The influence of vesicular-arbuscular mycorrhizae on uptake of 90Sr from soil by soybeans

Soil Bid. Biochem. Vol. 5, pp. 205-212. Pergamon Press1973. Printedin GreatBritain

THE INFLUENCE OF VESICULAR-ARBUSCULAR MYCORRHIZAE ON UPTAKE OF poSr FROM SOIL BY SOYBEANS N.

E.

The Ohio Agricultural

JACKSON,*

R. N. MILLER

and R. E.

FRANKLIN

Research and Development Center and The Ohio State University, Columbus, Ohio 43210, U.S.A. (Accepted 19 May 1972)

Summary-The Stanford-DeM~nt technique was used in a study of the influence of the vesicular-arbuscular (VA) mycorrhizal fungus Endogme mosseae on 9aSr uptake by soybean. Thirteen-day-old mycorrhizal soybean plants absorbed significantly more pOSr than control plants after 1, 3 or 7 days contact with 90Sr amended sterilized or nonsterilized soil. The same positive influence of Endogone mosseae on Sr absorption was observed in a second study which allowed for YYr uptake concurrent with mycorrhizal infection and development. In the Stanford-DeMent study, soil sterilization exerted a short (1 day) negative influence on the uptake of 90Sr by mycorrhizal roots.In the second study,mycorrhizal roots absorbed more gOSr from sterilized soil than from unsterilized soil while the reverse occurred with the control plants. Infected plants in both studies showed an early decrease in dry matter yield. INTRODUCTION

A REVIEW of literature on vesicular-arbuscular (VA> mycorrhi~e (~~~~g~~e spp.) by Gerdemann (1968) discussed the possible signi~cance of these fimgal endophyt~s in increasing growth and nutrient absorption by higher plants. He stressed the difficulties in evaluating the effects of mycorrhizal En&gone spp. on the host. Firstly, the obligate or near-obligate parasitic nature of Endogone makes it difficult to obtain pure inoculum. For this reason, doubt often remains whether the observed growth or nutrient absorption effects are due to mycorrhizal infection or to other associated microorganisms, Secondly, because mycorrhizal Endogone spp. are ubiquitous, partial or complete sterilization of soil is necessary to obtain control plants. Sterilization changes soil physical, chemical and biological properties so that interpretation of mycorrhizal responses is difficult. Most studies designed to show the influence of VA mycorrhizae on nutrient uptake by higher plants have been restricted to phosphorus, usually in pho~horus deficient soils (Gerdemann, 1968). There is evidence that infection by VA mycorr~za might also influence absorption of other nutrients. For example, Mosse (1957) reported that mycorrhizal apple seedlings had a higher K, Fe, and Cu content and a lower Mn content than nonmycorrhizal plants. In contrast, Gerdemann (1965) noted that mycorrhizal maize contained a lower concentration of K, Mg, B, and Mn than control plants, However, because of the greater growth of infected plants, larger quantities of these nutrients had been removed from the soil. Recently Ross and Harper (1970) found that mycorrhizal soybeans grown in previously fumigated field plots accumulated greater quantities of P, N, Ca, Cu, and Mn than nonmycorrhizal plants. In this paper we shall present results of our studies which describe the influence of a VA mycorrhizal fungus Endogone mosseae on gOSr uptake by soybean and show the applicability and advantages of the Stanford-DeMent technique (Stanford and DeMent, 1957) * Present address: Bookers Sugar Estates Limited, 22 Church Street, Georgetown, Guyana. 205

206

N. E. JACKSON,

R. H. MILLER

AND R. E. FRANKLIN

for short-term mycorrhizal studies. The technique seems par~cularly useful in overcoming some of the problems with inoculum and soil sterilization noted previously. MATERIALS

AND METHODS

Soil

The soil (Table 1) was from the Ap horizon of a silt loam collected on the Ohio State University Farm, Columbus, Ohio. It is classified as a Typic Hapludalf. The soil was airdried and sieved to pass a 2-mm screen. TABLE

f. CHEMICAL

AND

Sand (%)

Sib (%)

Clay (%)

Organic matter (%)

1.5

63

22

3.2

PHYSICAL

* P extracted with 0.03 N NH4F and 0.025 t K, Ca and Mg extracted with neutral 1

N N

PROPERTLES OF THE EXPERIMENTAL

P”

PH

l/3 bar moisture (%)

6.8

26.7

32

SOIL

K?

Cat

Mgt

4018

673

(kg/Ha) 167

HCI (Soil:extractant ratio of 10: 1). NH40Ac (Soil:extractant ratio of 3: 1 for K, and 10: 1 for

Ca + Mg). Sub-samples of the experimental soil were transferred to polyethylene bags and amended carrier free 90Sr e*Sr Cl, in dilute HCI) to provide 105 dpmfg soil. All subsamples were thoroughly mixed and equihbrated for at least 24 h before use. Soil sterilization was performed by autoclaving soil at 121°C for 1 h on each of 2 successive days.

with sufficient

Mycorrhizal inoculum

Soybean seeds (Glycine max var. Harosoy 63) were surface sterilized by shaking in a 7 % calcium hypochlorite solution [Ca(OCl),, 30-35x active chlorine] for 45 min and then washed in sterile distilled water. Twelve surface sterilized soybean seeds were planted into each porcelain crock containing 1300 g of sterile washed sand. An inoculum” of Eizdogone Ylfoxseaein the form of soybean roots mixed with soil was used to supply mycorrhizal inoculum. Ten grams of the root-soil inoculum of En~ogo~e ~o~~e~e was suspended in 100 ml of sterile water and dist~buted over the seeds at the rate of 20 ml/pot. The seeds were then covered with an additional 500 g of sterile sand. The seedlings were grown for 3 weeks in a growth chamber with no attempt to prevent contamination from airborne microorganisms. Portions of the roots were then examined to determine the presence of VA mycorrhizae. The remaining roots were washed, frozen at -2O”C, freeze dried, and ground in a Wiley mill to pass a 20-mesh screen. This formed a secondary inoculum (0.2 g of freeze-dried ground root material/20 ml HzO) which was then used in a repeat of the procedures described above to increase the supply of inoculum. After 3 weeks, the second crop of mycorrhizal soybean roots were harvested, washed, frozen, freezedriedground, and stored at -20°C. This root material served as the mycorrhizat inoculum in subsequent experiments. * Obtained from Dr J. W. Cerdemann, Illinois.

Professor

of Plant Pathology,

university

of Illinois, Urbana,

VA hlYCORRHIZAE AND UPTAKE OF g0Sr

207

An inoculum control consisting of freeze-dried ground roots was prepared in a similar manner without inoculation with a soil-root mixture containing ~ndugone mosseae. Microscopic examination of control inoculum showed it was free of Endogone spp. Studies on goSr uptake by soybeans The Stanford-DeMent technique (Stanford and DeMent, 1957) was used to compare the uptake of gOSr by mycorrhizal and nonmycorrhizal soybean plants. Plants were grown in acid-washed sand in nested 400 ml waxed cardboard containers. The bottom was removed from the inner container and placed within the intact outer container before filling with sand. Freeze dried mycorrhizal root inoculum (O-2 g) was placed in sterile Petri dishes with 20 ml of sterile distilled water. Eleven surface-sterilized soybean seeds were placed in each Petri dish and swirled with the inoculum. These seeds were transferred to the surface of 350 g of sand within the nested containers and the remainder of the suspension poured over the seeds. The seeds were covered with 250 g of sand and 70 ml of sterile distilled water was added. All containers were kept in the dark until the seedlings emerged. After emergence, the plants were thinned to lo/container and allowed to grow for 13 days within a growth chamber. The growth chamber provided a 16-h light period supplied by a bank of General Electric PG 17 cool white fluorescent bulbs supplemented with incandescent bulbs. Light intensity at plant height was 2.7 x lo4 lux. The temperature was 30°C during the light period and 23°C during the dark period. The plants were watered daily and alternately with either distilled water or sterile Hoagland’s solution, the quantity depending upon the age of the plants. Control plants were grown in the same way, except that control inoculum was used instead of mycorrhizal inoculum. After 13 days of growth a good root mat had developed and the inner container was removed and nested in another waxed cardboard container which contained 50 g of p”Sr amended soil. The plants were allowed to grow for 1,3 or 7 days with the root mats in contact with the soil. A split plot design was used with mycorrhizal and control inoculum as the main treatments and with nonsterilized and sterilized (autoclaved) soil as the subtreatments. Each treatment was replicated 4 times. A second experiment was designed to permit mycorrhizae to develop concurrently with gOSr uptake by the growing soybeans. All procedures were the same as the StanfordDeMent study except that 50 g of gOSr amended soil was placed beneath the sand layer of each container before planting soybeans. The plants were grown for 14 days after emergence. The experimental design was the same as in the first experiment. The effectiveness of the freeze dried inoculum of Endogone mosseae in producing consistent infection and the Iack of infection by control inoculum was evaluated microscopically before the main experiments. All procedures were identical to those described in the next section. Measurement of radioactivity At the end of the uptake period the plants were harvested and the roots were washed free of sand and soil. The plants were separated into leaves, stems, and roots for analysis. The samples were dried for 24 h at 7O”C, weighed and ashed at 500°C in glass counting vials (Packard Instrument Co.). Each sample was treated with 2 ml of a mixture of nitric, perchloric and sulphuric acids, (10:4: 1) and digested on a hot plate overnight. After evaporation to dryness, any remaining organic matter was oxidized with hydrogen peroxide. Each sample was dissolved in 8 ml of distilled water and 6 drops of 4 N HCl. The Cerenkov radiation was counted in a liquid scintillation counter (Packard Tricarb) after allowing 1 month for g”Y and 90Sr to reach secular equilibrium.

208

N. E. JACKSON,

R. H. MILLER

AND R. E. FRANKLIN

RESULTS

Experiment 1. Mycorrhizal studies using the Stanford-De&lent technique Thirteen-day-old soybean plants inoculated with Endogone mosseae absorbed more goSr than control plants when placed in contact with soil amended with gOSr for 1, 3 or 7 days (Fig. 1). The rate of gOSr uptake remained nearly constant for mycorrhizal and control plants between day 1 and day 3, but increased for mycorrhizal plants between day 3 and day 7. These data may be explained by assuming that the previously developed mycorrhizal:root

120

110 mycorrhiral

100

n

P G

90

50 40

30 20 IO

I I

I

I

3

7

Uptake

Period,

days

FIG. 1. The effect of inoculation of soybeans with VA mycorrhizae on uptake of “‘Sr from 90Sr amended soil using the Stanford-DeMent technique. Data shown are the means of main treatments expressed as Yir uptake (leaves, stems and roots) for 10 plants/container. The means differed significantly at the I % level. Inoculated plants were grown for 13 days before placing in contact with 90Sr.

mats continued to function throughout the 7-day uptake period. If the mycorrhizae had not remained functional, the two curves in Fig. 1 should have remained parallel throughout the entire uptake period since the new roots being developed in both the infected and control root mats are considered to be non-mycorrhizal. The content of gOSrin the leaves, stems and roots of mycorrhizal plants was greater than the controls at all times with one exception; gOSrcontent of the stems of mycorrhizal plants after 3 days were the same as control plants (Table 2). These data also show that g”Sr was translocated about as rapidly from mycorrhizal roots as from control roots. This means that gOSr absorbed by mycorrhizal hyphae reaches the plant vascular tissue as rapidly as gOSr absorbed directly by the root. Soil sterilization by autoclaving had a negative influence (P > 0.05) on the uptake of “Sr by mycorrhizal soybean roots after 1 day of root-soil contact (Fig. 2a). There was no

VA MYCORRHIZAE

AND

UPTAKE

OF 90Sr

209

TABLE 2. THE EFFECTOF VA MYCORRHIZAEON THE ?Gr CONTENTOF LEAVES,

STEMSAND ROOTSAFTERVARIOW TIMESOF ROOT-SOILCONTACT Uptake period (days)

?Sr uptake/l0 plants (cpm x 10m3) Plant part

Mycorrhizal

Control

1

Leaves Stems Roots

1.3** 4,0** 13.3**

0.47 1.8 6.9

3

Leaves Stems Roots

14.5** 12.5 17.5*

9.9 12.0 12.2

7

Leaves Stems Roots

37.4** 41.4** 39.8**

27.3 28.7 26.2

**** Indicates that the mycorrhizal and control treatment means differed significantly at the 1% and 5 % level respectively.

Mycorrhizal

_

Non sterilized

---Sterilized

soil

soil

Control

I

plants

plants

3 Uptake

7 period,

days

FIG. 2. The effect of soil sterilization on 90Sr uptake by mycorrhizal and control plants in the Stanford-DeMent study. Data shown are the means of each treatment expressed as 90Sr uptake (leaves, stems and roots) and of roots only, for 10 plants/container.

210

N. E. JACKSON,

R. H. MILLER

AND R. E. FRANKLIN

difference by day 3 and the effect was reversed by day 7 resulting in a positive influence of soil sterilization on gOSr uptake. Roots of control plants meanwhile showed a smalf but statistically insignificant increase in goSr uptake from sterilized soil at both day 1 and day 7 (Fig. 2b). These data suggest that the early negative effect of soil sterilization on gOSr uptake by mycorrhizal roots was due to some adverse influence on soil-hyphal contact. The difference in gOSr uptake by mycorrhizal roots between sterilized and nonsterilized soil by day 7 may reflect the absence of competition from other micro-organisms on the extension or function of mycorrhizae hyphae. Soil sterilization did not produce a consistent trend in the gOSr content of leaves and stems of the experimental plants. Differences in the goSr content of the total plant because of soil sterihzation were not statistically significant, but do reffect the responses shown previously by mycorrhizal and control roots (Fig. 2a and b). (P > 0*10)

Experiment 2. goSr uptake concurrent with the infection and de~el#p~le~ltof mycor&izai roots With the Stanford-DeMent technique it was possible to separate those soil and microbiological factors which influence infection and development of mycorrhizal roots from soil factors which influence the uptake and translocation of gOSrby mycorrhizal roots. The rationale for Experiment 2 was to compare gOSr uptake by soybeans using the StanfordDeMent technique (Experiment 1) with gOSr uptake when myconhizal infection and development proceeded concurrently. Mycorrhizal soybean pIants absorbed significantly more g”Sr from “Sr amended soil than did control plants (Table 3). These data are in agreement with the resuIts of Experiment

TABLE

3.

THE EFFECTOF VA MYCORRHIZAEAND SOTLSTERILIZATIONON TIONINSOYBEANS 14IIAySAFTEREMERGENCE

YSr uptake/l0 Mycorrhizal

Plant part Leaves

Stems

Roots

Total

x

10-3)

Control

Mean

54.7 b 61*8a 58,2*

46.0 c 38.8 d 42.4

Mean

48.2 b 55.0 a 51.6**

42.1 c 36.5 c 39.3

Mean

20.7 b 28.2 a 24.4

22.9 b 21-l b 22.0

124.0 b 145.0 a Mean 134*0*

lll.Oc 96.4 d 104.0

NS S

NS S

NS S

NS S

plants (cpm

90Sr~~~~~~~-

NS and S refer to plants grown in nonsterilized and sterilized soil respectiveIy. **** Indicates that mycorrhizal and control treatment means are significantly ditTerent at 1% and 5% level respectively. For each part, means not followed by a common letter differ sign~cantly at the 5% level using Duncans Multiple Range Test.

VA MYCORRHIZAE

AND UPTAKE

211

OF 90Sr

1 using the well developed root mats of the Stanford-DeMent technique. Strontium 90 in the leaves and stems but not in the roots of mycorrhizal plants were greater than in the controls. This lack of difference in the gOSr content of roots contrasts with the results of Experiment 1. Mycorrhizal soybean plants in this experiment absorbed more gOSr from sterilized soil than from nonsterilized soil during the 14-day growth period while the reverse was true for control plants (Table 3). These differences reflect a more extensive development of mycorrhizae in the absence of microbial competition in the sterilized soil. Also, control plants grown in nonsterilized soil became infected by indigenous VA mycorrhizal fungi and gOSr uptake increased somewhat by this association. It is these particular opposing events which tend to mask the recognition of mycorrhizal responses when working with nonsterile soil by the usual methods of study and which are eliminated in short term studies using the Stanford-DeMent technique. It should be noted, however, that the gOSrcontent of mycorrhizal soybean plants growing in nonsterilized soil still exceeded that of control plants, although the magnitude of this difference was not as great as in sterilized soil.

TABLE~DRYMATTERYIELDSOFMYCORRHIZALAND PLANTS

CONTROLSOYBEAN

Dry matter yield (g/10 plants) Experiment No.

Days

Mycorrhizal

Control

1

1 3 7

3.22** 4.05 6.54*

3.66 4.10 6.20

2

14

3.09**

4.31

**,* Indicates that mycorrhizal and control treatment significantly different at 1 y0 and 5 ‘A respectively.

means

are

Effect of mycorrhizal infection on dry matter yields

The data in Table 4 compares the dry matter yield of mycorrhizal and control plants for both Experiments 1 and 2. Soil sterilization had no effect on top and root growth in either experiment so the data are for main treatments only. Total dry matter yield of control plants in the Stanford-DeMent study grown in sand culture but after contact with soil for one day (14 days after emergence) were greater than that of mycorrhizal plants. This growth effect had largely disappeared by day 3 and had become reversed after 7 days. The soybean plants grown in soil in Experiment 2 (14 days after emergence) showed a definite and significant decrease in dry matter yield because of infection with mycorrhizae. In these studies adequate nutrition was supplied by nutrient solution so that the mycorrhizae did not contribute to plant nutrition. Under these conditions the normal pathogenic effects of a fungal infection might become evident. Alternatively it is interesting to speculate that early infection with mycorrhizae might be detrimental to plant growth. The positive influence on growth usually attributed to mycorrhizal infection might occur only during later stages of plant growth.

212

N. E. JACKSON,

R. H. MILLER

AND R. E. FRANKLIN

DKSCUSSION

Evidence is provided in this study that vesicular-arbuscular mycorrhizae can markedly influence the root absorption of a cation (90Sr) from solI. This increased absorption occurred even in no~lsteri~ized soil with normal anlo~lnts of Ca and Mg which have chemical and biological properties similar to Sr. Thus it seems reasonable to speculate that mycorrhizal infection might significantly influence nutrient absorption in soils of normal fertility, not just under conditions of low fertility or from relatively unavailable sources of nutrients (Gerdemann, 1968). Of considerable interest in this study was the very rapid infection and establishment of a functional mycorrhizal-plant symbiosis. Within 13 days from seedling emergence, and 17 days after inoculation and planting, infection could be observed microscopically and reflected functionally in 9oSr uptake. In most studies reported previously in the literature, mycorrhizal responses were not assessed until after at least 30 days of growth (Gerdemann, 1968). The earliest reported response to VA mycorrhizal infection was enhanced growth by 19-day-old tobacco plants grown in sand culture (Daft and Nicolson, 1966). The reason for the early response to 9oSr absorption may be only a refiection of the use of a radioisotope assay which is more sensitive than dry matter yields or plant analysis which would become significant only during later stages of plant development. Perhaps the volatile components of the freeze dried root material used in the inoculum may have stimulated spore germination and enhanced the metaboIic activity of Endogme in a manner analogous to that proposed for soil fungi by Gilbert and Griebel (I 969). Certainly these data on the earIy positive influence of mycorrhizal fungi on ion absorption, coupled with early negative influences on plant growth (Table 4) suggests a need for studies of the functional longevity and early development of mycorrhizal associations. The Stanford-DeMent technique employed in this study proved to be an excellent method for studying short-term mycorrhizal effects on higher pIants where dry matter production is not the parameter of concern. The technique is simple and economical to use and allows for adequate replication without much work. Only small quantities of soil are needed which can be easily amended with radioisotopes, chemical inhibitors, etc. without affecting the development of the mycorrhizal infection itself. Furthermore, because the infected plants develop independently of the soii system to be studied, non-sterihzed soil can be utilized which eliminates the adverse physical and chemical effects of soil sterilization which so often hinders interpretation of mycorrhizal studies. The main disadvantage is that soiI factors affecting mycorrhizal infection and development cannot be elucidated. Acknowledgement-Published with the permission of the Director of the Ohio Agr. Res. and Devel. Center as Journal Article No. 5-72. This research was supported in part by Public Health Service Research Grant RH 00124 from the Division of Radiological Health. REFERENCES DAFT M. J. and NICOL~ONT. H. (1966) Effect of En&gone mycorrhiza on plant growth. New Phytuf. 65, 343-350. GERDEMANNJ. W. (1965) Vesicular-arbuscular mycorrhizae formed on maize and tuliptree by Endogone fhsiculata. Mycologia 57, 562-575. GERDEMANN J. W. (1968) Vesicular-arbuscular mycorrhiza and plant growth. A. Rer. Phyropath. 6,397-418. GILBERTR. G. and GRIEBELG. E. (1969) Stimulation of soil respiration by volatiles from alfalfa. Soif Sci. Sot. Am. Froc. 33,2X!-273. Mossy B. (1957) Growth and chemical composition of mycorrhizal and non-mycorrhi~l apples. Nature, Land. 179,922-924. Ross J. P. and HARPERJ. A. (I 970) Effect of Endugone mycorrhiza on soybean yields. Phytopath. 60, 15521556. STANFORDG. and DEMENT J. D. (1957) A method for measuring short term nutrient absorption by plants. I. Phosphorus. Soil. Sci. Sm. Am. Proc. 21, 612417.