Springer-Verlag t995

Brent G. DeMars �9 Ralph E.J. Boerner

Arbuscular mycorrhizal development in three crucifers

Abstract To determine the developmental patterns of arbuscular mycorrhizae (AM) in three crucifers (Bras- sicaceae) of differing life histories, we inoculated seed- lings of the annual Capsella bursa-pastoris, biennial Hesperis matronalis, and the perennial Matthiola incana with Glomus intraradices. The plants were grown either alone or in a matrix of living roots of the mycotrophic grass Sorghum sudanense. The percent root length col- onized was greatest in C. bursa-pastoris and least in H. matronalis. Colonization was greater in plants grown in the grass matrix than in plants grown alone, and coloni- zation in grass matrix-grown plants continued to in- crease over the 90-day growth period, whereas coloni- zation leveled off or decreased near the end of the growth period in crucifers grown alone. No arbuscules were observed in crucifer roots at any time, which sug- gests that AM in these crucifers is nonfunctional. Fur- thermore, the increase in colonization only in pots with both crucifers and active mycotrophic roots suggests that AM development in crucifer roots is primarily the consequence of progressive root senescence in the cru- cifer and continued inoculum spread from the myco- trophic plant.

Key words Arbuscular mycorrhizae �9 Brassicaceae �9 Cruciferae �9 Life span �9 Glasshouse inoculation studies

Introduction

Although the family Brassicaceae is typically consid- ered to be nonmycotrophic (Gerdemann 1968; Trappe

B.G. DeMars 1 (N~) �9 R.E.J. Boerner Department of Plant Biology, Ohio State University, 1735 Neil Avenue, Columbus, OH 43210, USA Fax: (614) 292-6345; e-mail: ae870@dayton.wright.edu

Present address: 1 Division of Health and Science, Lakeland Community College, Kirtland, OH 44094, USA

1987), reports of arbuscular mycorrhizal (AM) devel- opment in crucifers are common (Medve 1983; Harley and Harley 1987; DeMars and Boerner 1994) from both field-collected and laboratory-inoculated specimens. Despite such reports, the functionality of such mycorr- hizae remains unknown, especially since few authors have observed arbuscules in the root segments exam- ined. The lack of arbuscules suggests that any mycorr- hizal development in the family is nonfunctional, since these structures serve as the interface for symbiotic nu- trient transfer.

Numerous hypotheses have been advanced to ex- plain the general nonmycotrophic nature of the Brassi- caceae (Schwab et al. 1983; Tester et al. 1987; Schreiner and Koide 1993), and the apparent occasional occur- rence of mycorrhizae (Hirrel et al. 1978; Ocampo et al. 1980; Miller et al. 1983; Peat and Fitter 1993; DeMars and Boerner 1994). The sporadic induction of AM in this family may occur when typically nonmycotrophic taxa are exposed to roots of normally mycotrophic plants. However , apparent mycorrhizal development in typically nonmycotrophic plants may result from a fail- ure to distinguish physiologically active roots from old- er, senescing roots (Hirrel et al. 1978).

The primary objective of this present study was to examine mycorrhizal development in three crucifers of different life histories over a period of time sufficient for arbuscule development. Additionally, we indirectly examined the difficulties in distinguishing functional and senescent roots.

Materials and methods

Three species of Brassicaceae were selected to represent the range of life span in the family. Capsella bursa-pastoris (L.) Me- dikus is a cosmopolitan annual, Hesperis matronalis L. is a com- mon biennial and Matthiola icana (L.) R. Br. is an often woody- based perennial. All three are native to Eurasia and naturalized in North America (Rollins 1993).

Several seeds of each crucifer were sown into acid-washed sand in 512-cm 3 plastic pots. To determine the effect of living roots of mycotrophic species in the vicinity of the crucifer roots,

406

one half of the containers were oversown with seeds of sudan grass [Sorghum sudanense (Piper) Stapf.]. All pots were inocu- lated with a root inoculum of Glomus intraradices Schenck and Smith at planting. The root inoculum was prepared from S. suda- nense roots grown in sand culture. The original fungal culture, isolated from agricultural field crops, was obtained from J. H. Gerdemann at the University of Illinois.

On day 10, the pots were thinned to one crucifer per pot, and for those oversown with sudan grass, three grass seedlings per pot. There were 120 pots per crucifer species, 60 of which also had grass seedlings in them.

The planted pots were placed in trays and randomly allocated to three glasshouse benches (blocks), and watered three times per week through the 90-day growing period. In addition, 250 ml of a low P (1.0 mg P/l) Ruakura solution (Smith et al. 1983) was added to each pot on days 0 and 45. No other nutrients were added dur- ing the experiment.

Plants were grown under ambient glasshouse light and tem- perature conditions through the spring and summer. On days 10, 25, 40, 60, and 90, four replicates from each block'crucifer spe- cies 'root matrix treatment (i.e. presence or absence of living su- dan grass in the pot) were harvested. Sudan grass roots were also harvested to facilitate comparison of the level of mycorrhizal de- velopment in the grass with that of the crucifers.

Roots were separated, cleared and stained with trypan blue (Phillips and Hayman 1970) and stored in plastic tissue cassettes in formalin:acetic acid:ethanol. Mycorrhizal colonization was de- termined microscopically in five random 2- to 3-cm root segments per plant and was defined as the presence of internal nonseptate hyphae, vesicles, arbuscules or chlamydospores. The percentage of root length colonized by mycorrhizal fungi was estimated for each segment (Giovanetti and Mosse 1980). Roots without cortex or roots with obvious damage to the cortex were not used.

The experimental design was a randomized complete block, with 3 blocks (glasshouse benches) '3 crucifer species'2 root ma- trix treatments (with and without sudan grass in the pot) '5 sam- ple dates. Each combination was replicated four times for a total of 360 pots. The mean percentage root length colonized (Giova- netti and Mosse 1980) for each plant within treatment combina- tions was analyzed by analysis of variance (ANOVA), with root matrix treatment, crucifer species, and harvest date and their sec- ond-order interactions as main effects (SAS Institute 1985). F- values were computed using the mean squares of the block inter- actions as a divider term.

Results

By day 90, the C. bursa-pastoris plants had flowered, set seed, and begun to senesce. In contrast, H. matron- alis, M. icana, and S. sudanense remained vegetative and actively growing throughout the 90-day growth pe- riod. The number of plants in each t rea tment group ex- hibiting A M development varied with crucifer species, root matrix t reatment , and harvest dates (Table 1). The frequency of A M development was greater in C. bur- sa-pastoris and H. rnatronalis than in M. icana, espe- cially in the later harvests, and was greater in plants grown with sudan grass than in plants grown alone in eight of nine species-by-harvest date comparisons for days 40, 60, and 90 (Table 1). A M development in cru- cifer roots was limited to hyphae and vesicles; no arbus- cules were observed in any of the 1800 root segments.

The dynamics of mycorrhizal development ex- pressed as the percent root length colonized also varied by species, root matrix t reatment , and harvest dates (Fig. 1). The percent root length colonized increased through day 90 in all t rea tment combinations except

Table 1 Number of plants colonized by mycorrhizal fungi by har- vest date. n = 12 for each crucifer species by root matrix treatment by harvest date combination

Species x treatment Day of harvest

10 25 40 60 90

Capsella bursa-pastoris Without sudan grass 0 5 8 10 10 With sudan grass 0 7 9 11 11

Hesperis matronalis Without sudan grass 1 7 6 10 10 With sudan grass 0 7 9 10 11

Matthiola icana Without sudan grass 0 0 2 4 8 With sudan grass 0 0 3 8 9

the C. bursa-pastoris grown without sudan grass (Fig. 1). A N O V A revealed significant effects of harvest date, root matrix t reatment , and the interaction of har- vest date and matrix t rea tment on percent root length colonized (Table 2). The species effect was statistically insignificant (P>0.08) ; however, among the three cru- cifer species, max imum mean colonization ranged f rom a low of 14.2% + 2.8 for H. matronalis grown alone to a high of 31.3% + 5.0 for C. bursa-pastoris grown with su- dan grass (Table 3). Overall, the crucifers grown with sudan grass had greater A M colonization than plants grown alone (27.1% + 5.5 and 16.0 + 3.5, respectively); however, the t ime required for this matrix effect to ap- pear differed among the crucifers. The difference ap- peared as early as day 25 for H. rnatronalis, on day 40 for M. icana, and not until day 90 for C. bursa-pastoris (Fig. 1, Table 3). Such differences were responsible for the significant root matrix t rea tment-by-harves t date interaction in the A N O V A (Table 2).

In contrast to the relatively low percent root length colonized in the crucifers, considerable A M develop- ment was already apparent at day 10 in the sudan grass (Fig. 1). A M development in sudan grass reached a peak of 68.2% + 4.0 of root length colonized on day 60. Again, in contrast to the crucifers, arbuscules were common in the sudan grass roots.

Discussion

Our data suggest two interpretat ions of A M develop- ment in these crucifers. First, true A M development oc- curred but development was sporadic, and was in- fluenced by the heavily mycorrhizal root matrix offered by the living sudan grass and its mycorrhizae (Ocampo et al. 1980; Miller et al. 1983; DeMars and Boerner 1994). Alternatively, the observed A M development is essentially an artifact of our inability to distinguish be- tween senescent roots and active roots (Hirrel et al. 1978), with fungal colonization linked to senescent

r o o t s no longer able to defend themselves. Although reports of A M development in crucifers

are common, only Ross and Ha rpe r (1973) and Tom-

407

Fig, 1 Mean percent root length colonized in three cru- cifer species: Capsella bur- sa-pastoris (annual), Hesperis matronalis (biennial), and MatthioIa icana (perennial) and the matrix treatment grass ~ Sorghum sudanense. Solid -~ squares represent the grass (S. .= sudanense) matrix treatment and open squares represent the nongrass matrix treatment. "6 Circles refer to the mean root s length colonized in S. suda- nense in pots containing cru-

o cifers

E

30 J Capsulla bursa-pastorLs

2 0 ~ _ 1 0 " ~

i i i i i i 10 20 30 40 50 60 70 80 90

30 60

20 45

10 30

i E ? " i i t i i ~ I

30"

20.

10

10

Matthiola icana

20 30 40 50 60 70 80 90

20 30 40 so 60 70 80 90 10 2'0 3'0 ,,;0 s'o do 7'0 8'0 90 Day Day

Table 2 ANOVA ofmean root length colonized by mycorrhizal fungi in three crucifer species

Source df SS F-value P > F

Glasshouse bench (block) 2 55.4 0.2 0.7927 Crucifer species 2 2476.3 10.2 0.0840 Root matrix 1 2533.4 20.9 0.0296 Harvest date 4 20426.0 42.2 0.0001 Species * root matrix 4 296.0 1.2 0.2934 Species * harvest date 8 1411.5 1.5 0.1679 Root matrix * harvest date 4 1213.5 2.5 0.0409 Model 23 28412.4 10.27 0.0001 Error 336 40406.9

merup (1984) have reported arbuscule development. Since no arbuscules were observed in any of the root segments we examined here, or in a comparative study of 600 crucifer taxa (B.G. DeMars and R.E.J. Boerner, unpublished work), it is probable that the normal mechanisms leading to the establishment of a function-

al mutualism are not present in these plants and, hence, the AM development should be classified as "appar- ent" only.

Other evidence exists for the interpretation of cru- cifer mycorrhizal development as nonfunctional. Glenn et al. (!985) showed that hyphae from several Glomus spp. (but not Gigaspora spp.) robustly penetrated the roots of Brassica spp., provided that dead epidermal or cortical cells were present. In these cases, arbuscules never formed but vesicles were common and all inter- nal hyphae were intercellular. Reports of "host-non- host" mycelial interconnections between mycotrophic sorghum and nonmycotrophic cabbage also support this nonfunctional view (Ocampo 1986). Although, my- corrhizal development in nonhost plants has been ob- served in the presence of a mycorrhizal host (Hirrel et al. 1978; Ocampo et al. 1980), Ocampo (1986) showed that no nutrient transfer occurred between the myco- trophic and typically nonmycotrophic individuals. These results and ours also suggest that the apparent

Table 3 Percent root length colonized by mycorrhizal fungi by harvest date. N = 12 for each crucifer species by root matrix treat- men t by harvest date combination. Standard errors of the mean

are indicated within parentheses. Values followed by different lowercase letters within a crucifer species by matrix treatment by harvest date indicate significant differences at P > 0.05

Species x treatment: Day of harvest

10 25 40 60 90

C. bursa-pastoris Without sudan grass 0.0b (0.0) 8.3 a b (3.6) 11.7 a b (3.0) 18.8 a (3.9) 17.5 a (3.1) With sudan grass 0.0 c (0.0) 8.3 b c (2.6) 14.6b (3.5) 20.4 a b (3.9) 31..3 a (5.0)

H. matronalis Without sudan grass 0.8 b (0.8) 0.0b (0.0) 4.6b (1.8) 13.8 a (3.2) 14.2 a (2.8) With sudan grass 0.0b (0.0) 12.1 a b (3.7) 15.8 a (4.3) 20.4 a (4.1) 24.2 a (5.4)

M. icana Without sudan grass 0.0b (0.0) 0.0b (0.0) 1.7 b (1.1) 4.6 a b (2.1) 16.3 (4.5) With sudan grass 0.0b (0.0) 0.0b (0.0) 6.3 b (3.9) 12.5 b (4.0) 25.8 a (6.2)

408

AM development in crucifers may be parasitic (Ander- son 1988).

In the present study, apparent AM development consistently increased over time in crucifers growing in the presence of living sudan grass roots. In contrast, AM colonization leveled off in crucifers grown without a sudan grass matrix. Furthermore, the rate of increase in the percent of root length colonized in these crucif- ers through day 90 suggests to us that colonization was likely to continue to increase. We suspect that this pat- tern is the result of two interacting factors: inoculum availability and fine root turnover.

Although estimates of the rate of fine root turnover for herbaceous species are scant, Garwood (1967) showed that root turnover in perennials can be as high as 81% per year. Thus, it is quite likely that some of the roots of both H. matronalis and M. icana began senesc- ing during the study period. Since the study plants were grown in initially sferile sand cultures, low rates of de- composition of senesced roots would be expected, even though fine root decomposit ion can be rapid in the field (Fahey and Hughes 1994). Thus, the amount of senescent (and even dead) roots in the crucifer pots should have increased over time.

The mycorrhizal inoculum potential of the sand in the pots in which crucifers were grown alone was sup- plied solely by the spores and hyphae added a t the be- ginning of the experiment. In these pots, the inoculum potential should decrease over time, as spores germi- nate and sporlings die for lack of suitable hosts. In con- trast, the soil inoculum potential in pots with living su- dan grass roots should continue to increase throughout the experiment as the hyphal network from the grass roots explores more of the soil volume. Thus, in pots with crucifers and sudan grass, increase in both the availability of senescent roots and the inoculum poten- tial led to a steady increase in the percent length of col- onized roots. In contrast, in pots without the grass, the steadily decreasing inoculum potential led to a leveling off in the rate of development (except for M. incana), despite a continuing increase in senescent roots over time.

Unpublished data (e .G. D e M a r s ) f r o m the wood- land crucifer Cardamine concatenata (Michx.) Scharz also support the hypothesis that apparent mycorrhizal development is due to colonization of senescent roots. During the active growth period in March and April, C. concatenata roots showed no indication of AM devel- opment. At senescence (prior to root shedding), in con- trast, roots had a mean percent root length colonized of 18%.

The present results demonstrate that future studies must incorporate analysis of root turnover and inocu- lure potential to fully understand the dynamics of AM development in this family. Furthermore, techniques such as fluorescein diacetate staining (Cooper 1984) or radiolabelling must be applied to assess functionality of the mycorrhizae formed in this family.

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