The effect of soil fumigation on microbial recolonization and mycorrhizal infection

Soil

Bid.

Biochem.

Vol.

4, pp.

295-305.

PergamonPress 1972.Printedin Great Britain

THE EFFECT OF SOIL FUMIGATION ON MICROBIAL RECOLONIZATION AND MYCORRHIZAL INFECTION E. H. CSIRO

RIDGE

and C.

THEODOROU

Division of Soils, Glen Osmond, South Australia, 5064 (Accepted 15 January 1972)

Summary-The fungal populations of two forest nursery soils were greatly reduced by fumigation with methyl bromide or dazomet (Basamid). Fungal recolonization was rapid, but original numbers were not attained even 7 months after fumigation. Some fungi not detected in untreated soil colonized the fumigated soil. Seedling pine roots were always colonized by larger numbers of fungal species and individuals in control than in treated soil. Infection of roots by a seedinoculated mycorrhizal fungus was increased by fumigation at one site but not at the other. Aerobic bacteria in fumigated soils were initially reduced in number but rapidly rose to 10 times that of control soil. Dazomet had a more persistent effect on bacteria than did methyl bromide. Counts of fluorescent pseudomonads from soils treated with methyl bromide, after an initial fall, rose quickly to constitute up to 78 per cent of the total aerobic count, then slowly declined. Bacterial spore numbers were initially reduced 50-90 per cent by methyl bromide but not by dazomet. The effectiveness of methyl bromide differed in the two soils.

INTRODUCTION

IN EXPERIMENTS where nurseries were inoculated with mycorrhizal fungi Theodorou (1971) found that, as well as stimulating growth of Pinus radiata D. Don, fumigation enhanced mycorrhiza formation by the inoculated fungus. This was attributed to elimination of competitors and antagonists to the introduced fungus. A knowledge of the effectiveness of fumigants in eliminating micro-organisms and of the pattern of microbial recolonization of soil is desirable when fumigated soils are to be inoculated with mycorrhizal fungi, since this would indicate microbial conditions under which the introduced fungus would have to become established in the soil in order to colonize the host roots. The studies reported here examined microbial recolonization of soils of two South Australian forest nurseries after treatment with methyl bromide or with dazomet (Basamid granular, BASF). Microbial colonization and mycorrhizal infection of roots of P. radiuta seedlings grown in these soils were also investigated. MATERIALS

AND METHODS

Soils Two soils were used (Table 1). One was a terra rossa, pH 5.7, at the Forest Research Institute, Mount Gambier, S.A. The other was a sandy loam, pH 5 *3, overlying a clay sub-soil, at the Kersbrook nursery of the South Australian Woods and Forests Department. Soil sterilization Methyl bromide (98 per cent with 2 per cent chloropicrin) (MB), at the rate of 244 g/m2 was used at Mount Gambier and Kersbrook and “Basamid granular” [98 per cent w/w 295

296

E. H. RIDGE

AND C. THEODOROU

TABLE1. NURSERY SOILCHARACTERISTICS Particle size sand (%)

Fine sand (%I

Coarse

Organic C (%)

Bicarb-soluble P (parts/106)

Silt (%)

Clay (%) 6 7

Mount Gambier

0.81

0.061

63

26

65

1

Kersbrook

0.95

0.065

136

45

38

7

dazomet (3,5-dimethyl tetrahydro-1,3,5,2H thiadiazine-2-thione), supplied by courtesy BASF Australia Ltd.] at 68 g/m2 at Mount Gambier only. MB was applied to the soil surface under airtight plastic sheets and the beds left covered for 48 h. Dazomet granules were spread on the surface and the soil then cultivated to a depth of 8 cm, but left uncovered. Untreated plots were also left uncovered. P. radiata seed was sown at Mount Gambier 35 days and at Kersbrook 20 days after fumigation. Mycorrhizal inoculation At Mount Gambier the seed was inoculated with basidiospores of Rhizopogon luteolus Fr. and Nordh. (Theodorou, 1971) and at Kersbrook with mycelium of R. luteolus grown in Perlite medium (Theodorou, 1967). At both sites inoculated and uninoculated seed was sown in both fumigated and untreated plots. Sampling procedure Soil. Samples of soil from 0 to 10 cm depth were taken from within 20 cm radius of a fixed peg in each of 4 replicate control and fumigated plots. The samples (total 300 g/plot), from areas at least 20 cm from any sown pine seed, were placed in plastic bags which were closed immediately and transported to the laboratory as quickly as possible. They were stored at 5°C until used for microbiological examination within l-4 days of collection. At Kersbrook one sample per plot from 22 cm deep was also taken 20 days after fumigation to test the effectiveness of MB fumigant at that depth. Pine roots. When root samples were required, whole pine seedlings, including root systems with attached soil, were placed in plastic bags, stored and handled as for soils. Media For fungal counts and isolations, Czapek-Dox agar plus 0.5 per cent yeast extract, pH4 *5, with chloramphenicol(80 pg/ml), novobiocin (80 pg/ml) and penicillin (100 units/ml) was used. For bacterial counts three media were employed. “Total” aerobic counts were made on yeast extract-peptone-soil (YPS) agar (Bunt and Rovira, 1955); counts of fluorescent pseudomonads on the selective NPC agar medium of Sands and Rovira (1970) supplemented by chloramphenicol (12 yg/ml) and now called NPCC (Simon, Sands and Rovira, unpublished); and bacterial spores were counted on a yeast extract-glucose-peptone agar medium, M32 (Ridge and Rovira, 1971). Before inoculation, media were dried at 60°C for 15-20 min. Fungal isolation From soil. Fungi were isolated by the soil-dilution and soil plate (Warcup, 1950) methods. To attempt

plate (Waksman, 1927; Warcup, 1960) to recover Basidiomycetes, not usually

MICROBIAL

RECOLONIZATION

AFTER

SOIL FUMIGATION

297

isolated by these methods, the hyphal isolation technique (Warcup, 1955) was used. The inoculum for the soil plates was 10 mg soil/plate. Plates were incubated at room temperature (1%23°C). From roots. In addition to dilution plates of root washings, root segments were washed 30 times with sterile distilled water (SDW) (Harley and Waid, 1955), fragmented aseptically by scalpel to 0 - 5 mm pieces and plated. Incubation was as for soil isolations. Fungal identification

Identification was to genus except in a few cases where organisms were identified to species. All colonies different in appearance were identified. Where there were a number of colonies of similar type, 4 random colonies were examined microscopically for identification, based on the keys of Gilman (1957), Barnett (1960) and Barron (1968). Fungai and bacteriaf counts One gram of moist soil was shaken for 15 min in 10 or 100 ml SDW with beads and further IO-fold dilutions were made in 0.01 M Tris-HCl buffer (pH 7.2). For counts of fungal propagules 1 ml per dilution (lo- ’ to 10V5) was inoculated into duplicate pour plates. For bacterial counts 0.1 ml volumes of appropriate dilutions were spread on the surface of dried agar media. To assess the numbers of bacterial spores, dilution tubes were heated in an 80°C water bath for 10 min, cooled quickly in tap water and 0 * 1 ml from each was spread on a plate of M32. For fungi, plates were counted at 3 and 7 days; bacterial counts were made after 2 days at 3O”C, with a recount of YPS plates at 6-7 days. In microbial investigation of roots 2 or 3 lateral roots (with very little adhering soil), to a total length of 3-4 cm, were removed from each seedling, shaken in 10 ml SDW with beads, and subsequent dilutions and plate inoculations made as for soil samples. Assessment of mycorrhizal infection

From each plot 5 plants at random were sampled. The intensity of infection was expressed as the percentage of short lateral roots which had become mycorrhizal (“percentage mycorrhiza”) as indicated by dichotomous root forking or the presence of a fungal mantle. RESULTS

Fungi

Numerical data from dilution plates on fungal recolo~zation at the two locations are included in Table 2. At first, MB practically eli~nated all fungi at Mount Gambier. At 35 days, when dazomet-treated plots were first sampled (after allowing 2-3 weeks for effective fumigation from the granules) plots treated by both. fumigants showed about 1000 propagules/g. At the final sampling, nearly 7 months after treatment, the number of fungal propagules in the treated soils was still only about 3 per cent (- 1.5 log,,) of those in the untreated plots. At Kersbrook MB initially reduced fungal numbers to 440/g or to almost O- 1 per cent of that of untreated soil and recolonization was more rapid than at Mount Gambier. By 145 days the fungi detected in the fumigated soil rose to over 60 per cent of the count in untreated soil. At Kersbrook MB decreased fungal numbers at 22 cm depth but not as markedly as in the O-10 cm region. Fungi isolated more than once on any one occasion at Mount Gambier and Kersbrook respectively are named in Tables 3 and 4. The lists do not include Basidiomycetes, which

E. H. RIDGE

298

OF FUMIGATION

TABLE 2. THE EFFECT

AND C. THEODOROU

ON NUMBERS

OF FUNGAL. PROPAGULES

Fungal propagules counted on Czapek-Dox medium + antibiotics Days after fumigant applied

C*

2 35 68 107 135 201

5.83t 5.70 5.92 5.84 ND 5.77

4 11

5.50 ND ND 5.32 5.48 5.54 5.52 5.56

MB

AND

BACTERIAL SPORESIN SOIL

Bacteria (spores) surviving 80°C 10 min, counted on M32 medium

D

C

MB

D


ND 2.84 4.24 4.10 ND 4.18

5.74 5.75 5.77 5.78 5.81 5.69

4.75 4.65 5.12 5.17 4.95 4.58

ND 5.85 6.12 5.98 5.86 5.73

2.64 ND ND 4.46 3.57 4.77 4.41 5.39

** -

5.77 5.71 5.62 5.64 5.58 5.59 5.60 5.75

5.46 5.34 5.20 5.28 5.11 5.50 5.60 5.22

** -

Mount Gambier

Kersbrook

(22 cm depth)

z8 34 54 92 145

* C-untreated control. MB-methyl bromide treatment. D-Dazomet (Basamid) treatment. t Log,, mean counts/g dry soil taken from O-10 cm depth in 4 replicate plots. ND-Not determined. **-Not treated.

TABLE 3. THE MORE ABUNDANT FUNGI IN MOUNT GAMBIER NURSERYSOIL BEFOREAND AFTER FUMIGATION

Fungus

Untreated soil 0

AspergilIus Fusarium Penicillium Stemphylium Verticillium

Sterile, dark Gliocladium Humicola Ulocladium Acrostalagmus Mucor Trichoderma Cephalosporium

* Detected. ** Moderately abundant. *** Plentiful. - Not detected.

*** *** *** ** ** ** * ** ** ** ** **

Time of isolation (days after fumigant applied) Methyl bromide Dazomet 2

35

68

107

201

_ _ _

*

*

-

-

* ** * * * .-

* * ** *

-

** ** ** * * _

-

**

-

-

35

68

*

_

_

* *

**

**

*

_ _

* ** *

**

-

-

-

-

* *

*

* -

_ *

_

107

201

*

* * *

** *

*

**

*

MICROBIAL TABLE

4. TIIE MORE

Fungus

RECOLONIZATION

ABUNDANT

Untreated soil 0

Aspergillus Cladosporium Fusarium Mucor Peniciflium Rhizopus S~emphylium Trichoder~ V~rlici~~~urn

Sterile, dark

** * ** * *** ** ** t* +*

*

Monilia

-

Sterile, grey, fluffy

-

FUNGI

AFTER

IN KERSBRWK FU*MIGATION

4

SOIL FUMIGATION NURSERY

SOIL BEFORE

AFI-ER

145

* ** t ** + * -

* * ** *** ** * ** -

** * -

-

**

-

**

-

-

-

-

;a

AND

Time of isolation (days after MB applied) 34 54 92 ** ** ** ** * * *

-

299

**

*

* * -

* *** ** * * -

* **

*

*

* Detected. ** Moderately abundant. *** Plentiful. - Not detected.

were not isolated but which were known to be present since mycelium with clamp connections was observed microscopically on the roots of plants growing in both fumigated and non-fumigated plots. Also, infection by myco~hi~l Basidiomycetes occurred on all seedlings. With this limitation of the fungal isolation methods (Warcup, 1960) borne in mind, these lists show that a variety of fungi recolonized the fumigated soils. The earliest genera to colonize the Mount Gambier soil were Penicillium, Fusarium, Aspergillus and Stemphylium, while Trichoderma, Penicillium, and a grey, sterile fungus (with raised fluffy colony) were the first to recolonize at Kersbrook. In both soils some genera present in the untreated soil were eliminated by sterilization and did not reappear, while other genera not detected in the untreated soil were found after fumigation. In general, the fungi which colonized the roots of pine seedlings in the untreated and fumigated soils were those found in the corresponding soils themselves. However, in MB-fumigated plots at Mount Gambier, Ha~pospo~ium was found on the roots while at Kersbrook roots from MB-treated soil bore PapuZaspo~a, although neither genus was detected in the corresponding soil. Mycorrhizal infection occurred on al1 seedlings in all treatments. At Mount Gambier infection was greatest on the seedlings inoculated with spores of Rhizopogon luteolus and grown in plots fumigated with MB. The reverse was true at Kersbrook (Table 5). The inoculated fungus, R. luteolus, forms a multi-branched mycorrhiza having a definite white fungal mantle. The presence of this type of mycorrhiza may be an indication of the success of inoculation, although other fungi and R. luteolus already in the soil may form the same type. Table 5 shows that inoculation was successful only in MB-fumigated soil at Mount Gambier. In dazomet-treated soils mycorrhizal infection was similar for both inoculated and uninoculated seed. The plots fumigated by dazomet had to be resown after most of the seedlings of the first sowing had been killed by residual toxic effects of the fumigant. Hence results from the two sowings (control and fumigated) were not strictly comparable.

300

E. H. RIDGE AND

C. THEODOROU

TABLE 5. MYCORRHIZALINFECTION ON SEEDLINGROOTSGROWNIN MB-FUMIGATEDAND IN UNTREATED SOIL Treatment

Mount Gambier

Kersbrook

White mycorrhizat (%)

Mycorrhiza* (%)

Mycorrhiza* (%)

White mycorrhizat (%)

Fumigated soil: Uninoculated seed Inoculated seed

48 84

15 96

60 54

44 28

Untreated soil: Uninoculated seed Inoculated seed

64 59

8 21

74 71

29 11

* Results are from examination of 20 plants per treatment. t Percentage of mycorrhizal roots bearing white fungal mantle. TABLE 6. NUMBERSOF VI~LE BACTJXRIA AND FUNGI ON ROOTSEGMENTS OF PINE SEEDLINGS FROMFUMIGATED AND UNTREATED SOILS Days after fumigant applied Mt. Gambier Kersbrook Organisms counted and medium used CT 3.87$ Fungi on Czapek-Doxfantibiotics 5.60 Aerobic bacteria on YPS <2-o Fluorescent pseudomonads on NPCC 3.60 Bacterial spores (after 80°C for 10 min) on M32

135 days (loo)* MB

D

3.50 7.15 3.0 3.26

3.53 5.72 -2-O 3.48

92 days (72) C MB 4.72 6.67 5.23 3.49

4.05 6.26 3.20 3.40

145 days (125) C MB 4.46 6.32 -3.7 2.95

4.18 5.23 <2.5 2.90

* In brackets-age of seedlings in days. t C-from untreated soil. MB-from soil treated with methyl bromide. D-from soil treated with dazomet. $ Log,, mean count/cm root from 2 replicates, each containing 34 cm lateral roots.

Included in Table 6 are data on the numbers of fungal propagules obtained per cm root. Numbers were consistently greater on roots grown in untreated soil than on those from fumigated plots at both sites. Bacteria

Included in Table 2 are counts of bacterial spores (i.e. surviving 80°C for 10 min) at both sites made on M32, on which medium total counts had been found comparable with YPS counts. There were differences in the severity of the effect of MB at both sites, and also differences at Mount Gambier between the effects of dazomet and of MB. Fumigation by the latter at Kersbrook produced an immed.iate drop of 50 per cent in spores germinating, which thereafter did not fall below 30 per cent of control numbers. At Mount Gambier there was a 90 per cent reduction (-0.99 log,,) in initial numbers after MB treatment, with a range of 7-25 per cent of control numbers over the sampling period. Dazomet treatment resufted in no reduction in viable spores and may even have given a slight increase over the controls at up to 107 days. The effect of fumigation with dazomet and MB at Mount Gambier on total populations of general aerobic soil bacteria (as counted on YPS medium) and of fluorescent pseudo-

MICROBIAL

RECOLONIZATION

AFTER

SOIL FUMIGATION

301

monads (counted on NPCC) is illustrated in Fig. 1. This shows that, immediately after removal of the plastic covers at 2 days, the total viable numbers were reduced by MB to 5 per cent and fluorescent pseudomonad numbers to 0.2 per cent of control samples. But counts of fluorescent pseudomonads in MB-treated soil increased some lOO,OOO-fold by 35 days to a total (2-O x 107/g) which was 700 times larger than for the untreated soil, while, at that same sampling, the total count was 10 times higher for treated than for untreated soil. After similar differences in both counts at 68 and 107 days, the numbers in treated samples declined to approach those in the untreated soil.

Counts

on YPS

medium

Counts on NPCC (fluorescent

(total)

C = Untreated

medium pseudomonads)

D = Dazomet ND=

2

68

35 Days

After

Fumigant

Applied

soil

MB = Methyl bromide No1 detected

107 ( Day 0 = 22

135 July

treatment

treatment (
201

1

FIG. 1. Mount

Gambier. Comparison of total bacterial counts (on YPS) and fluorescent pseudomonad counts (on NPCC) of untreated and fumigated soils, O-10 cm depth.

Dazomet exerted a more persistent early effect, with greatly lowered fluorescent pseudomonad counts up to 68 days, probably due to the residue of fumigant granules. At 107 days the total count for treated soil (4.4 x lO’/g) was some 20-fold higher than for the untreated, and, while the pseudomonad count at 2.5 x 104/g was 30 times higher than for the untreated soil it indicated that fluorescent pseudomonads did not then constitute a significant fraction of the increased total aerobic population. At the final sampling (201 days) on 8 February, i.e. in mid-summer, all fluorescent pseudomonad counts had been reduced in the dry conditions to less than 1000/g in treated and untreated soils, but the total counts from the dazomet and MB treatments were still higher than for the untreated. The response to MB fumigation at Kersbrook was similar to that found at Mount Gambier (Fig. 2). Again the 4-day samples, taken just after fumigation, showed a drop in total aerobic bacterial numbers to 12 per cent of those of the control soil, while the fluorescent pseudomonad count fell to less than 0.4 per cent of that of the control. But, even one week later, the total count of MB-treated samples was 3 times higher than for the control, while the pseudomonad count of the treated soil rose by about lO,OOO-fold over that period to a total of 8.7 x 106/g, or some 60 times higher than the control. Further increases in both total and pseudomonad counts were recorded at 20 days, when samples from 22 cm depth gave the same trend but with lower numbers of bacteria. Numbers thereafter steadily declined, with NPCC (fluorescent pseudomonad) counts from treated soil paralleling, but

302

E. H. RIDGE

Counts % u1

Counts

? 0

on YPS on NPCC

AND C. THEODOROU

medium

(total)

C = Untreated MS=

medium

(fluorescent pseudomonads)

soil

Methyl bromide treatment

ND = Not

detected (-=lOs/gt

? * z 5 I!! 8.00 B 3B 600 .z c.

400

:: E 0

2.00

8 _)

4

34

20

54

Days After Fumigunt Applied

92

145

(Day 0 = 20 August)

FIG. 2. Kersbrook. Comparison of total bacterial counts (on YPS) and fluorescent pseudomonad counts (on NPCC) of untreated and fumigated soils, O-10 cm depth.

still l~l~times higher than, falling control counts for pseudomonads in the spring-surer dry period (sampling at 92 days was on 20 November). Figure 3 highlights the changes in the ratio of counts on NPCC medium to counts on YPS medium (i.e. fluorescent pseudomonads to “total” aerobic population). Expressed as percentages, this ratio for control soils at Mount Cambier fell from 5 to O-04 per cent at 107 days and then remained fairly steady. The ratio fell even lower (to
Untreated

Days FIG.

3.

From

Application

Of

soils

l ----*

Kersbrook

A---A

Mt. Gambier

A----A

Kersbrook

Fumigant

Changes with time in ratios of counts of fluorescent pseudomonads (on NPCC medium) to total counts of aerobic bacteria (on YPS), expressed as percentages.

MICROBIAL

RECOLONIZATION

AFTER

SOIL FUMIGATION

303

sampled at 20 days, it might have shown a maximum nearer to that found for Kersbrook. The ratio then declined for MB-treated soils at both sites and was reduced to 0.1 per cent or less at the final samplings in summer. Table 6 presents a summary of the bacterial and fungal counts made from root samples. At Mount Gambier the trend was for total bacterial and fluorescent pseudomonad numbers to be lower on roots from the control than from the treated plots (especially for MB treatmerit)-or the same general effect as found in the soils alone. However, the two samplings at Kersbrook gave generally higher numbers counted in both media for roots from control soil-paralleling fungal counts at both sites. The counts of bacterial spores from roots in treated soils were generally slightly lower than those of control roots. DISCUSSION

Although the fungal lists given are not complete, they indicate the range of fungi which could be expected to recolonize fumigated soils. At both sites some fungal genera which were not detected in untreated soil colonized the fumigated plots. These, probably occurring in low numbers in untreated soil, could have remained undetected in the presence of more numerous fast-growing fungi on the plates; or fumigation could have created better conditions for their growth by reduction or removal of their bacterial or fungal antagonists. Trichodermu viride, which is often a common early colonizer of fumigated soil (Warcup, 1957) was not found in Mount Gambier soil after treatment. The effect of dazomet was generally not as rapid nor as potent as that of MB. Being a granular product, dazomet apparently persists so as to restrain recolonization and could also offer some hazard if treated areas are sown too soon after application, especially when soil temperatures are low. The rapid increases in fluorescent pseudomonad numbers immediately after MB fumigation indicate that they arose from survivors rather than from populations introduced after fumigation. Mostly they are poor competitors with other bacteria (Ridge, unpublished) but readily proliferate on fragments of organic matter (Rovira and Sands, 1971). Hence, in the presence of killed bacteria, fungi and other organisms, conditions were especially favourable for their multiplication. It is possible that the increase in the number of fluorescent pseudomonads in a treated soil, relative to their number in untreated soil at, say, 10-20 days after fumigation, could be an index of the effectiveness of fumigation. Reference to Fig. 3, in conjunction with Figs 1 and 2, indicates that by about 90 days the fluorescent pseudomonads, which had formed a high proportion (at some stages a majority) of total countable bacterial numbers in MB-treated soils for at least 35 days, had fallen to be an inconsequential fraction. After 3.5 days some other classes of bacteria must have proliferated so that, even during the dry summer period, “total” counts from treated soils remained substantially above those for the untreated. Possibly there were present or introduced other competitors which could multiply and survive more readily in conditions which had greatly reduced the pseudomonad number. To determine the genera concerned in this later increase requires the use of other selective media. From our experience, it is unlikely that Bacillus contributed significantly since counts of Bacillus-like colonies on YPS medium were seldom greater than the spore numbers counted on M32. Some recent work (Ridge and Rovira, unpublished) indicates that the effects on bacterial populations of the chloropicrin component alone equal those of a 50 : 50 MB-chloropicrin mixture. Hence some of the changes we found could have been due to the relatively smafl but significant (45 kg/ha) level of chloropicrin added with our MB.

304

E. H. RIDGE

AND C. THEODOROU

On pine roots from fumigated soil at Kersbrook the numbers of fluorescent pseudomonads in the rhizospheres were as few as 1 per cent of the counts on roots from control soii, whereas their numbers in the treated soil alone had been at least 10 times that of controt soil. Hence, these organisms were not as effective in colonizing roots as at Mount Gambier, where their numbers on the roots more nearly reflected the soil situation. Fumigation may of itself result in improved plant growth [Wilhelm, 1966). Warcup (1957) and Martin (1963) have indicated some explanations for this improvement but Ingestad and Molin (1960) and Ingestad and Nilsson (1964), after work with spruce and pine, found that increased growth could not all be ascribed to the release of nutrients, nor to elimination of pathogens, although both may occur. They suggested that the growth increases resulted from new populations of micro-organisms colonizing the fumigated soil. These studies showed that bacterial and fungal recoIoni~tion of fumigated soil and subsequent colonization of pine roots in them were quite rapid and that the reintroduced microflora may be effective competitors with inoculated mycorrhizal fungi. Thus, at Kersbrook in MB-treated soil, mycorrhizal infection was due to native mycorrhizal fungi surviving or recolonizing in the fumigated soil rather than to inoculation, whereas at Mount Gambier the apparently more effective soil treatment with MB, in conjunction with seed inoculation, greatly increased the percentage of typical “white mycorrhizae” compared with inoculation alone. However, in areas where naturally-occurring mycorrhizal fungi are few (Bowen, 1963), the inoculated fungus may not be faced with this competition from naturally-occurring mycorrhizal fungi (Theodorou, 1971), though it might still suffer from the presence of other microbial competitors which have recolonized the treated soil {Brian, Hemming and McGowan, 1945; Levisohn, 1957). The success of inoculation after fumigation therefore depends on the types of microbes recolonizing the soil and establishing themselves on the roots. Acknowledgements-We thank officers of the Research Section, South Australian Woods and Forests Department, for provision and care of trial plots, Mr A. R. P. CLARKEfor soil analyses and Mrs N. WILLOUGHBY for competent technical assistance. Dr J. H. WARCUP has provided helpful criticism of this paper. One of us (EHR) was supported by the Australian Wheat Industry Research Council; some financial assistance was also made available from a fund provided by a number of Australian forestry organizations.

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CSIRO Division of Soils, Adelaide, Australia, Divisionai Report 6/63 12 pp. BRIANP. W., HEMMINGH. G. and MCGOWANJ. C. (1945) Origin of a toxicity of mycorrhiza in Wareham Heath. Nature, Land. 155, 637-638. BUNT J. S. and ROVIRAA. D. (1955) Microbiological studies of some sub-antarctic soils. J. Soil Sci. 6,119-128. GILMAN3. C. (1957) A Manual of Soil Fungi. Iowa State College Press, Ames, Iowa. HARLEYJ. L. and WAID J. S. (1955) A method of studying active mycelia on living roots and other surfaces in the soil. Trans. &it. mycol. Sot. 38, 104-l 18. INGESTADT. and MOLIN N. (1960) Soil disinfection and nutrient status of spruce seedlings. Physiol. Plant. 13,90-103. INGESTAD T. and NILSSONH. (1964) The effects of soil fumigation, sucrose application and inoculation of sugar fungi on the growth of forest tree seedlings. PI. Soil 20, 74-84. LEVISO~YI. (1957) Antagonistic effects of Alternaria tenuis on certain root-fungi of forest trees. Nature, Lo&. 179, 1143-1144. MARTIN J. P. (1963) Influence of pesticide residues on soil microbiological and chemical properties. Res. Reu. 4,96-129.

MICROBIAL

RECOLONIZATION

AFTER

SOIL FUMIGATION

305

RIDGE E. H. and ROVIRAA. D. (1971) Phosphatase activity of intact young wheat roots under sterile and non-sterile conditions. New &t&gist 70, 1017-1026. ROC?RAA. D. and SANDSD. C. (1971) Fluorescent pseudomonads-a residual component in the soil microflora. J. appl. Bact. 34, 253-259. SANDSD. C. and Rovm~ A. D. (1970) Isolation of fluorescent pseudomonads with a selective medium. Appl. Microbial. 20, 513-514. THEODOROU C. (1967) Inoculation with pure cultures of mycorrhizal fungi of radiata pine growing in partially sterilized soil. Amt. For. 31, 303-309. THEODOROUC. (1971) Introduction of mycorrhizal fungi into soil by spore inoculation of seed. Amt. For. 35,23-26.

WAKSMANS. A. (1927) Principles of Soil Microbiology. Williams and Wilkins, Baltimore. WARCUP J. H. (1950) The soil-plate method for isolation of fungi from soil. Nature, Lord. 166, 117. WARCUP J. H. (1955) Isolation of fungi from hyphae present in soil. Nature, Lord. 175, 953. WARCUP J. H. (1957) Chemical and biological aspects of soil sterilization. Soils Fertil. 20, 1-5. WARCUP J. H. (1960) Methods for isolation and estimation of activity of fungi in soil. In The Ecology of Soil Fungi, (D. Parkinson and J. S. Waid, Eds) pp. 3-21. Liverpool University Press. WILHELMS. (1966) Chemical treatments and inoculum potential of soil. Ann. Rev. Phytopath. 4, 53-78.