Colonization of wheat roots by Gaeumannomyces graminis inhibited by specific soils, microorganisms and ammonium-nitrogen

00)%0717/7830603 -0175xlz.09/r1

COLONIZATION OF WHEAT ROOTS BY ~~~U~~~~~~YC~~ GRAMlNlS INHIBITED BY SPECIFIC SOILS, MICROORGANISMS AND AMMONIUM-NITROGEN RICHARD W. SMILEY Department of PIant Pathology, CorneIl University, Ithaca, N.Y. 14853, U.S.A. (Accepted 28 Nouembm

1977)

Summary-Lineal extension of Gaeumannomyces graminis var. tritici hyphae along roots of intact wheat plants growing in soils was measured. Hyphal growth rates were lower in soils treated with NHf-N than with NO;-N. In a soil that is suppressive to the take-all disease, the controlling influence of NH:-N was eliminated by soil fumigation fmethyi bromide), and reintroduced to fumigated soil by additions of 1% nonsterife soil. Effects of fumigation on hyphal growth were absent in a nonsuppr~sive soil, and in NO;-treatments of the suppressive soil. When inocuia of selected groups of wheat rhizo-

plane microflora were reintroduced into a fumigated or a soil-reinoculated soil via a root-food base, the Pseudomonas spp. consistently appeared more suppressive in NH; -N treatments than the general bacterial flora, Bacillus spp. spores. streptomycetes, JNTRODUCFION

Take-all of wheat (Triticum aestivum L.), caused by Gaeumannomyces graminis &cc. v. Arx & Ofivier var. tritici J. Walker, formerly referred to as Ophiobolus gruminis Sacc., has been controlled in the field and in the glasshouse with fertilizers which supply N to the roots, largely but not entirely, as NH:-N (Huber et af., 1968; Huber and Watson, 1972; Smiley and Cook 1973). NOT-N is generally ineffective for suppression of take-all, Studies of this disease control based upon manipulation of N-forms supplied to wheat (through use of nitrogen stabilizers) indicates that suppression of disease may be linked to changes in the composition of rhizoplane and rhizosphere microfloras (Smiley and Cook, 1973; Smiley, 1978). Smiley (1978) showed that apparent populations of bacteria and streptomycetes were similar in rhizoplanes of wheat grown in NH:- vs NO;-N treated soils, but that shifts did ticur in the proportions of Pseudomonas and Streptomyces species that were antagonistic in vitro toward C. gr~~njs. Extensive inve~igation has been made of microbial antagonists of G. graminis. These reports have been reviewed (Baker and Cook, 1974; Butler, 1961; Garrett, 1970; Gerlagh, 1968). Previous investigations have implicated various fungi, streptomyces, and bacteria as being important specific antagonists of G. graminis in take-all suppressive soils. Since the take-all decline, and the disease differences due to plant uptake of Nra- vs NO;-N appear to be mediated through microbial interactions with the pathogen in the rhizoplane, it appeared profitable to pursue additional work on antagonistic microorganisms. Much of the early work was based solely upon in vitro presumptive tests, or upon cultural observations in which few if any mjcroorganisms were isolated for study. Trends of recent studies have been toward selective techniques which inhibit some components of the soil microflora more so than others. Gerlagh (1968) and Shipton et al. (1975) have, for instance, shown that the responsible component of the microflora is eliminated by soil treatments with aerated steam at

and fungi.

4@-6O”C,and by methyl bromide fumigation. Sporogenous bacteria and fungi, and most streptomycet~, survive treatment of 60°C (Broadbent ef al., 1971; Smiley, unpublished), and may thus be thought not to be active in the decline phenomenon. Asporogenous bacteria and fungi are among those organisms killed at 60°C. Much of the earlier work suggested a strong role for fungi as the dominant antagonists, however, recent evidence (Baker and Cook, 1974; Cook and Rovira, 1976; Pope and Jackson, 1973; Zogg and Jaggi, 1974) suggests that asporogenous bacteria are likely to be most important groups. The predominance of Gram-negative, asporogenous short rods on the rhizopfane (Gyllenberg, 1955; Holding, 1960; Rovira, 1956), and the restricted activity of most actinomycetes and Basilic spp. in the rhizosphere (Clark, 1939; Gyllenberg, 1955) and in acid environments (Clark, 1939), give additional emphasis to more detailed studies of the asporogenous rhizoplane microflora. Evidence for in vitro antagonism of G. graminis by ~s~d~rno~ spp. (Smiley, 1978), and especially among isolates from a take-all suppressive soil. have led to the in vivo study reported here. MATERIALS AND METHODS Soils and fertilizers

The soils and their treatments were described (Smiley. 1974 and 1978). Briefly, the alkaline Wimmera grey clay from the Longerenong Agricultural College near Horsham, Victoria has been recropped to wheat since 1916, and is thought to be in an advanced state of the take-all decline phenomenon (Shipton et al., 1975). The acidic Rosedale sandy clay loam from the South Australian Department of Agriculture’s Turretfield Research Centre, Rosedale, S.A., has had a variable cropping history. The soils were passed through a 3mm sieve and then burned lime was added to Rosedale soil at the rate of 2.2 g. kg-‘, which was shown in previous work to increase the soil pH to about 6.5 (sat’d paste id lOmM CaCl,). Each soil was moistened to a water

176

RICHARD

content approximating - 1 bar suction (32 and 17% for the Wimmera and Rosedale soils) and stirred frequently for 1 week. One portion of each soil was fumigated for 3 days (l-4 March) with methyl bromide (450 kg.ha- ‘), left open in a stream of nearly sterile flowing air for 3 days, and then reinoculated (1% w/w) on 7 March with nonfumigated portions of the same soil. Frequent stirring and aseptic additions of water as needed were then made during the 2 weeks following reinoculation with soil. A second soil portion was fumigated for 3 days (9-12 March) and left uncovered an additonal 3 days to coincide with the end of the 2-week incubation for reinoculated soil. The soils were then treated with fertilizers which consisted of blending dry commercial fertilizer grade crystals of (NH&jO, and prills of Ca(NO& into soil, at the rate of 1OOmg N kg- 1 soil. The nitrification inhibitor N-Serve 24 was applied to both N sources at the rate of 2% (w/w-based on N) before their application to soil. Inoculum

Microbial inocula were added to soils on 18 March, one day after their treatment with fertilizers. Inoculum was prepared as follows. Halberd wheat seeds were surface disinfected in a chlorox solution and then plated on seed germination agar medium. After seed germination, the plates were inspected, contaminated cultures were rejected, and the sterile seeds were transferred to moist sterile sand in Petri plates: 5 seeds/plate. The inoculum was prepared by suspending in one tube of sterile water per microbial group, 20 randomly selected colonies of Pseudomonas spp. (10 fluorescent and 10 nonfluorescent isolates). Bacillus spp., streptomycetes. and the general bacterial flora (pseudomonads, bacilli, and streptomycetes had been segregated out of this group). All isolates had been collected (Smiley, 1978) from rhizoplanes of NH: -treated Wimmera soil. Twenty morphologically dissimilar fungi were also selected (on 0.3% Tryptic Soy Agar medium plus 0.5% erythromycin) from a collection of 73 rhizoplane isolates and of 58 isolates from macerated roots which had been washed five times by shaking with glass beads in 10m~ Tris buffer. Fifteen representative rhizoplane and five root isolates were retained for the study. Dr Peter Merriman (Victorian Plant Research Institute, Burnley. Vict.) has kindly identified 13 of these fungi as Aspergillus spp.-4. Harposporium spp.- 1, Fusariurn spp.-2, F. avenaceum-2, F. oxysporum-2. Mortierella sp.-1, and a phycomycete-1. Other fungi died in culture before being identified. Bacterial suspensions were adjusted to about lO*cells ml-’ and dispensed over sterile wheat roots at 2ml/plate. Fungal infested agar medium was suspended in a blender and dispensed over the wheat roots at about 3ml/plate. Two days after adding the microbes to wheat roots growing in sterile sand, the wheat seeds and coleoptile were severed from roots and removed to prevent further growth. Roots from each culture plate were inspected microscopically to ensure colonization by the inocula, or absence of apparent contamination in noninoculated controls. The microbial-root food base preparation was then separated from most of the sand, macerated, and one quarter of the roots/plate were blended into appro-

W.

SMILEY

priate soil treatments. Subsequent tests for in vitro antagonisitic ability of these inocula toward G. graminis, as described previously (Smiley, 1978), revealed that the numbers of antagonists in each group of twenty were pseudomonads- 12 (fluorescent-5 and nonfluorescent-7) general microflora-1. streptomycetes-1. bacilli spores-O. and fungi-O. Potting. incubation and samphg

Four days after adding the microbial inocula to fumigated soil or to fumigated soil reinfested with natural soil, the soils were placed in 0.6 1 plastic pots; 650 g/pot with five replicates per treatment. Six small holes about 1Scm deep were made in the surface soil of each pot, and a 5 mm dia. agar plug infested with G. graminis isolate 8a-2i (Smiley, 1978) was placed at the bottom. A surface disinfected wheat seed was placed over each agar plug, the soil was smoothed, and a 1 cm layer of dry Perlite was spread over the top to inhibit evaporative water loss. The growing conditions were: 12 h day length, 1860 lux at plant height, 20°C days and 15°C nights, soil water suction about - 1 bar. Plants emerged in 3 days (26 March) and were harvested 21 days after emergence. Sampling consisted of gently removing the soil from roots and measuring under a microscope (40x) the maximum distance to which darkened ectotrophic hyphae of G. graminis extended down the seminal root axes (Holden. 1976; Smiley and Cook, 1973). Three roots/plant were inspected for each of 30 plants per treatment. Vascular discoloration of roots near the inoculum plug appeared directly proportional to the growth of runner-hyphae but the extent of discoloration was not measured. The plant shoots had no visual symptoms of disease then they were removed from soil after 21 days. Numbers of Bacillus spp. spores, Pseudomonas spp., streptomycetes. and the general microflora in the rhizosphere were estimated (Smiley, 1978) at the conclusion of the experiment, and the percentages of antagonists were determined. Enumeration of microorganisms in soils that were not fertilized nor planted to wheat, but otherwise treated the same. were also made throughout the study. RESULTS

Gaeumannomyces-colonization of roots

Hyphal growth rates for G. graminis on roots of intact soil-grown wheat plants were strongly influenced by the form of N used as a fertilizer and, in the Wimmera soil, by soil fumigation (Table 1). The growth rate was always less in NH,-treated soils, as compared to the NO;-N treatments. This response was most evident in the nonfumigated Wimmera soil, and in the fumigated treatments where small amounts of nonfumigated soil were reintroduced. Hyphal growth rates were significantly increased in the NH;treated Wimmera soil as a result of fumigation, but the additional growth capability was almost completely eliminated upon reintroduction of native soil inoculum. Fumigation and fumigation plus reinoculation with soil did not influence hyphal growth rates in NH:-treated Rosedale soil, or in either of the soils when NO;-N was utilized. A G. graminis suppressive entity that is active in rhizoplanes of NH:-treated

Colonization

by Ga~mannamyces

Table 1. Growth of G~eumunnomyces gr~~nis hyphae along wheat roots growing in soils that were fumigate

(methyl bromide), fumigated and reinoculated with 1% nonfumigated soil, or left nonfumigated and then treated with Ca(N0J2 or (NH&SO4 f N-Serve 24 (1OOmg N.kg-’ Soil preparation Nonfumigated Fumigated Fumigated + reinoculated

soil)

Rosedale soil NOT-N NH;-N

Wimmera soil NO;-N NH;-N

13.2 b” 17.5a

7.4 c l.Oc

16.7 ab 15.6 ab

14.5 ab

5.4 c

i8.6a

3.7c 13.9 b 6.6 c

’ Hyphal extension (mm/23 days) down seminal root axis from G. gr~jnjs colonized PDA plug placed in contact with wheat seeds at time of planting. Means followed by the same letter are not significantly different at the 99% confidence level of Duncan’s Multiple Range Test.

Wimmera soil, but not the Rosedale soil, was apparently eliminated by fumigation. Additions to soil of food bases (young wheat roots) colonized by selected microbial groups also influenced the hyphal growth rate of G. gruminis on wheat roots grown in fumigated soils, and those which were fumigated and reinoculated with l”i, nonfumigated soil (Table 2). Again, the hyphal growth rate was greatest in NO;-treated soils. In fumigate Wimmera soil. the hyphal growth rat& was significantly reduced only by Pseudomonas spp. in NHftreatments, and by none of the microbial inocula in NO;-treatments. Perhaps of equal importance is the observation that growth was increased significantly by Pseudomonas spp., the general bacterial flora, and by fungi in NO;-treatments, and only by the fungi in NHf-treatments. In fumigated-reinoculated Wimmera soil, all except the fungi and Bacillus spp. decreased hyphal growth in NO;-treatments. and Pseudomonas spp. was the only group that did not significantly increase the growth rate in NH~-treatments. Relationships between the hyphal growth rates of

177

G. ~r~fnis (Table 2) and the numbers of microorganisms in the rhizosphere Fable 3) 21 days after plant emergence were evaluated by linear regression analysis. Although none of the correlations were statistically significant, several trends were apparent, and three such relationships closely approached the 95% level of significance. Two strong trends were that the growth of G. graminis was negatively related to pseudomonad numbers and positively related to numbers of the total bacteria. The strongest relationship (r = - 0.593) occurred between the fungus’ growth rate and the pseudomonad numbers in Wimmera soil. There was almost no relationship between the growth of G. gruminis and numbers of streptomycetes and bacilli spores in either soil. not with pseudomonads in Rosedale soil. Tests for in vitro antagonistic ability of randomly-picked representatives of the rhizosphere microflora revealed that the numbers of antagonists and numbers of tests conducted for each group were pseudomonadsout of 169, general microfloraout of 158, streptomycetes4 out of 72, and bacilli spores-l out of 170. Measurements of G. graminis growth in fumigated and soil- or microbe-amended soils, relationships between the growth rates and numbers of specific groups of microorganisms in the rhizosphere, and comparative in vitro antagonistic abilities of the microorganisms each suggest that Psuedo~n~ spp. were more closely associated than the other microbial groups with the growth of G. graminis in Wimmera soil. For fumigated and fumigated-reinoculated Rosedale soils, significant reductions in hyphal growth rates were absent in the presence of microbial inocula where NH:-N was supplied. On the other hand, reductions in growth were given by all groups except the general bacterial flora and Bacillus spp. in NO;treated, fumigated Rosedale soil, and by fungi, Bacillus spp. and the general bacterial flora in the fumigated-reinoculated portion. The lack of influence by microbial inocula on growth of G. gruminis in NHf-treated Rosedale soil coincides with a lack of fumigation or reinoculation effect, as compared to

Table 2. Influence of soil fumigation (methyl bromide), reinoculation with 1% nonfumigated soil, and inoculation of soil with specific groups of microorganisms on the hyphal growth of Gaeumannomyces graminis along wheat roots in two soils treated with Ca(NO& or (NH&SO, + N-Serve 24 (1OOmg N.kg-’ soil) Microbial inoculum” Control Pseudomonas spp. Baciflus spp.

Other bacteria Streptomycetes Fungi Bact. + strept. All of above

Rosedale soil Fumigated Reinoculated NO;-N NH:-N NO;-N NH;-N 17.5b !0.6* 16.4 29.7* 10.2* i1.1* 19.9 11.6*

7.0 8.0 5.8 7.9 5.1 6.8 7.8 9.8

14.5 9.7+ 14.2 12.6 8.2’ 17.0 lO.o+ 14.1

5.4 3.9 4.2 9.3* 3.4 5.0 5.9 4.5

Wimmera soil Fumigated Reinoculated NO;-N NH:-N NOT-N NH:-N 15.6 24.3% 18.6 20.6* 18.5 32.6* 31.4* 20.5*

13.9 8.7* 10.6 12.4 9.8 30.3* 15.3 16.4

18.6 14.8’ 15.5 14.7* 12.9’ 21.5* 9.0* 11.7*

6.6 1.5 14.7* 8.5* 12.4* 11.4* 8.8* 6.9

“Inoculum consisted of coarsely chopped wheat roots colonized by 20 mixed rhitoplane isolates of each microbial group. “‘Other bacteria” included a random selection of wheat rhizoplane isolates from which Bacillus, Pseudomonas, and streptomycetes were removed. b Hyphal extension (mm/23 days) down seminal root axis from G. graminis colonized PDA plug placed in contact with wheat seeds at time of planting. Growth is significantly different (99% level of confidence) from the noninoculated control when so indicated by an asterisk (*). Paired means do not differ when underlined.

RICHARD

178

W.

SMILEY

nonfumigated soil. Pseudomonas and Streptomyces spp. were implicated as having influential roles in reducing hyphal growth rates in NO;-treated Rosedale soil. When significant effects of individual microbial groups on pathogen growth were considered without respect to soil type or fumigant treatment, decreases occurred in most instances where pseudomonads (4/5) and streptomycetes (3/4) were introduced, and increases occurred in most instances where fungi (4/S) and the general bacterial flora (4/5) were added. Bacillus spp. had little influence (except in one event). Of the eight instances where growth was affected by the individual groups in NHf-treated soils, the growth was decreased in only one instance and that was by additions of Pseudomonas spp. Growth was significantly altered in 19 instances in NO;-treated soils. and these events were shifted toward more decreases (12 instances) than increases (7 instances). Pseudomonads and streptomycetes suppressed pathogen growth in the presence of NO;-N. except in the fumigated Wimmera soil where an increase was noted for the pseudomonads, and there was no significant effect of streptomycetes.

to persist in the soil after being introduced on the wheat-root food base. Estimates of total bacterial populations remained relatively stable in the nonfumigated soils, but increased with time to very high levels in fumigated soils. This also occurred in the fumigated soils reinoculated with soil or the general bacterial flora. Final populations in the Rosedale soil were higher where the bacterial flora was selectively reintroduced than where native soil was used as an inoculum source, and the reverse was true in the Wimmera soil. Populations of Pseudomonas spp. remained fairly stable in nonfumigated soil, and did not become excessive during recolonization of fumigated soil. The populations did, however, reach very high numbers in both soils where they were selectively reintroduced. and where soil reinoculum was added to Rosedale soil. Streptomyces spp. and Bacillus spp. spores were more effectively reestablished in fumigated soil by introduction of native soil than by the wheat root food base. Numbers of these genera were not rapidly reestablished in fumigated soil.

Microbial populations in soil

Interactions of the soil microflora in the inhibition of G. graminis hyphal growth by NH:-N. but not NOT-N, is strongly suggested by these in oiro gfasshouse studies. The influence of different N-forms was

DISCUSSJON

Populations of bacteria and streptomycetes were estimated (Table 3) to evaluate their short term ability

Table 3. Soil and wheat-rhizosphere populations of bacteria and streptomycetes from soils which were not fumigated (NF). fumigated with methyl bromide (F), fumigated and reinoculated with cultures of the microbial group indicated on 18 March (F + M), or fumigated and reinoculated with 1% nonfumigated soil on 7 March (F + R) Wimmera soil

Rosedale soil F

Sample location

NF

Date 5 March 12 March 23 March 4 April 23 April 23 April

bulk soil bulk soil bulk soil bulk soil NO3 -rhizosphere NH, -rhizosphere

5.8 13.4 19.6 16.0 16.7 46.2

2.1 223.0 4.5 4.2

5 March 12 March 23 March 4 April 23 April 23 April

bulk soil bulk soil bulk soil bulk soil NOa -rhizosphere NH,-rhizosphere

0.3 1.3 1.6 0.5 0.1 0.3

0 0 0 0.7 0.4 0.4

5 March 12 March 23 March 4 April 23 April 23 April

bulk soil bulk soil bulk soil bulk soil NO,-rhizosphere NH,-rhizosphere

1.3 2.0 7.1 7.4 1.6 1.7

0 0 nd. 34.0 1.3 0.4

5 March 12 March 23 March 4 April 23 April 23 April

bulk soil bulk soil bulk soil bulk soil NOa -rhizosphere NH,-rhizosphere

2.4 3.4 5.3 6.9 0 0

0 0 0.1 2.0 0.5 0.7

n.d. = not determined.

F+M 0

0

F F+R NF Total bacteria/g. soil ( x 106)

n.d. n.d. 32.5 455.3 3.4 4.9

n.d. 224.0 186.9 208.9 0.6 0.7

n.d. 76.0 110.2 428.0 0.4 1.1

soil (x 105) 0.5 0 0.1 0 0 0.5 0 I.2 0.1 0.8 0.6 0.5

n.d. nd. 53.1 98.0 0.6 7.5

n.d. 0.7 2.5 2.5 0.2 1.2

Streptomycetes/g. soil (X 105) nd. n.d. 7.1 0.3 n.d. 146.0 21.3 0 n.d. 348.0 53.1 n.d. 101.0 150.5 53.3 76.0 0.7 11.0 0.1 4.7 2.7 2.4 0.2 14.0

n.d. n.d. n.d. 8.8 0.4 0.2

n.d. 263.0 376.0 178.0 1.2 2.6

Bacillus spp. spores/g. soil (x 105) n.d. 2.9 0.1 n.d. 60.8 1.3 0.1 0.1 31.1 3.8 0.1 5.0 33.4 I.2 0.5 0.1 0.6 0 0.1 0.5 0.6 0 0.2

nd. n.d. 0.3 1.2 0.1 0.1

n.d. 61.2 95.8 35.5 0.3 1.7

n.d.

0 0 46.0 275.2 19.8 4.5

F+R

n.d. n.d. 46.3 248.5 6.0 3.6

Pseudomonads/g. nd. n.d. n.d. 308.0 247.6 375.0 427.5 319.0 1.6 0.2 4.8 0.4

11.9 6.0 13.3 13.5 5.8 9.6

F+M

Colonization

by

greatly modified by fumigation of the take-all suppressive Wimmera soil, but not of the Rosedale soil. Reintroduction of native soil inoculum reversed the fumigation effect in NH:-treated Wimmera soil, thus supporting previous observations in take-all suppressive soils (Cook and Rovira, 1976; Gerlagh. 1968; Pope and Jackson, 1973; Shipton et al., 1975). The NO;-N system in Wimmera soil was more nearly like the nonsuppressive soils such as Rosedale. In the latter, suppression of hyphal growth by NH:-N could be attributed to excess acidity acting directIy to inhibit parasitic growth (Smiley, 1974; Smiley and Cook. 1973). Smiley and Cook (1973) have suggested that growth rates of G. grounds hyphae on wheat roots are more affected by the rhizosphere pH than by soil properties or the form of N absorbed by roots. This hypothesis was later questioned (Smiley, 1978) because growth rates varied in NHf- vs NO;-N treated Wimmera soil, but reductions in rhizosphere pN in the presence of NH:-N were not measured. It appears possible in the latter study that the rhizoplane microflora responded to pH changes at the root surface in Wimmera soil, but that these changes are not measurable in the rhizosphere due to the very highly buffered nature of this soil. Of the microbial groups studied in virro (Smiley, 1978) and in ~iv5.the pseudomonads have been more consistently ~plicat~ as possible specific antagonists of G. graminis than other microbial groups. These groups were selected as composites of twenty isolates to avoid as much as possible the limitations of variations in potential antagonistic abilities among isolates. Although streptomycetes are very effective producers of antibiotics, they are not thought to be highly competitive in the rhizosphere, and their in vitro and in vivo antagonistic ability was greater in NO;- than in NH:-treated soils. Isolates of the general asporogenous microflora (minus pseudomonads) and the sporogenous Bacillus spp. have consistently failed to appear as stong suppressants of parasitic growth by G. graminis. Results presented here, therefore, support the hypothesis of Cook and Rovira (1976) that Ps~udamonas spp. are important specific antagonists of this pathogen of wheat. The pseudomonads as a group have only recently been studied for antagonistic properties toward the take-all fungus (Ridge, 1976), possibly because (1) a high proportion of isolates which are antagonistic upon fresh isolation from soil lose this activity after a few weeks of storage as agar cultures, and (2) a medium has only recently been devised which permits large numbers of these bacteria to be easily and selectively isolated from root surfaces and soils (Simon er al.. 1973). The pseudomonads. even in low total numbers, are strategically located on the root surface and given the correct environmental conditions, have the physiologic potential of being effective inhibitors of the parasitic colonization of roots by G. graminis. These studies suggest that suppression of take-all through manipulation of the nitrogen fertility in the wheat root zone is caused at least in part by the antagonism of G. graminis by Pseudomonas SPP. Acknowledgements-The author wishes to thank the CSIRO Division of Soils, Adelaide, South Australia for

179

Gaeumannomyces

hosting the NATO Post Doctoral Fellowship under which this work was completed in 1973. Special appreciation is given for expert advice, technical assistance, or manuscript review by A. D. Rovira, J. K. Martin, E. H. Ridge. G. D. Bowen. A. Simon and MS J. Price. REFERENCES BAKERK. F. and CCKIKR. J. (1974) Biological Control of Plant Parhogens. Freeman, San Francisco. BROA~RENTP.. BAKERK. F. and WATERWORTH Y. (1971) Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Ausr. J. hial. Sci. 24, 925-944. BUTLERF. C. (1961) Root and Foot Rot Diseases of Wheat. N.S.W. Department of Agricultural Science Bulletin No. 77. CLARK F. E. (1939) Effects of soil amendments upon the bacterial populations associated with roots of wheat. Trans.

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SMILEYR. W. (1978) Antagonists of Gaeumannomyces graminis from the rhizoplane of wheat in soils fertilized with ammonium- or nitrate-nitrogen. Soil Biol. Biochem. This issue p. 169. SHILEYR. W. and COOKR. J. (1973) Relationship between take-all of wheat and rhizosphere pH in soils fertilized with ammonium- vs nitrate-nitrogen. Fhytopathology 63, 882-890. ZOGG H. AND

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