Vesicular-arbuscular mycorrhiza and growth in barley: Effects of irradiation and heating of soil

Soil Birtf. Bi~~~~~. Vol. 14. pp. 171 to 178, 1982 Printed in Great Britain. Ail rights reserved

VESICULAR-ARBUSCULAR MYCORRHIZA AND GROWTH IN BARLEY: EFFECTS OF IRRADIATION AND HEATING OF SOIL I. JAKOBSEN* Botanical

Institute,

University

of Aarhus.

Nordlandsvej

68, DK-8240

Risskov.

Denmark

and A. J. ANDERSCN Agricultural

Research

Department.

Risa National Laboratory, DK-4000 Roskilde. (kcpred

Denmark

1 October 1981)

Summary-The influence of soil irradiation (0.25-4.0 Mrad) and soil heating on mycorrhizal survival, establishment and development after reinoculation, and on plant growth, was investigated. The lowest radiation dose applied. completely eliminated the infectivity of a soil with a high number of mycorrhizal propagules. Mycorthiza developed more slowly after maculation in irradiated soils than in untreated soils. This could have been due to the small amounts of inocuium used, hut the high concentrations of n~~trients released by irradiation of the soil were probably of greater significance particularly the increased amounts of plant-available N as indicated by incubation experiments. Inorganic N was increased to similar levels by the various treatments, Available soil P increased with increasing irradiation dose. Incubation of inoculum in soil for 40 days before sowing Increased mycorrhizal infection, INTRODUCTION

Subsequent plant growth is affected to varying degrees by irradiation and heating of soil. In general, soil irradiation will increase plant growth (Cawse. 1975) whilst heating sometimes produces certain unidentified phytotoxic compounds (Rovira and Bowen, 1966). Different groups of organisms differ in their susceptibility to irradiation, fungi being known to be the most radiosensitive group of soil microorganisms (Cawse, 1975). This offers the possibility of using irradiation in a selective way in studies on soil microorganisms. Both irradiation, and heat-treatments such as steaming and autoclaving, have been widely applied to soils used for research on vesicular-arbuscular mycorrhiza (VAM). where it has been necessary to eliminate the indigenous mycorrhizal propagutes to achieve controlled experimental conditions (Mosse. 1973). The dose of radiation generally applied to soils to eliminate mycorrhizal propagules is 0.8 1.0 Mrad. A lower level might well be adequate. According to Cawse (1975) it is sometimes difficult to introduce microorganisms into irradiated and heatsterilized soils. There are no reports on the effects of irradiation on mycorrhizal establishment and development in inoculated soils. Even if soil irradiation does not influence later mycorrhizat development, a plant growth response to mycorrhizal infection might well be biased by the increased plant growth often caused by soil irradiation. A. Jensen and 1. Jakobsen (unpublished) had difficulties in es~blishing vigorous mycorrhizal infection in barley grown in three irradiated low phosphorus

soils compared to infection in non-irradiated soils. Also irradiation produced from 1.31-to 2X-fold incrcascs in yield of plants with no mycorrhiza formation. Our aim was to seek the reasons for these findings and to determine the radiation level lethal to VAM-propagules in soil. MATERIAIS

Exprrirntwt

171 141

PI

METHODS

1

The aim of this experiment was to whether or not incubation of irradiated

* Present address: Agricultural Research Department. Riser National Laboratory, DK-4000 Roskilde, Denmark. 5””

AND

The loamy soil used in these experiments was sampled from the plough layer in Rise experimental field. The soil was air-dried and sieved ( t5 mm) before mixing with sand (1: 1). The mixture had the chemical properties given in Table 1 and was prepared in the same way in Expts 1 and 2. but chemical analysis was carried out only on the mixture used in Expt 2. The high amount of CO,-C (0.S2”/Q) originated from the sand mixed into the soil. The experiments were done in 2 kg pots in the greenhouse with barley. var. Rupal. as test plant. The plants were watered when necessary. After harvesting, dry weight and P content of tops were determined. For P the molybdenum blue method after wet digestion was used. Washed root samples were cleared and stained (Phillips and Hayman. 1970) and mycorrhizai infection. based on 100 intersections, was assessed at x 40 and x 100 magnification using a line-intersection method with the compound microscope (Ambler and Young. 1977). Differences in infection intensities at intersection points were taken into account by applying categories 1- 3 for recording of infection. The formula used for calculating percentage infection was: VAM-infection = (a + 2b + 3c/300) x JO”.,. where a + b + c = 100 (total number of intersections). determine soil sub-

171

and A. J.

I. JAKOBSEN

Table 1. Some chemical

properties

of the soil:sand

AUDEKWN

mixture

before application

of soil treatments

P*

PH 0.01 M CaCI, 7.7

0.5 N NaHCO, @gg-‘) 13

0.3 N

Resin @gg-‘) I‘l

H2S0, @gg-‘) 123

* For descriptions of the different BondorlT (1950). respectively.

K 0.5 M NH,.k (pgg-‘) 56

extraction

Ca l.o.M

c

NH,CI !“<,I

Total (“,A

Org. 1”,,1

Kjeldahl (“,,I

100 g soil

0.29

0.82

0.30

0.094

10.5

methods.

to inoculation and before planting increases mycorrhizal development in barley roots. Mycorrhizal inoculum was obtained from a white clover stock plant with Glomus mo.s.se~tc (Nicol. and Gerd.). Rothamsted strain. Inoculated pots received log crude inoculum (infected roots. sporocarps and spores mixed with sand) containing ca. 300 sporocarps. Inoculum was mixed into the pot soil in a 2 cm layer 5 cm below soil surface. Irradiated soil (10 keV electron beam. 4 Mrad) was filled into pots 40 days before sowing and placed on the greenhouse bench. Inoculum was added to l/3 of the pots while the remaining pots received mycorrhiza-free leachings only, obtained by washing inoculum on a 38pm pore sieve. For comparision. pots with untreated soil were included in the experiment. Half of these pots were inoculated 40 days previous to sowing. During the 40 days of incubation all pots were kept moist. After the incubation time half the uninoculated pots with irradiated soil were inoculated. Thus Experiment 1 included the treatments shown in Table 3. Five plants were grown per pot, with 16 replicates per treatment. Pots were randomized in a 4 block design. To each pot 100mg N was added as NH,NO,-solution at 1, 4 and 7 weeks after sowing. The experiment was performed from January to April with additional lighting and heating, the air temperature being l&20/12- 15 C (day/night), Four pots from each treatment were harvested at 4, 7, 9 and 13 weeks after sowing. Besides the records mentioned above. mycorrhizal infection was assessed in roots from both the upper and lower halves of the pots at the last three harvests. sequent

Plant cxprrirnwt. This experiment was to investigate the extent to which different radiation levels and heat treatments of soil would affect plant growth and mycorrhizal development with and without subsequent inoculation. Before application of the relevant treatments. the soil and roots from a 2 kg pot from Expt 1. with well-established mycorrhizal infection, were chopped and thoroughly mixed into the soil-sand mixture to ensure a high “indigenous” VAM-population. Experiment 2 included nine treatments with the addition of inoculum leachings ( ~38 pm) only and seven treatments with the addition of crude inoculum of G. I~IOS.WU~ (15 g pot- ’ containing ca. 150 sporocarps). The various treatments are given in Table 4. The pots, with 4 plants in each, were randomized on the greenhouse bench. N being added after 35 days

CEC N

c

see Olsen

c’t ul. (1954). Sihbesen

m-equi\

(1977) and

as NH,NO, (200mg N,‘pot). The experiment was conducted from May to August. air temperatures being 2&30/12- 18 ‘C (day/night). After 5. 8 and 11 weeks, four pots were harvested from all VAM inoculated treatments and from the non-inoculated treatment with untreated soil. Other treatments were harvested after 11 weeks. Changes in available soil P as a result of the different soil treatments were determined by an anion exchange resin method (Sibbesen. 1977). N-rl~in~,rali-_utioll experiment. This experiment was intended to investigate the levels of plant available N as affected by the different soil treatments. Soil given the nine treatments listed in Table 4 was mixed with quartz sand (1: 1) to achieve well aerated conditions and 40g samples were incubated in the dark at 26 ‘C and IO”, water content. Prior to incubation, leachings ( < 38 ltrn) from fresh soil were added to ensure the presence of microorganisms involved in N transformation in soil. Samples with sand and sievings only were included as controls. At 0. 7, 14 and 28 days, three samples per treatment were extracted with 100 ml 1 N KCI, filtered, and the extracts stored frozen. Finally, each extract was analysed for NH:, NO; and NO, using a Chemlab autoanalyser. RESULTS

Incubation of mycorrhizal inoculum in soil for 40 days before sowing had a beneficial effect on mycorrhizal development in barley roots (Fig. 1). Throughout the experiment in ,irradiated soil. mycorrhizal infection in pots with incubated inoculum was significantly higher than in pots without incubated inoculum. However, mycorrhizal spread was rather slow, especially from 4 to 9 weeks in irradiated soil compared to in untreated soil. where infection spread rapidly along the roots after 4 weeks. The uninoculated controls in irradiated soil did not become infected, while mycorrhiza established rather slowly in controls in untreated soil during the first 7 weeks. after which it spread rapidly, Roots from the upper halves of the pots were generally more infected than roots from the lower halves (Table 2). This difference was most pronounced in the treatment without inoculum incubation. This is illustrated by the calculated ratios given in the table. Plant growth was much affected by soil irradiation (Table 3) while mycorrhizal infection had no significant influence.

Soil irradiation,

50

IRRADIATED x-_-x

inoculated

-adaptatm

e---a

moculated

+adaptation

SOIL

d-A

not

,_b

inoculated

and plant growth

173

Comparing the dry weights from irradiated and untreated soil, the following growth increases after 4, 7,9 and 13 weeks due to irradiation were calculated:8, 38. 57 and SS”,:. The corresponding figures for P-uptake were:- 64. 105, 83 and 46%.

T

control

o---o

UNTREATED

40-

SOIL

VA-mycorrhiza

Inoculated

Experiment

2

P/ant rxprrimmt. No mycorrhizal found in the non-inoculated treatments

infection

was

after 11 weeks except for 12% in dry heated soil. Thus all radiation doses and autoclaving had eliminated the infectivity of the mycorrhizal propagules present Mycorrhizas developed very rapidly in pots with untreated soil and were not influenced by inoculation (Fig.

-I Weeks

Fig. 1. Development of VAM-infection in barley roots from Expt 1, as affected by irradiation of soil, mycorrhizal inoculation and incubation of inoculum. Vertical bars represent k SE.

2). In contrast

they

developed

very

slowly

dur-

ing the first 5 weeks in both irradiated and heattreated soils. After 5 weeks, mycorrhiza spread rapidly in heat-treated soil and somewhat more slowly in irradiated ones. The significant difference between the two groups at 8 weeks was disappearing by 11 weeks, and remained significant only for autoclaved compared to the 4 Mrad treatment. At this time infection spread was as rapid as in roots from untreated soils, but infection intensities still differed widely (22-37x compared to 60%). As in Expt 1, irradiation significantly increased plant yield (Table 4), all doses doing so to a similar extent (67-877; after 11 weeks). Autoclaving had a similar effect, while the increase from dry heating was much smaller (non-significant after 11 weeks). Mycorrhizal inoculation did not seem to affect plant growth (compare dry weights in Table 4 with corresponding figures in italics).

Table 2. Mycorrhizal infection in barley roots from upper (LJ) and lower(L) pot halves in Expt 1 at 7. 9 and I3 weeks after sowing VAMintensity (“,) Treatment

7

9

13

Xsin-‘L

Irradiated soil: Inoculated, incubation without inoculum

U L

I2 1

15 I

26 10

4.2

Inoculated, incubation with inoculum

U L

10 16

16 7

23 13

1.4

control

U L

4 3

21 13

46 37

I.4

Inoculated, incubation with inoculum

U L

21 8

39 27

56 51

I.4

Untreuted soil: Uninoculated

Table 3. Dry weight and P-uptake Treatment Weeks after sowing Irradiated soil: Uninoculated control Inoculated, incubation Inoculated. incubation Untreated soil: Uninoculated control Inoculated, incubation LSD (P = 0.05)

X sin-‘U

without inoculum with inoculum

with inoculum

in tops of barley

from Expt 1 sampled

four times after sowing P-uptake

Dry weight (g pot-‘)

(mg pot- ‘)

4

7

9

I3

4

7

9

13

0.9 0.7 0.9

4.7 5.3 5.3

10.9 12.0 10.2

27.6 26.3 27.1

3.5 3.5 3.7

13.9 16.8 14.3

18.7 20.1 19.2

29.0 32.6 29.3

0.8 0.7 0.2

3.5 3.8 0.9

6.7 7.3 1.9

16.5 17.8 4.3

2.3 2.0 0.8

7.1 7.4 2.2

9.8 11.1 2.1

21.3 20.3 4.2

174

I. JAKOBSEN and A. J. ANDEKSEN

60

50

_ x-x

untreated,

o-o b--b

untreated 0 25 Mrad

O-0 l -m

0 75 M rod 20 Mrad

a-_*

LO

0-o

autoclaved

.+

dry

uninoculated

Mrad heated

5 Weeks

Fig. 2. Development of VAM-infection in barley roots from Expt 2 as affected by irradiation and heating of soil. Vertical bars represent LSD-values (P = 0.05). The P contents of plants at 5 weeks were significantly increased by irradiation and autoclaving (Table 4). However P content fell in plants from treated soils from 5 weeks onwards but was more constant in plants from untreated soil. P content of plants increased slightly with increased radiation dose, most markedly at 5 weeks. After 5 and 8 weeks, P uptake

per pot increased significantly in plants from treated soils (Table 4). At 11 weeks. P uptake was similar in all treatments. All treatments increased available soil P. dry heating most (Table 4). In general. a positive correlation existed between radiation doses and P concentrations. N-minrrahtion esprriment. The concentration of NH; in the soil increased with increasing radiation dose. except for 4 Mrad. which produced less NHf than expected (Fig. 3a. 0 days). Of the heat treatments, autoclaving produced the highest increase in NH;. During the first 7 days of incubation. NH: increased in the heat treatments and the three highest radiation doses. After 7 days. NH: decreased in all treatments and was as low after 28 days as in untreated soil throughout the incubation period. At certain times rather high NO, concentrations were present in some of the treatments (Fig. 3b): especially in autoclaved soil after 14 days. The soil treatments hardly influenced NO; concentrations (Fig. 3c, 0 days). During the first 7 days of incubation the rate of NO; formation was inversely proportional to the radiation dose. After 28 days’ incubation the different radiation doses had produced equal amounts of NO;. Autoclaving nearly tripled the amount of NO, formed compared to untreated soil. while a near doubling of NO; concentration was found in the other treatment. The overall effect of the soil treatments on soil N mineralized is seen in Fig. 3d. where the N in its various forms has been totalled for each treatment. In the irradiation and dry heating treatments the N concentrations were practically equal at all times. as indicated by the small standard errors in the means. Note that differences between treatments were mainly produced during the first 7 days of incubation. Net mineralization after 2X days can be read from Fig. 3d, as follows (pm01 g- ’ soil):- untreated soil. 0.72; mean of irradiated and dry heated treatments. 1.30: autoclave treatment, 1.98. DISCUSSION The minimum dose of irradiation required to eliminate mycorrhizal infection may differ between soils. It

Table 4. Available soil phosphorus before and after soil treatments, and dry weight, P-content barley from Expt 2 sampled three times after sowing Treatment

Soil-P

(pgg-‘) Weeks after sowing None 0.25 Mrad 0.50 Mrad 0.75 Mrad 1.00 Mrad 2.00 Mrad 4.00 Mrad Autoclaving (121 C, 2 h) Dry heating (85 C. 2 h) LSD (P = 0.05)

Dry weight (g pot - ‘)

Plant-P

8

5

8

2.8 2.x 3.7 ND 3.9 ND 4.1 4.3

3.2 3.3 2.4 ND 2.4 ND 2.5 2.5

3.3

2.1

9.7 2.9 2.2 0.3

2.3 0.2

5

11

14.0 16.3 16.6 16.3 17.3 17.7 19.9

1.1 1.1: 1.9 ND 2.0 ND 2.1 2.0

3.5 3.3 7.2 ND 6.4 ND 6.9 7.2

7.9 13.3 ND 14.4 ND 15.1 14.5

16.8

2.2

7.1

15.2 14.5

22.2

2.0 0.3

5.6 0.6

* Italics refer to non-inoculated ND = not determined.

treatments.

8.9 2.2

8.4 14.0 14.9 15.6 15.7 15.2 15.2

(mg P g-

’ dry wt)

and P-uptake

P-uptake 5

11

in tops of

(mg P pot- r) 8

11

3.1 3.4 7.0 ND 7.8 ND 8.6 8.6

11.3 10.7 21.0 ‘1.5 17.0 21.4 ‘(1.I ND ND 23.7 24.8 XV 15.6 ND 25.Y ND ‘7.1 -70.7 17.4 ‘6’._ __. 734 18.0

1.5 1.4

7.1

14.5

22.5 X1)

2.2 -‘.I 0.3 0.2

5.8 1.3

12.8 2.2

19.6 IV.7 2.8 4.(J

2.7 1.6 ND 1.8 ND 1.8 1.8

-7.6 I.5 1.6 1.7 1.7

I.4 I.6

Soil irradiation.

VA-my,corrhiza

175

and plant growth I o0-o .-

untreated outoclaved dry heated

.-. *-_*I.0

20

Days

0

7

14

Mrad Mrad

28

Days

Fig. 3(a). 0.5

,

0.6 = 70.3 IN

o-m o--o .-.

untreated autocloved dry heated

-

.

20

Days

Fig. 3(b).

176

I. JAKOBSEN

3 _

0

-0

untreotE.2

o-o

autoclaved

.-.

dry

and

A.

J. ANDERSEN

heated

0

14

7

20

Days

14

d’

0.25

o--o

OSOMrad

M rad

O-O ~-6 n -m

075Mrad Mrad 10 20 Mrad

+-_*

LO

Mrad

28

Days Fig. 3(c).

0

0

o-o -0

untreated outoclaved

*-=

means

tSE

of

other

7

treatments

1L

I 28

Days Fig.

3(d).

Fig. 3. Transformation of mineral N in soil during 28 days of incubation as affected by irradiation and heating of soil. (a) NH:-concentrations, (b) NO;-concentrations, (c) NO;-concentrations and (d) NH:. NO; and NO; totalled for each treatment.

117

Soil irradiation, VA-mycorrhiza and plant growth

might also be influenced by soil moisture and number and form of mycorrhizal propagules in the soil. If. however, the 0.25 Mrad found in this study or an even lower dose proves generally sufficient, it would be a desirable level for use in future mycorrhizal work in order to reduce disturbance to the general soil microflora and soil nutrient status to as little as possible. The increased mycorrhizal infection after 40 days of incubation of inoculum in treated soil confirms the results obtained by Ocampo and Hayman (1981). They found that inoculum left in moist soil in pots with no plants in the glasshouse for 10 weeks produced a much more rapid build-up of infection in barley, lettuce and maize than the same inoculum preserved at 2’C. These findings may be explained on the basis of some saprophytic activity by mycorrhizal fungi (Warner and Mosse, 1980). The generally poor mycorrhizal development in irradiated soil in Expt 1 suggested that the sterilizing dose of 4 Mrad had produced adverse conditions for mycorrhizal development. possibly by releasing large amounts of nutrients, which are known to decrease mycorrhizal infection (Hayman, 1975: Jensen and Jakobsen. 1980). The slow development of infection in Expt 2 may also be explained by this hypothesis. Alternatively, the amount of mycorrhizal inoculum used in Expt 2 may have been too small. The higher infection levels in heated than in irradiated treatments at 8 weeks suggest an additional inhibiting factor produced by irradiation. The amounts of mineral N and available P released after heat treatments were not less than released after irradiation (Fig. 3d and Table 4). but infection in heated soils was still 17722’);. compared to only 69% in irradiated ones. According to McLaren (1969) no toxicity to soil microorganisms has been noted following irradiation. However, other workers have sometimes found recolonization of irradiated soils to occur slowly, and this has been related to cytotoxicity produced by the radiolytic breakdown of sugars (Cawse, 1975). The rapid changes in form of inorganic N during incubation (Fig. 3a, b and c) did not influence the progress of mycorrhizal infection. NH; is more inhibitory to mycorrhiza than NO; (Chambers et al.. 1980) but persistent high NH: concentrations in the incubation experiment were probably prevented by nitrifiers added with the soil leachings. Such large plant growth responses to soil irradiation as are reported here (67787”,b) are unwanted in mycorrhizal research, as they may obscure growth responses to mycorrhiza. The real response to irradiation might well have been even greater in these experiments, as plants in the untreated soils were heavily mycorrhizal. Therefore in studies on growth response to soil irradiation. plants should be used which are known not to be hosts to mycorrhiza, e.g. members of the family Cruciferae. The growth increases due to soil irradiation were probably produced by elevated concentrations of both plant-available soil P and N. Available soil P was somewhat increased after irradiation (Table 4) but differences in P concentrations between plants from irradiated and untreated soils after 5 weeks of growth were even more pronounced. than would be expected from these soil P analysis. Besides, the

increasing dry matter production with time, accompanied by declining P-concentrations in plants from irradiated soils and the results from the N-mineralization experiments points to the importance of mineral N release after irradiation, as in the results of Bowen and Cawse (1964) and Jenkinson et al. (1972). The large amount of N released even at the lowest radiation dose may be explained by the fact that only few fungi survive 0.25 Mrad (Cawse. 1975) and that fungi contributed an average of 75”” to the total soil microbial biomass in 17 soils (Anderson and Domsch, 1980). The small increases in plant yields at 8 weeks and 11 weeks in dry heated soil were surprising. as the same amounts of mineral N were produced in the incubation experiment after dry heating and irradiating soils. Much more available P was present in the soil after dry heating than after autoclaving (Table 4); probably because some of the P released during autoclaving was able to react in aqueous solution with the soil colloids; this would not happen during dry heating. Other workers have found growth responses due to soil irradiation ranging from 1.17-to 4.00-fold (see Cawse, 1975); even as small a dose as 5 krad producing a significant growth response in lettuce (Bowen and Cawse. 1964). To sum up, our work suggests that a lower irradiation dose than is usual should be used for eliminating infectivity in experiments on mycorrhizas. The undesirable side-effects produced by irradiating the soil point to the possible advantages of killing mycorrhizal propagules by alternative methods such as fumigation with methyl bromide or fungicides. Untreated soil free of mycorrhizal propagules should be used by preference.

Ac~norvledgrments~This

work was supported by The Danish Agricultural and Veterinary Research Council. We thank Dr K. Sehested. Rise National Laboratory, for irradiation of soils, MS J. Bargholz and Mr J. D. Thomsen for their technical assistance, and Dr C. M. Hepper. Rothamsted Experimental Station. England. for comments on the draft.

REFERENCES

AMBLER J. R. and YOUNG J. L. (1977) Techniques for determining root length infected by vesicular~arbuscular mycorrhiza. Journul. Soil Science Society of‘ Amrricu 41, 551-556. ANDERSON D. P. E. and DOMSCH K. H. (1980) Quantities of plant nutrients in the microbial biomass of selected soils. Soil Science 130, 211-216. BONDORFF K. A. (1950) Studier over jordens fosforsyreindhold. V. En ny fremgangsmade ved undersegelsen af jordens fosforsyreindhold. Tidsskrift ,fiv Plantearl 53, 336342. BOWEN H. J. M. and CAWSE P. A. (1964) Effects of ionizing radiation on soils and subsequent crop growth. Soil Science

97, 252-259.

CAWSE P. A. (1975) Microbiology and biochemistry of irradiated soils. In Soil Biochernistrv. Vol. 3. (E. A. Paul and A. D. McLaren. Eds). pp. 213-267. Marcel Dekker. New York. CHAMBERSC. A.. SMITH S. E. and SMITH F. A. (1980) Effects

I. JAK~HSEN and A. J. ANDERSW

178

of ammonium and nitrate ions on mycorrhizal infection. nodulation and growth of Trjfi~liwtl srrhtorc~r~t~um. Nrw Phytoloyist

85. 47-62.

HAYMAN D. S. (1975) The occurrence of mycorrhiza in crops as affected by soil fertility. In Endonl~~~orrhi~as (F. Sanders. B. Mosse and P. B. Tinker. Eds). pp. 495-505. Academic Press, London. JENKINSON D. S., NO~AKO~SKI T. Z. and MITCHELL J. D. D. (1977) Growth and uptake of nitrogen by wheat and ryegrass in fumigated and irradiated soil. Pltmf md Soil 36. 149- 1%. JENSEN A. and JAKOBSEN 1. (1980) The occurrence of vesicular-arbuscular mvcorrhiza in barley and wheat grown in some Danish soils with different fertilizer treatments. Plunt und Soil 55, 403-414. MCLAREN A. D. (1969) Radiation as a technique in so11 biology and biochemistry. Soil Bioloy~~ & Biochcnlistry I. 63-73.

MOSSE B. (1973) Advances in the study of vesicular-arbuscular mycorrhira. .Arlnuu/ Rrricrv of’ Ph~~fop~tho/oy~~ 11, 171-194. OCAMPO J. A. and HAYMAN D. S. (1981) Influence of plant

interactions on vesicular--arbuscular mycorrhizal infections, II. Crop rotations and residual effects of non-host plants. ,\‘CLYPhytoloyi.st 87, 333-344. OL.SEN S. R.. COLE, C. V.. WATANABE F. S. and DEAN L. A. (1954) Estimation of avadable phosphorus in soils by extraction with sodium bicarbonate. Circular No. 939, U.S. Department of Agriculture. Washington. DC. PHILLIPS J. M. and HAYMAK D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Tnrn.vut~rio~s of’ thr British Myc~ologicul Society 55, 158-160. RUVIRA A. D. and Bowby G. D. (1966) The effects of microorganisms upon plant growth. II. Detoxication of heat sterilized soils by fungi and bacteria. PImt md Soil 25, 129-142. SIBBESEN E. (1977) A simple ion-exchange resin procedure for extracting plant avaIlable elements from soil. Pht md Soil 46, 665 669. WARNER A. and Mossp. B. (1980) Independent spread of vesicular-arbuscular mycorrhizal fungi in soil. Trunsuctiom oj’ the Brrrish

M~cvloyicul

Sociery

74. 407-410.