Studies on soil fumigation—II

solI hoI. Bdwn.

Vol. 8. pp. 249 to 253 Pergamon Press 1976.Printed m Great Britiin

STUDIES

ON SOIL FUMIGATION-II EFFECTS

ON BACTERIA

E. H. RIDGE CSIRO,

Division

of Soils, Glen Osmond. (Acceptetl

South

Australia

5064

I Decrinher1975)

Summary-In a South Australian wheat-field soil the viable counts of “total” aerobic bacteria and of Ruorescent pseudomonads were initially greatly depressed by fumigation with 220 kg’ha- I chloropicrin (CP) or with a combined application of 220 kg’ha-’ of chloropicrin and 220 kg’ha-’ methyl bromide (F). Bacterial numbers rose sharply within IO days of the completion of fumigation. For a further 14 days the fluorescent pseudomonads formed the major part of the aerobic bacterial population counted and over 5 months later their numbers in F-treated soil remained about IO times higher than in untreated soil. Numbers of aerobic spore-formers rose more slowly after CP or F treatment. but then remained significantly higher over the 159 days of the trial. Fumigation with 220 kg’ha- ’ of methyl bromide alone (MB) had little effect on soil bacterial numbers, A check of random isolates revealed a predominance of Gram-negative organisms in soil treated with CP, this dominance decreasing with time, whereas MB treatment did not result in any detectable

change. Fluorescent pseudomonads from rhizospheres of wheat plants in soil fumigated with CP contained smaller nronortions of strains antagonistic in t’itro to Gaeumannomyces graminis var. tritici than isolates from Mb-treated soil or from untreated soil.

INTRODUCTION

Australia, and, unless otherwise indicated, were taken from areas covered for 4 days after fumigation. The Most microbiological studies on fumigated soils have fumigants were chloropicrin at 220 kg. ha- ’ (CP220). been concerned with the elimination of or re-invamethyl bromide at 220 kg’ha- ’ (MB220) and comsion by fungi, especially pathogens. While there have bined chloropicrin-methyl bromide, each at 55 or 220 been many investigations into the fate of bacterial kg. ha- ’ (Fl 10 or F440). Pooled soil samples (tX10 populations after fumigation, these have often dealt cm) were taken from each of 2 or 3 replicate plots with bacteria in total (Martin, 1963) although some for control and F440 treatments. In the MB220. workers, e.g. Reber (1967a. b), have followed actinoCP220 and Fl IO treatments duplicate pooled samples mycetes, nitrifying and denitrifying bacteria and were taken from separate areas of the single plots spore-formers. involved. Improved plant growth which occurs after fumigaFor bacterial counts, 1 g from each pooled sample tion, even in the absence of pathogens (Wilhelm, was shaken for 10 min in 20 ml sterile distilled water. 1966) has been attributed in some degree to stimulaSubsequent dilutions were made in Tris-HCl buffer tory action by pseudomonads (Altman and Tsue. (0.01 M, pH 7.2). The surface of pre-dried agar 1965) or by non-pathogenic microorganisms generally medium was inoculated by a syringe and calibrated (Altman, 1970). The development of a medium selecneedle with one drop (20 ~1) per dilution. spread over tive for fluorescent psuedomonads (Simon et al., 1973) one-half of each 10 cm dish. Between samples the encouraged an investigation of these organisms in a syringe assembly was rinsed several times with boiling fumigated wheatfield soil. especially after Ridge and water and cooled. Agar cultures were incubated at Theodorou (1972) found that such pseudomonads 30°C and first counted after 2 days; final counts formed the major bacterial component of the soil of were made after a further 4-5 days at room temperature a pine forest nursery after fumigation with high con(20-25°C). (Early counts were found necessary centrations of methyl bromide (2400 kg. ha- ‘). because the occasional presence of spreading The present study also covered bacterial comorganisms could make a later count uncertain.) ponents of the rhizosphere in fumigated soil and inThe agar media used were (a) M32 (Ridge and vestigated potential antagonism towards Gueur~~~~~o- Rovira, 1971) a non-selective medium, for counts of tnycrs gr-uminis (Sacc.) von Arx and Olivier var. tritici “total” aerobic bacteria and (b) NPCC (Simon t’t
30

E. H. RIDGE

Dishes were incubated at 25°C. The zones of bacterial inhibition of the fungus were those measured at the third reading. taken when the outermost growth of fungus had reached the edge of the Petri dish (about 12 days). The few instances where there was no zone of inhibition of growth, but the fungus had been lysed in the region occupied by a bacterial colony, also been included as evidence of antagonism.

In general, the soil bacterial population was little affected by methyl bromide applied at 220 kg. ha-’ (MB220) whereas chloropicrin at the same rate (CP220) or combined with methyl bromide, both at 230 kg. ha- 1 (F440) greatly altered the numbers and composition of the bacterial flora (Fig. la). After a pronounced drop of 1.3 log,,, (or about OY’,,). in “total” numbers of aerobic bacteria during the 4-day fumigation period under covers the population of plots treated with CP (alone or with MB) rose rapidly to become about 10 times that of control plots bq 10 days later. These numbers gradually decreased over the next 4 months, but even in mid-summer (235 days after fumigant application, and not shown in the figure) the soil treated with F440 contained nearly 3 times as many countable bacteria as the untreated soil. Chloropicrin (alone or combined with MB) produced a more marked effect on numbers of fluorescent pseudomonads counted on the selective NPCC medium (Fig. 1b). The initial depression was about

II 4

I I4

I

I

28

OAYs CTER

APPLATION

Fig. 1.

I

,

117

159

of FUMIGANT

Log,, number g-’ solI

have

RESULTS

0

Table I. Effects of fumigant treatments and covering on numbers of fluorescent pseudomonads (O--l0 cm depth) counted 14 days after application

tluorcscent pseudomonads counted on NPCC medium Uncovered Covered*

Treatment Nil Methyl bromide, 220 kg’ ha(MB220) Chloropicrin-methyl bromide each at 55 kg. ha(FllO) Chloropicrin, 220 kg. ha- ’ (CP220) Chloropicrin-methyl bromide each at 220 kg. ha-

’

5.97 5.69

6.08 6.48

6.72

7.3x

7.60

8.00

7.80

8.05

0.46

0.46

1

’

FW L.S.D. (p = 0.05)

* Plots covered by plastic sheeting, 4 days after fumigant applied.

sealed at edges

for

1.5 log,, (or 97’x) but by IO days later the numbers had risen over l,OOO-fold, so that fluorescent pseudomonads then constituted over 80% of the countable total aerobic population, compared with 10% or less in the control soil. In CP-treated soil the numbers of these pseudomonads fell by about 90%) (I log,,) in the next 32 days, followed by a slower decline. However, samplings in summer at 203 and 235 days after fumigation (again not included in the figure) showed that the numbers of fluorescent pseudomonads in soil treated with F440 were still over 10 times those in untreated soil. When MB alone was used the numbers of fluorescent pseudomonads (Fig. 1b) rose after an initial depression to about the control level then became significantly less after 28 days. In its effect on counts of fluorescent psuedomonads (and of “total” aerobes-not shown in the graphs) FllO was intermediate between the control and the CP220 or F440 treatments (Fig. 1b). By day 4 there had already been some recovery from the initial kill of the pseudomonads by Fl IO and their numbers rose to a peak 20 times higher than in control soil, then declined to remain not significantly different from the control soil values from 46 to 159 days. The CP220 and F440 treatments were again rather similar in their effect on aerobic bacterial spores (Fig. lc). The initial reduction (0.5 log,, or 66%) in spore numbers in these two treatments was comparatively small, but again MB220 had no noticeable effect. Spore numbers in CP- (or F440-) treated soil reached their peak more slowly than the fluorescent psuedomonads in the same treatments. The eventual level of bacterial spores counted in CP treatments was 2.5-10 times the number in untreated or MB-treated soils. In most of the samplings for aerobic spores, (Fig. lc) as well as for “total” bacteria (Fig. la) the effects of CP and of F440 could not be statistically separated, but were significantly different (p = 0.05) from the MB and the nil treatments, which again were generally not distinguishable from each other.

Fumigants Table 2. Short-term Days after fumigant applied

effects of chloropicrinmethyl

Treatment

and soil bacteria-II

bromide

“Total” aerobic 224 cm 12-14 cm 22-24’cm

4

Control Fumigated-U/C$ Fumigated-C

6.65* 6.61 5.86

14

Control Fumigated-U/C Fumigated-C

6.92 7.79 1.79

6.28 5.69 5.73 LSD 6.04 7.03 6.70 LSD

5.81 5.82 5.59 0.43 ND ND ND 0.75

mixture

251

(F440) on numbers

of soil bacteria

at three depths

Fluorescent pseudomonads Aerobic spore-formers 24 cm 12-14 cm 22-24 cm 2-4 cm 12-14 cm 22-24 5.43 5.75 3.34

4.28 <3.00 <3.00 LSD 6.09 3.93 7.68 6.98 1.52 7.06 LSD

3.12 <3.00 <3.00 0.82 ND ND ND I .65

6.25 6.01 5.86

5.98 5.68 5.79

5.50 5.15 5.31 0.29 ND ND ND 0.61

LSD 6.36 6.07 6.43

cm

5.81 5.2t 5.32 LSD

* Means of log,,- transformed counts. 8 U/C-Plots left uncovered after fumigant applied. C-Plots covered with plastic sheeting for 4 days after fumigant application, LSD-Least significant differences (p = 0.05) for comparison of values within each cell of the table.

Effects of covering

and of different

rates of fumigation

The general effect of lower fumigant dosage or lack of covering during fumigation may be gauged by comparing the numbers reached by fluorescent pseu-

domonads 14 days after the field soil had been fumigated (Table 1). There was no significant effect from the addition of MB to CP whether the treatments were covered or not. In fact, there was no significant difference between covered and uncovered treatments when CP was used, but covering the MB treatment resulted in a significant increase in numbers at 14 days. However after 28 days from application of the fumigant no difference was found between covered and uncovered MB-treated areas. When samples were taken at different depths it was found that in untreated soil the decline in “total” numbers from near the surface (24 cm) to the 22-24 cm depth was about 70% (Table 2). For fluorescent pseudomonads this change was nearer 99%, and for spores about 60% (Table 2). When fumigated (F440) soil was left uncovered for the 4-day treatment there was either a smaller kill or faster recovery near the surface so that numbers of all three classes of bacteria were not significantly different from the corresponding counts in untreated 24 cm soil. At 12-14 cm, the depth of injection of the fumigants, the “total” counts showed approximately the same kill (- 70%) in covered or uncovered Table 3. Gram-reaction

Treatment* NIL MB220 FllO CP220 F440

of random

Soil (S) or Rhizosphere (R)

areas at 4 days whereas at the 22 cm depth the initial kill for all three classes of bacteria was minimal, irrespective of covering. These results, taken in conjunction with the 60% reduction in “total” numbers in the covered treatment at 24 cm depth, point to a quick, early kill at and above the depth of injection as a consequence of the volatility of the fumigants. Whether there was a later bacterial kill at depths below the injection site cannot be stated, as only one series of samples was taken from 22-24 cm depth. Sampling at the 2 and 12 cm depths 10 days later showed that “total” and fluorescent pseudomonad numbers had increased in fumigated soil (covered and uncovered) at the upper level to 7-27 times those in control soil but much more sharply at the 12 cm depth where fluorescent pseudomonads, not detectable on the 4th day (at the removal of covers), had increased by 14 days to about lO’g_’ or about 1,COO times higher than in untreated soil at the same depth. At the 12 cm depth there was no effect due to covering during fumigation, the soil above this depth apparently having delayed the dispersion of the fumigant. Changes in staining

bacterial isolates from untreated and fumigated sampled 69 days after fumigant application No. Examined? 56 57 63 54 69 66 49

No. Gram-positive Large rods Total 19 6 27 35 12 7 II

characteristics

of soil bacteria

Gram-staining of 50-70 colonies randomly selected from “total” count plates (M32) for soil sampled at 69 days showed that CP220 and F440 treatments

10

29 5 9 23 21 20

the sum

15 38 9 15 49 46 30

27 (40)$ 67 (ND) 14 (46) 28 (65) 71 (92) 70 (93) 61 (ND)

of Gram+ ve and

in the same

treatments

rhizopheres,

Percentage of isolates which were Gram-ve

No. Gram-negative Small rods Total

40 16 47 39 19 20 19

* For type and rate of fumigation see Materials and Methods. t Discrepancies between the number of isolates examined and due to Gram-variable isolates which are excluded from table. $ In brackets, percentages of Gram-negative organisms found application.

soil and corresponding

Gram-ve

at 28 days

isolates after

are

fumigant

E. H. RIUGE

252 Table

4. Occurrence

of antagonism in vitro to G. yruminis among random rhizosphere 188 days after fumigant applied to corresponding soil

No.

Soil treatment*

tested

Fluorescent No. inhibitory

(a)

pseudomonads ex NPCC Lysis Antagonists (a + b)

“Total”

isolates

bacteria?

only (b)

as ‘>~A of those tested

Per cent antagonists

from

roots

Spore formersl

Per cent antagonists

Control

41

26

2

6X

‘0

7

MB440 Fl IO CP220 F440

35 35 15 24

30 20 1 4

0 3 4 3

86 66 33 29

21 26 4 8

0 0 0 0

* For type and rate of fumigation see Materials and Methods. ,’ Number of Isolates whose colonies were surrounded by zone of inhibition h Number of isolates causing lysis of fungus without a zone of inhibition. t “Total” bacteria isolated from non-selective M32 agar. $ Aerobic spore-forming bacteria which survived 8O”C,‘IO min.

markedly increased the percentage of Gram-negative organisms from 2704 (untreated soil) to some 70% (Table 3). Between 28 and 69 days in the F440-treated soil “total” numbers declined from 8.08 to about 7.5 log, 0 while countable fluorescent pseudomonads fell from 7.76 to about 7.0 log,, (Fig. la, b). Hence these psuedomonads, as a percentage of the “total”, fell from 50 to 30%. Other Gram--negative organisms which had reinvaded the fumigated soil therefore constituted by 69 days about 40% of the countable “total”. These other Gram-negative bacteria were not identified in this study. The use of MB resulted in little change in the Gram-staining character of isolates compared with control soil. In untreated soil. rhizosphere isolates showed a reversal of Gram-stain characters compared with isolates from the bulk soil.

Tests of antagonism in vitro on random isolates of the three classes of organism from the rhizospheres of wheat plants from the field showed that a much higher proportion of the fluorescent psuedomonads tested were antagonistic to G. grumirzis than of the “total” population or of the aerobic spore formers (Table 4). These results also showed that the proportion of in vitro antagonists among the fluorescent pseudomonads was reduced by fumigation with the higher concentration of CP (CP220 or F440). The high incidence (68:;) of antagonists in the rhizospheres of plants in untreated soil was paralleled by the proportions (X6’:/,, 66Y;, respectively) found in rhizospheres from soil treated with MB or with combined MB-CP in the low rate treatment, FIIO. But the inclusion of CP220 in the soil fumigant decreased the occurrence of antagonistic fluorescent pseudomonads in the rhizospheres by half to 33% and 29”/,. Furthermore. the pseudomonad isolates from CP220 or F440 treatments showed a smaller average zone of fungal inhibition. The proportion of antagonists from rhizosphere isolates, plated on M32 as “total” bacteria or as spore-formers, also decreased with the higher rates of CP fumigation. from 2@26”,, to 4% and 8x:,. These effects were found for plant roots taken 188 days after fumigation commenced, so that higher rates of CP fumigation may have quite persistent effects on the

of fungal

growth

sampled

after

12 days at 25’C.

characteristics of a class of soil bacteria such as the fluorescent pseudomonads. It is important to note that, because of the much higher total numbers of pseudomonads after CP fumigation. there were, in absolute terms, many more of these antagonistic bacteria present in the fumigated soil than in untreated areas. DISCUSSION

Because fluorescent pseudomonads are primarily associated with soil organic matter (Rovira and Sands, 1971) they are more likely to be protected from fumigant effects by “organic shielding” (Kreutzer, 1965) than some other sections of the soil population, even though the initial kill is quite drastic (up to 97% in this study). The greatly increased supply of available organic matter resulting from the death of practically all the biomass provides the fluorescent pseudomonads, arising from survivors or from reinfestation from less affected areas, with a substrate which they can more rapidly utilize than can other survivors (including the spore-formers) or casual contaminants. Again, I have noted that fluorescent pseudomonads, though generally only a small part of the countable population of normal soil, multiply and spread rapidly in soil where few other organisms exist. Hence, by their combined abilities to survive, multiply and spread in the presence of plentiful substrate and in the absence of nearly all microbial competition, these organisms become a dominant, or at least a significant. factor in well-fumigated soil, as already noted qualitatively by Martin (1963). The other class of survivors expected. the aerobic spore-formers, showed a slower rise in numbers after fumigation and reached a comparatively lower peak. Nevertheless, the increase in their numbers after CP220 or F440 fumigation persisted from some 6 months, although these counts are subject to two important limitations. Firstly. an unchanged number over a period may indicate a steady turnover of organisms, with the regular production of fresh spores, or may merely reflect a fairly constant number of dormant spores surviving from the earlier flush due to fumigation. Secondly, heating at 80°C during counting procedures to remove non-sporing organisms and vegetative cells probably activates germination of

253

Fumigants and soil bacteria-- II some spores which would otherwise not form colonies on the count medium (Evans and Curran, 1943). Thus there were occasions, especiaily in fumigated soil, when the number of sDores counted after heating was higher than the app&ent “total” number of liable aerobic bacteria, not subjected to heating but counted on the same non-selective M32 medium. The elimination of whole classes of bacteria. such as the nitrifiers, by fumigation has been frequently reported (Martin, 1963). Changes in the composition of individual classes of surviving bacteria have been observed less often. In a short-term experiment Reber (1967a) found that the composition of the actinomycete population, measured by pigmentation, changed after fungicidal doses to soil of three different fumigants. In the present study the increased fluorescent pseudomonad population which arose after treatment with at least 220 kg. ha- * of chloropicrin as one fumigant contained a reduced proportion of organisms antagonistic irl ~:itro to G. graminis (Table 4). an indication of a selective action within species by certain strengths and types of fumigant, operating directly on the bacteria or indirectly through changes in competition or available substrate in the soil environment. The magnitude of the bacterial changes caused by fumigation seems to be related to the severity of the treatment. Support for this view, quoted by Martin (1963) as “The greater the initial kill. the greater the subsequent peak in numbers,” is given by the generally parallel, but less marked, effects of the low rate (Fl 10) treatment compared with the high (F440) both of which contained chloropicrin (Fig. 1). Furthermore, the effects of chloropicrin on the rise in pseudomonad numbers have been paralleled by greatly increased doses of methyl bromide (Ridge and Theodorou. 1972). In contrast to short-term laboratory investigations such as conducted by Reber (1967a) the work here reported applies to farming soil treated under field conditions. This longer term study and work in subsequent seasons (unpublished) show that marked increases in numbers of fluorescent pseudomonads and of spore-formers are a common feature of South Australian field soils treated with effective rates of fumigant (here CP220 and F440) in seasons of average rainfall.

Acknowledgrnlerlts---I

am indebted to A. Simon and Ms. technical assistance, to A. D. Rovira for helpful consultation. and to R. L. Correll (CSIRO Division of mathematics and Statistics) for statistical analyses.

J. Price for competent

REFERENCES

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and decreased plant growth responses resulting from soil fumigation. In Rout Diseases nnd ~~j~-~~~~~~ Put~~og~r~,~ (T. A. Tousson. R. V. Bega and P. E. Nelson, Eds.). pp. 216-221, University of California Press, Berkeley. U.S.A. ALTMAX J. and TSUE K. M. (1965) Changes in plant growth with chemicals used as soil fumigants. PI. Dis. Reptr. 49. 600-602. EVANS F. R. and C~IRRAN H. R. (1943) The accelerating effect of sublethal heat on spore ~ermin~ttion in mesophilit aerobic bacteria. f. Baci. 46. 513-523. KREUTZER W. A. (1965) The reinfestation of treated soil. In Ecology ofSoiLBorne Planr Pathog~~s (K. F. Baker and W. C. Snyder, Eds.), pp. 495-508, llniversity of California Press, Berkeley, U.S.A. MARTIN J. P. (1963) Influence of pesticide residues on soil microbiological and chemical properties. Rrsi&e Rrr. 4. 9h- 129. REI~ERH. (1967a) Vcrglcichende Ll~~ters~~ci~t~ngenzur Toxizitit und Sclectivitit van Entseuchungsmittein fiir Bodenmikroorganismen. 2. qpKrrx/lkh. FyPath. I_ltlSchutz. 14. 414-426. R~B~R H. (1967b) Ilntersuchungen iiber die Wiederbesiedlung eines chemisch entseuchten Bodens. Z. PfiPCrnrlkk. ~~~f~f~l. ~~se~~~(r~ 74. 427-438. RIDGE E. H. and R~VJRA A. D. (1971) Phosphatase activity of intact young wheat roots under sterile and non-sterile conditions. New Phytol. 70. 1017-1026. RIDGE E. H. and THEDUOROU C. (1972) The effect of soil fumigation on microbial recolonization and mycorrhizal infection. Soil Biol. Bioclwm. 4. 295--3(X. ROVIRA A. D. (1976) Studies on soil fumigation-I. Effects of ammonium. nitrate and phosphate in soil and on the growth. nutrition and yield of wheat. Sail Bid. B~~~~~I~~J~?. 8. 241- 247. ROVIRA A. D. and SANDS D. C. (1971) Fluorescent pseudomonads-a residual component in the soil microflora’! J. oppl. Bact. 34, 253-259. SIMW A.. ROVIRA A. D. and SANX D. C. (1973) An improved selective medium for isolating fluorescent pseudomonads. f. uppi. Bnct. 36. 141-145. WILHELM S. (1966) Chemical treatments and inoculum potential of soil. An/z. Rcr. Phytopc~tlt. 4. 53-78.