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Wheat-rhizoplane pseudomonads as antagonists of Gaeumannomyces graminis
WHEAT-RHIZOPLANE PSEUDOMONADS AS ANTAGONISTS GAEUMANNOMYCES GRAMINIS
OF
RICHARD W. SMILEY Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Accepted 10 March 1979)
Summary-The role of rh~opiane-inhabiting P~e~~~rn~~~sspp as inhibitors of take-ah on wheat was investigated. Apparent numbers of p~udomonads in wheat rhizoplanes and numbers that were antagonistic in oitra toward Gaeu~nnumyces gram~n~~ var. tririci did not differ when wheat was supplied with NH:-N or NO;-N. More intense antagonism was expressed by colonies selected from soil treated with NH:-N than with NOT-N, and from isolation media prepared at pH 5.5 rather than at 7.0. Antagonists were not recovered from methyl bromide-treated soil. Highly antagonistic pseudomonads were recovered from a wheat-monoculture soil which is considered suppressive toward the pathogen in the field, and were not recovered from a “nonsuppressive” soil. Pseudomonad antagonism ratings were inversely correlated with take-all severity in the suppressive soil, but not in the nonsuppressive soil. Pseudomonads were considered to be antagonists of G. graminis on rhizoplanes of wheat in a soil exhibiting the “take-all decline” uhenomenon, but the significance of this interaction remains to be determin&l.
INTRODUCFION F~udomonads have been proposed to be spe~i~~Iy inhibitory to the ~tablishment of ectotrophic hyphal networks by Gue~~nnomyces gram&is Sacc. v. Arx & Ohvier var. tririci J. Walker (Cook and Rovira, 1976; Smiley, 1978b), the causal agent of take-all of wheat (Triricum aestivum L.). However, not all pseudomonads are antagonists toward G. graminis; the highest proportions of antagonistic vs nonantagonistic pseudomonads occur on rhizoplanes of infected roots growing in soils which have supported Iongterm wheat monoculture. In one such soil it was found (Smiley, 1978a,b) that take-all was significantly less severe following treatment with NH:-N fertilizer than with NO;-N. The apparent pseudomonad population on the rhizoplanes of infected wheat did not differ in the NH:- vs. NO;-N ~eatment~ but significantly greater proportions of isolates from the NH:-N treatments, compared to NO;-treatments, were antagonistic in vitro toward G. graminis. The magnitude of in vivo antagonism by pseudomonads reintroduced onto rhizoplanes of intact wheat was also significantly greater in the NH:-N than in the NO;-N treatments. These results suggest that the differential absorption of predominantly NHf- or NO;-N may have caused a redistribution to occur among the dominant species or biotypes within the pseudomonad population on wheat rhizoplanes. Alternatively, if the composition of pseudomonads was not changed, some members may have been altered in antagonistic capability by changes in rhizosphere pH (Smiley, 1974), in the quality or quantity of root exudates (M. E. Brown, personal communication), or in other rhizoplane properties. Although it is difficult to separate the individual effects of pH and other environmental properties, enumeration and culture of Pseudomonas species on acidic and neutral media could provide information concerning the relative importance of acidity to the Pseudomonas-Gaeumannomyces interaction 371
in NHf- vs NO;-treated soils. This is especially important in view of the intolerance of G. graminis to acidic environments (Smiley and Cook, 1973), and to the management of wheat affected by take-all. I report the numbers and species or biotypes of pseudomonads isolated at pH 5.5 and at 7.0 from rhizoplanes of wheat grown for 3 wk in soils treated with NH:-N, NO;-N, methyl bromide, with or without an inoculum of G. graminis. The in vitro antagonism of G. graminis by Pseudomonas spp. was also measured.
MATERIALSAND METHODS Soils and fertilizers Details of the soils and treatments used have been described elsewhere (Smiley, 1974, 1978a). Soils included Wimmera grey clay and Rosedale sandy clay loam. The 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 (Cook and Rovira, 1976; Shipton, 1972). The Rosedale sandy clay loam from the South Australian Department of Agriculture’s Turretfield Research Centre, Rosedafe, S.A., has supported a variable cropping history, including cereals, clover pasture and fair low. The saturated paste pH values (using 1OmM CaQ) are 8.0 and 5.5 for the Wimmera and Rosedale soils respectively (Smiley, 1978a). Fertilizer treatments consisted of blending dry commercial fertilizer-grade crystals of (NH&SO.+ and prills of Ca(NO,), into soil at the rate of 0.10 g N kg-’ soil. The nitrification inhibitor N-Serve 24 [2-chloro-&(trichloromethyl)pyridine] was applied to both N sources at the rate of 2% (w/w-based on N) prior to their application to soil. Where indicated, a portion of each soil was fumigated with methyl bromide (450 kg ha-‘) 2 weeks before adding fertilizers.
Populations P~u~ff~na.s spp (x IO3 cells/mg air dry root) Pseudomonas spp (% of total bacteria) Fluorescent pseudomonads (“/, of PseiIdo~o~as spp) P. putida; all sets and biotypes3 (% of fluor. pseudo.) P. jluorescens; all sets and biotypes (% of fluor. pseudo.) P. jhorescens; set 30, biotype G (% of P, juorescms) P. jhorescens; other sets & biotypes4 (7; of P. fluorescens)
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
isolation medium PH 168b’ 16.5b 1.47ab 1.28b 31b 46ab 37ab 31ab 63a 63ab lOOa 83a Ob 17b
NO: 14.9b 14.8b 0.89b l.lOb 27b 33b 25b 50a 75a 5Ob 86a 89a 14b lib
NH,
Wimmera
1.7c 2.oc 0.03c 0.03c Oc oc ND ND ND ND ND ND ND ND
Fum f NH, 16.3b 12.lb 1.04b 1.89ab 70a 77a 5c 13c 95a 87a Ob 15b lO&% 85a
NO,
Inoculated soils
15.6b 17.lb 0.83b 1.34b 80a 70a 21b 5c 79a 95a 1OOa 87a Ob 13b
NH,
Rosedale
0.3c O.lc O.Olc O.Olc Oc oc ND ND ND ND ND ND ND ND
Fum + NH,
32.4ab 35.lab 4.51a 4.29a 32b 36b 26b 32ab 14a 68ab ND ND ND ND
NO,
59.8a 62.la 4.11a 3.93a 2lb 28b 19bc 38ab 81a 62ab ND ND ND ND
NH&
3.lc 3.6~ 1.04b 1.13b 60a 64a 17bc Ilc 83a 89a ND ND ND ND
NO,
1.7c 2.3~ 1.09b 0.92b 61a 59a 8c 22b 92a 78a ND ND ND ND
NH&
Noninoculated soils Wimmera Rosedale
Table 1. Pseud~~onus spp isolated at pH 5.5 or 7.0 from rhizoplanes of wheat growing in soils treated with NH.+- or NO,-N, methyl bromide + NH&-N, or inocu~um uf Gaeumannompces graminis; and their in vitro antagonism toward C. graminis
F 9
F
r
6
E
p~udomonads
other sets & biotypes
P. juorescens,
set 30, biotype G
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
7.0 5.5 7.0
5.5
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
3.8a 1.4ab 3.8a 0.2b Oa Oa 5.2a 0.2b 5.2a 0.3b ND Ob
67a 46ab 75a 18b Ob Ob 1OOa 25b lOOa 2lb ND oc 4.0a 2.2a 4.4a O.lb 0.3a Oa 6.la Ob 7.la Ob 4.8a Ob
55a 65a 15a lob 30a 13b 1OOa Oc 83a Ob 80a oc Ob Ob ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND O.lb Ob O.lb Ob Oa Oa O.lb Ob ND Ob 0.2b Ob
lob oc Ob Ob 1OC oc ND Ob 33b OC
6c 4c
Ob Ob Ob Ob Oa Oa O.lb Ob O.lb Ob ND Ob
llb 3b ND 2c
SC
8b 5bc IOb Ob llc
DC
O.lb Ob ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND
oc
0.4b 0.2b Ob Ob ND ND ND ND ND ND ND ND
2c 4c oc Oc ND ND ND ND ND ND ND ND
ND ND
ND
4.2a 1.6ab 3.9a 0.4b ND ND ND ND ND
34b 30b 31ab 27ab ND ND ND ND ND ND ND ND DC
Ob Ob Ob Ob ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
oc oc
OC
Ob Ob Ob Ob ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
oc oc oc oc
r Ca(NO& or (NHJ2S04, both with N-Serve 24, were applied at the rate of O.lOg N kg-’ soil. ’ Averages of 4 replicates. Averages within paired rows of isolation pH 5.5 and 7.0 that are followed by the same letter are not significantly different at the 0.05 level of Duncan’s Multiple Range Test. 3 gets 33, 3.5, and 57 for Wimmera-NH,; sets 33, 59, and 62 for Wimmera-NOJ; sets 57, 60, 62 and 64 for Rosedale-NH,; and sets 47, 49 and 64 for Rosedale-NO,. 4 Set 17 for Wimmera-Nap; set 13 for Wimmera-NON; set 13 for Rosedale-NH,; and sets 13, 29 and 32 for Rosedale-NO,. ’ Antagonism Rating = decimal percentage of antagonists x zone of inhibition distance in mm. ND = no determination.
P. fiuorescens, other sets & biotypes
P. fluorescens,
P. jhorescens
P. putida
Fluorescent p~udomonads
Antagonism Rating5 Nonfluorescent pseudomonads
set 30, biotype G
P. fluorescens,
P. jluorescens
P. putida
Fluorescent pseudomonads
Antagonists (70 Nan~uores~nt
374
RICHARD V?. SMILEY
Inaculum, incubation, and sampling
Smiley (1978a) described techniques used in these investigations. Inoculum of G. graminis-colonized crushed oats was blended into the soil at a rate of 0.05% (w/w), or the soil remained noninoculated. TWO “Halberd” wheat plants were grown in pots filled with treated soils that were packed into 0.6-I plastic pots and placed in a controlled environment chamber. Treatments were replicated four times and sampling was 3 weeks after seedling emergence. Roots were gently shaken from the soil and the remaining soil adhering to roots was retained for measurement of pH and N concentrations. Roots were then washed gently in sterile water to remove remaining soil particles, transferred to 100 ml of sterile water containing glass beads, and vigorously shaken on a wrist-action shaker for 10min to dislodge the majority of rhizoplane-inhabiting microorganisms. Roots were dried at 25°C and then weighed. The rhizoplane suspension was diluted in a 5-fold dilution series with 10 mM Tris [(2-amino, 2-hydroxymethyl, 1,3-propanediol)-HCl] buffer; pH 7.2. One drop (18 ~1) of each suspension per half plate was spread on NPCC medium (Simon et af., 1973) prepared at pH 5.5 or 7.0. Incubation was at 30°C. Pseudomonads were counted after 2 days, with ~uorescent species counted under U.V.radiation. Twelve pseudomonad isolates (6 fluorescent and 6 nonfluorescent) from each of four replicates of each treatment for each soil were selected 7-10 days after plating on NPCC at pH 5.5 or 7.0 (e.g. 960 isolates), and transferred to potato-dextrose-agar (PDA) medium for tests of antagonism toward G. graminis (Smiley, 1978a). The basis for selection of antagonists was inhibition of growth; the mechanism (e.g. antibiotic production, medium acidi~catio~ nutrient competition, etc.) was not evaluated. All fluorescent pseudomonads from two of the replicates of nonfumigated G. graminis-inoculated Wimmera and Rosedale soils were further identified to species (P. jluorescens or P. putida), biotype, and set (Stanier et al., 1966), following the diagnostic testing procedure outlined by Sands and Rovira (1970). The tests included gelatin hydrolysis, levan production, denitrificatio~ ethanol utilization trehalose utilization, and sorbitol utilization. Fluorescent isolates from noninoculated soils were identified only to species. Most of the study was repeated, and the results were similar to comparable treatments of the first study. All data were evaluated (P = 0.05) according to Duncan’s Multiple Range Test. RESULTS ln~uen~e of Gaeumannomy~s
graminis Numbers of pseudomonads on rhizoplanes differed for healthy and G. graminis-infected roots (Table 1). Pseudomonads were as much as four times more numerous on infected roots than on healthy roots in the Rosedale soil, and the opposite relationship was found in the alkaline Wimmera soil. The number of rhizoplane pseudomonads on roots parasitized by G. gruminis was nearly equal in the two soils, but the numbers on healthy roots differed greatly. This presumably resulted from nutritional and pH similarities at the root:soil interface for infected roots, and to
dissimilar rhizoplane properties in the two soils when roots were intact. Although the proportions of the detected fluorescent and nonfluorescent pseudomonads were not changed by the presence or absence of the pathogen, much higher proportions of the Wimmera-soil pseudomonads were antagonistic to G. graminis if recovered from the inoculated soil rather than the noninoculated treatments. This inoculum effect was small or nonexistent in the Rosedale soil. Influence of soil type
Pseudomonads were equally numerous on the rhizoplanes of diseased wheat in the inoculated Wimmera and Rosedale soils, but in the absence of the pathogen, the numbers in Wimmera soil were significantly higher than in the Rosedale soil (Table 1). The pseudomonad component of the population of rhizoplane bacteria in inoculated Wimmera and Rosedale and noninoculated Rosedale soils was similar, but the population was about three times higher in noninoculated Wimmera soil. The proportions of pseudomonads that produced fluorescent pigments were two-fold higher in the Rosedale than in the Wimmera soil. Pseudomonas putida isolates comprised less than half of the fluorescent pseudomonads in the rhizoplanes, with the lowest proportions being in the Rosedale soil; a reciprocal relationship occurred for P. jhorescens since isolates of P. aeruginosu were not detected in either soil. The NPCC pseudomonad isolation medium of Simon et al. (1973), which is designed for the selective isolation of fluorescent species from soil, did not effectively prevent the isolation of nonfluorescing species. The medium satisfied its purpose best with the acidic Rosedale soil that characterizes the South Australian soils for which the medium was prepared (Sands and Rovira, 1970). Wheat rhizoplanes in the two soils generally harbored different physiological sets of P. jiuorescens and P. putida, although P. jhorescens set 30 was clearly the dominant set in both soils. Set 17 of P. fluorescens was found only in Wimmera soil, sets 29 and 32 were only found in Rosedale soil, and set 13 was common to both soils. For P. putida, the sets found only in Wimmera soil included sets 33, 45 and 59 whereas only Rosedale soil yielded sets 47, 49, 60 and 64. Sets 57 and 62 were common to rhizoplanes in both soils, The proportion of the Pseudomonas isolates that were antagonistic in oitro toward G. graminis were greater for Wimmera than for Rosedale soil (Table 1). The percentage differences in most inoculated soil treatments were significant for each pseudomonad group except P. putida, where few antagonists were recovered from either soil. Most of the antagonistic fluorescent pseudomonads in both soils were P. jhorescens: however, higher proportions of both P. jluorescens and P. putida were antagonistic when isolated from Wimmera soil rather than from the Rosedale soil. In inoculated soils, a large proportion of the antagonists were identified as set 30 of P. jhorescens. Although several other sets also contained a high percentage of antagonists, their magnitude of antagonism on PDA was generally weak, resulting in low antagonism ratings. The tendency for different sets to occur in these soils, therefore,
Pseudomonads
had little or no apparent gonism ratings, except for of set 17 which occurred in NH:-treated Wimmera
and Gaeumannompces
importance to the antahighly antagonistc isolates at a low percentage only soil.
The pseudomonad ~mponents of bacterial populations estimated from wheat rhizoplanes in the Wimmera and Rosedale soils were not significantly altered when N was supplied primarily as NH:-N or as NOT-N (Table 1). Generally, different sources of N also did not alter proportions of the composite fluorescent and nonfluorescent groups that were antagonistic toward G. graminis or their antagonistic ability. Two exceptions included the occurrence of highly antagonistic isolates of set 17 of P. Jluorescens only in NH:-treated Wimmera soil, and the generally higher antagonism ratings of set 30 from NHftreated, than from NO;-treated, Wimmera soils. Fumigation of soil with methyl bromide caused significant reductions in the populations of all pseudomonads, and eliminated the detectable fluorescent component for at least 6 weeks. No antagonistic pseudomonads were detected in the fumigated soils. InJluence of isolation
medium pH
The isolation of pseudomonads on PDA adjusted to pH 5.5 or 7.0 had little or no effect on the apparent numbers or proportions of pseudomonad groups, species, or physiological sets found in wheat rhizospheres. The isolation medium pH also did not affect the apparent proportions of antagonistic nonfluorescent pseudomonads, but it had a very large effect on the activity of fluorescent species. Greater proportions of fluorescent species were ~tagonisti~ and antagonism ratings were much higher among pseudomonads isolated at pH 5.5 than at 7.0, even though the antagonism tests were all conducted on media adjusted to pH 7.0. The antagonism of G. gram~nis by fluorescent pseudomonads was, therefore, very responsive to changes in pH, but not to the prevalence of a specific form of N. There also appears to be variable antagonistic capabilities within specific fluorescent pseudomonad sets that separates them further than the biochemical and nutritional tests used in this study. The severity of take-all on 7-week-old plants (Smiley, 1978a) was inversely correlated with the antagonism ratings for pseudomonads in the inoculated Wimmera soil. Regression coefficients and their significance for isolations at pH 5.5 and 7.0 were -0.904 (P = 0.01) and -0.554 (NS), respectively. There were no correlations between disease and pseudomonads in the Rosedale soil. DISCUSSION
Colonization of wheat roots by Gaeumannomyces var. tritici is accomplished by production of infection hyphae which arise from ectotrophie “runner” hyphae. Penetration of roots by G. graminis and the enlargement of infection sites is dependent upon the ability of the pathogen’s runner hyphae to endure biotic and abiotic adversities in the rhizoplane. An example is the pathogen’s sensitivity to rhizoplane acidity that is caused by absorption of NH:-N by graminis
375
roots (Smiley and Cook, 1973; Smiley, 1974). Most of the NH:-induced inhibition of G. graminis growth on wheat roots is eliminated by soil fumigation (methyl bromide), and is reinstated by small additions (ly/, w/w) of certain nonsterile soils to the fumigated soil (Smiley, 1978b). This effect apparently results from pH-mediated interactions between the microorganisms and the pathogen. Pseudo~~s species appear well adapted as potential antagonists of this root parasite. They are highly competitive colonizers of the ecological niche in which G. graminis must compete (Marshall and A!exander, 1960; Vasantharajan, 1969) because they are selectively stimulated near wheat roots (Sands and Rovira, 1970), are tolerant of antibiotics (Simon et al., 1973), and have high growth rates (Rouatt and Katznelson, 1961). They are also very tolerant of acidic environments (Chan and Katznelson, 1961; Jensen, 1963) and are highly antagonistic toward other root parasites (Broadbent et al., 1971). Cook and Rovira (1976) and Smiley (1978b) have suggested that pseudomonads could have a role in the suppression of take-all in some soils. These hypotheses resulted from observations that treatments of soil with aerated steam (Baker, 1970) at 4@-6o’C or with methyl bromide increased take-all severity and the pathogen’s growth rate on roots (Shipton, 1972; Smiley and Cook, 1973), and also nearly eliminated the pseudomonad component of the rhizosphere microflora (Broadbent et al., 1971; Rovira and Ridge, 1973; Smiley, unpublished). Each of these effects was absent in soils treated with chloropicrin (Rovira and Ridge, 1973). When selected microbial species or groups were reintroduced into fumigated soils (Smifey, 1978b) the p~udomonads were more consistently associated with decreased pathogen activity than were the other soil microorganisms. Results of my study support the hypothesis that pseudomonads in some soils are antagonistic toward G. graminis, and it further identifies the pseudomonads which are primarily responsible for this disease-suppressive effect. Antagonism of G. graminis by Pseudomonas species appears to be very dependent upon environmental conditions in the rhizoplane. The conditions studied here included soil type, and pH and nutrition. The antagonism was most effective where isolations were made at low pH, and at this pH, a negative correlation existed between the antagonism ratings and disease severity. Isolates from healthy roots were antagonistic only if they came from NH:-treated Wimmera soil, but where roots were infected by G. graminis, there were no antagonism differences among These isolates from the NH:- and NO;-treatments. results suggest that a pH-nutritional interaction may occur in the rhizoplane of healthy roots, and that the nutritional effects alone are unimportant, at least after roots have become infected. I have demonstrated (Smiley, 1978b) that pseudomonads can reduce the rate of this pathogen’s ectotrophic growth. The acidifying influence of NH: -N may therefore depend more upon a slowing of the increase in infection site size than upon prevention of infections. This supports the observation (Smiley and Cook, 1973) that the numbers of infection sites on roots in NH:- and NOT-N treated soils are equal, and that they enlarge rapidly in the NOT-treatments, but not in the NH:-
376
RICHARDW. SMILEY
treatments. Rhizoplane conditions that are more favorable to the pathogen than to the antagonistic potential of Pseudomonas species may therefore lead to greater losses from take-all. The Wimmera soil has been considered suppressive of take-all and the Rosedale soil as nonsuppressive (Cook and Rovira, 1976). In these growth chamber studies, however, the disease was more severe in Wimmera than in Rosedale soil (Smiley, 1978a). Factors that were uniformly favorable to disease in each soil were the temperature, moisture, immaturity of seedlings, and an abundance of G. gruminis inoculum (Smiley, 1978a). The alkalinity of Wimmera soil was also very favorable, whereas the acidity of Rosedale soil was not. The pseudomonads in Wimmera soil were very inhibitory toward G. graminis, but they were incapable of countering the combined diseasefavoring factors in this growth cabinet study. Treatment of Wimmera soil with NH:-N apparently assisted in disease control by increasing the antagonistic capability of the pseudomonads and by discouraging growth by this acid-sensitive pathogen. The greater acidity of Rosedale soil than Wimmera soil appears to be largely responsible for the lower level of disease in Rosedale soil. However, the presence of pathogen-suppressing influence of NH;treated Rosedale soil, which could be eliminated by fumigation (Smiley, 1978aj, could not be attributable to antagonism by pseudomonads. This could have resulted from a reduction of antagonism by other members of the microflora (Smiley, 1978a,b), or to a factor that was not studied. This uncertainty in Rosedale soil indicates that a full interpretation of the negative correlation between antagonistic pseudomonads and disease severity in the Wimmera soil must await further investigation. Differences in take-all suppression among the two soils and their three treatments may have resulted from differences in the composition of pseudomonads in the rhizoplane, but this was not conclusively demonstrated. One highly antagonistic set (No. 17) was isolated only from the NH:-N treatment of Wimmera soil, where the growth of G. graminis was also inhibited most. Highly antagonistic pseudomonads were never isofated primarily from treatments where G. gramin~s growth was favored. Most of the set 30, biotype G isolates of P. Jfuorescens were antagonistic if isolated on acidified agar medium from Wimmera soil, and were not antagonistic if isolated from this soil on agar at pH 7, or if isolated from Rosendale soil. Antagonistic capabilities within a given set probably cannot be irreversibly altered by isolation medium pH, but differences in the medium’s acidity could selectively favor the active growth and therefore the isolation of a particular set or “sub-set”. These results are interpreted to mean that additional divisions existed within P. Juorescens set 30, and that these divisions were detectable only by their differences in antagonism toward G. graminis. Acknowledgements-This work was conducted while I was a Visiting Research Scientist, CSIRO, Division of Soils, Glen Osmond, South Australia, 5064. I thank A. D. Rovira, J. K. Martin, E. H. Ridge, G. D. Bowen, A. Simon, and MS J. Price, all of CSIRO, for expert advice and assistance; and the Departments of Agriculture of Victoria and
South Australia for supplying the soils used in these investigations. REFERENCES BAKERK. F. (1970) Selective killing of soil microorganisms by aerated steam. In Root Diseases and Soil-Borne PUQIOgens (T. A. Toussoun, R. V. Bega and P. E. Nelson. Eds), pp. 234-239. University of California Press, Berkeley. BROADRENT P., BAKERK. F. and WATERWORTH Y. (1971) Bacteria and actinomycetes antagonistic to fungal root nathogens in Australian soils. Australian Journal of Bioiogicai Science 24, 925-944. CHANE. C. S. and KATZNEL~N H. (1961) Growth interac. tions of Arthrobacter globiformis and Pseudomonas sp. in relation to the rhizosphere effect. Canadian Journal of ‘~~robiolog~ 7, 759-767. COOKR. J. and ROVIRAA. D. (1976) The role of bacteria in the biological control of G~eu~~~~yces graminis by suppressive soils. Soil Biology & Biochemistry 8, 269-273. GERLAGHM. (1968) Introduction of 0. graminis into new polders and its decline. Netherlands Journal of Plant Pathology 74, (Suppl. 2) l-97. JEN!ENV. (1963) Studies on the microflora of Danish beech forest soils. 3. Properties and composition of the bacterial flora. Zenrra~b~arr ftir Bak~er~o~ogie, AhteiIungen 116, 593611. MARSHALLK. S. and ALEXANDER M. (1960) Competition between soil bacteria and Fusurium. Pianr and Soil 12, 143.-153.
ROUATTJ. W. and KATZNEL~VN H. (1961) A study of the bacteria on the root surface and in the rhizosphere of crop plants. Journal of Applied Bacteriology 24, 164-171. ROVIRAA. D. and RIDGEE. H. (1973) The use of a selective medium to study the cology of Pseudomonas spp. in soil. Brflleiin Ecofoqical Research Communications, Sfockholm 17, 329-335. I SANDS D. C. and ROVIRAA. D. (1970) Pseudo~nas jfuorescens Biotype G., the dominant fluorescent pseudomonad in South Australia soils and wheat rhizospheres. Journal of Applied Bacteriology 34, 261-275.
SHIPTONP. J. (1972) Take-all in spring sown cereals under continuous cultivation; Disease progress and decline in relation to crop succession and nitrogen. Annaks of Applied Biology 71, 3346. SIMON A.,
ROVIRAA. D. & SANDSD. C. (1973) An improved selective medium for the isolation of fluorescent pseudomonads. journal qf Applied Bacrerjo~0g.v 36, 141-145.
SMILEYR. W. (1974) Rhizosphere pH as influenced by plants, soils, and nitrogen fertilizers. Proceedings of the Soil Science Society of America 38, 795-799. SMILEYR. W. (1978a) Antagonists of Gaeumannomyces graminis from the rhizoplane of wheat in soils fertilized with ammonium- vs. nitrate-nitrogen. Soil Biology & Biochemistry 10, 169-174. SMILEYR. W. (1978b) Colonization of wheat roots by Gaet~mQ~no~~~~ces graminjs inhibited by specific soils, microorganisms, and ammonium-nitrogen. Soil Binlog!: & Biochemistry to, 175-179. SMILF.Y R. W. and Coon R. 1. (1973) Relationship between take-all of wheat and rhizosphere pH in soils fertilized with ammonium- vs. nitrate-nitrogen. Phytopatholoa\ 1. _ 63, 882-890. STANER R. Y.. PALLERONI N. J. and DOUDCIROFF M. (1966) The aerobic pseudomonads: a taxonomic study. Journal of Generaf Microbiology 43, 159271. V A~NTWARAJAN V. N. (1969) Investigations into the pattern of microbial colonization of early rhizosphere with model systems. Journal of the Indian institute of Science 52, 148-154.
OF
RICHARD W. SMILEY Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Accepted 10 March 1979)
Summary-The role of rh~opiane-inhabiting P~e~~~rn~~~sspp as inhibitors of take-ah on wheat was investigated. Apparent numbers of p~udomonads in wheat rhizoplanes and numbers that were antagonistic in oitra toward Gaeu~nnumyces gram~n~~ var. tririci did not differ when wheat was supplied with NH:-N or NO;-N. More intense antagonism was expressed by colonies selected from soil treated with NH:-N than with NOT-N, and from isolation media prepared at pH 5.5 rather than at 7.0. Antagonists were not recovered from methyl bromide-treated soil. Highly antagonistic pseudomonads were recovered from a wheat-monoculture soil which is considered suppressive toward the pathogen in the field, and were not recovered from a “nonsuppressive” soil. Pseudomonad antagonism ratings were inversely correlated with take-all severity in the suppressive soil, but not in the nonsuppressive soil. Pseudomonads were considered to be antagonists of G. graminis on rhizoplanes of wheat in a soil exhibiting the “take-all decline” uhenomenon, but the significance of this interaction remains to be determin&l.
INTRODUCFION F~udomonads have been proposed to be spe~i~~Iy inhibitory to the ~tablishment of ectotrophic hyphal networks by Gue~~nnomyces gram&is Sacc. v. Arx & Ohvier var. tririci J. Walker (Cook and Rovira, 1976; Smiley, 1978b), the causal agent of take-all of wheat (Triricum aestivum L.). However, not all pseudomonads are antagonists toward G. graminis; the highest proportions of antagonistic vs nonantagonistic pseudomonads occur on rhizoplanes of infected roots growing in soils which have supported Iongterm wheat monoculture. In one such soil it was found (Smiley, 1978a,b) that take-all was significantly less severe following treatment with NH:-N fertilizer than with NO;-N. The apparent pseudomonad population on the rhizoplanes of infected wheat did not differ in the NH:- vs. NO;-N ~eatment~ but significantly greater proportions of isolates from the NH:-N treatments, compared to NO;-treatments, were antagonistic in vitro toward G. graminis. The magnitude of in vivo antagonism by pseudomonads reintroduced onto rhizoplanes of intact wheat was also significantly greater in the NH:-N than in the NO;-N treatments. These results suggest that the differential absorption of predominantly NHf- or NO;-N may have caused a redistribution to occur among the dominant species or biotypes within the pseudomonad population on wheat rhizoplanes. Alternatively, if the composition of pseudomonads was not changed, some members may have been altered in antagonistic capability by changes in rhizosphere pH (Smiley, 1974), in the quality or quantity of root exudates (M. E. Brown, personal communication), or in other rhizoplane properties. Although it is difficult to separate the individual effects of pH and other environmental properties, enumeration and culture of Pseudomonas species on acidic and neutral media could provide information concerning the relative importance of acidity to the Pseudomonas-Gaeumannomyces interaction 371
in NHf- vs NO;-treated soils. This is especially important in view of the intolerance of G. graminis to acidic environments (Smiley and Cook, 1973), and to the management of wheat affected by take-all. I report the numbers and species or biotypes of pseudomonads isolated at pH 5.5 and at 7.0 from rhizoplanes of wheat grown for 3 wk in soils treated with NH:-N, NO;-N, methyl bromide, with or without an inoculum of G. graminis. The in vitro antagonism of G. graminis by Pseudomonas spp. was also measured.
MATERIALSAND METHODS Soils and fertilizers Details of the soils and treatments used have been described elsewhere (Smiley, 1974, 1978a). Soils included Wimmera grey clay and Rosedale sandy clay loam. The 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 (Cook and Rovira, 1976; Shipton, 1972). The Rosedale sandy clay loam from the South Australian Department of Agriculture’s Turretfield Research Centre, Rosedafe, S.A., has supported a variable cropping history, including cereals, clover pasture and fair low. The saturated paste pH values (using 1OmM CaQ) are 8.0 and 5.5 for the Wimmera and Rosedale soils respectively (Smiley, 1978a). Fertilizer treatments consisted of blending dry commercial fertilizer-grade crystals of (NH&SO.+ and prills of Ca(NO,), into soil at the rate of 0.10 g N kg-’ soil. The nitrification inhibitor N-Serve 24 [2-chloro-&(trichloromethyl)pyridine] was applied to both N sources at the rate of 2% (w/w-based on N) prior to their application to soil. Where indicated, a portion of each soil was fumigated with methyl bromide (450 kg ha-‘) 2 weeks before adding fertilizers.
Populations P~u~ff~na.s spp (x IO3 cells/mg air dry root) Pseudomonas spp (% of total bacteria) Fluorescent pseudomonads (“/, of PseiIdo~o~as spp) P. putida; all sets and biotypes3 (% of fluor. pseudo.) P. jluorescens; all sets and biotypes (% of fluor. pseudo.) P. jhorescens; set 30, biotype G (% of P, juorescms) P. jhorescens; other sets & biotypes4 (7; of P. fluorescens)
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
isolation medium PH 168b’ 16.5b 1.47ab 1.28b 31b 46ab 37ab 31ab 63a 63ab lOOa 83a Ob 17b
NO: 14.9b 14.8b 0.89b l.lOb 27b 33b 25b 50a 75a 5Ob 86a 89a 14b lib
NH,
Wimmera
1.7c 2.oc 0.03c 0.03c Oc oc ND ND ND ND ND ND ND ND
Fum f NH, 16.3b 12.lb 1.04b 1.89ab 70a 77a 5c 13c 95a 87a Ob 15b lO&% 85a
NO,
Inoculated soils
15.6b 17.lb 0.83b 1.34b 80a 70a 21b 5c 79a 95a 1OOa 87a Ob 13b
NH,
Rosedale
0.3c O.lc O.Olc O.Olc Oc oc ND ND ND ND ND ND ND ND
Fum + NH,
32.4ab 35.lab 4.51a 4.29a 32b 36b 26b 32ab 14a 68ab ND ND ND ND
NO,
59.8a 62.la 4.11a 3.93a 2lb 28b 19bc 38ab 81a 62ab ND ND ND ND
NH&
3.lc 3.6~ 1.04b 1.13b 60a 64a 17bc Ilc 83a 89a ND ND ND ND
NO,
1.7c 2.3~ 1.09b 0.92b 61a 59a 8c 22b 92a 78a ND ND ND ND
NH&
Noninoculated soils Wimmera Rosedale
Table 1. Pseud~~onus spp isolated at pH 5.5 or 7.0 from rhizoplanes of wheat growing in soils treated with NH.+- or NO,-N, methyl bromide + NH&-N, or inocu~um uf Gaeumannompces graminis; and their in vitro antagonism toward C. graminis
F 9
F
r
6
E
p~udomonads
other sets & biotypes
P. juorescens,
set 30, biotype G
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
7.0 5.5 7.0
5.5
5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0 5.5 7.0
3.8a 1.4ab 3.8a 0.2b Oa Oa 5.2a 0.2b 5.2a 0.3b ND Ob
67a 46ab 75a 18b Ob Ob 1OOa 25b lOOa 2lb ND oc 4.0a 2.2a 4.4a O.lb 0.3a Oa 6.la Ob 7.la Ob 4.8a Ob
55a 65a 15a lob 30a 13b 1OOa Oc 83a Ob 80a oc Ob Ob ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND O.lb Ob O.lb Ob Oa Oa O.lb Ob ND Ob 0.2b Ob
lob oc Ob Ob 1OC oc ND Ob 33b OC
6c 4c
Ob Ob Ob Ob Oa Oa O.lb Ob O.lb Ob ND Ob
llb 3b ND 2c
SC
8b 5bc IOb Ob llc
DC
O.lb Ob ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND
oc
0.4b 0.2b Ob Ob ND ND ND ND ND ND ND ND
2c 4c oc Oc ND ND ND ND ND ND ND ND
ND ND
ND
4.2a 1.6ab 3.9a 0.4b ND ND ND ND ND
34b 30b 31ab 27ab ND ND ND ND ND ND ND ND DC
Ob Ob Ob Ob ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
oc oc
OC
Ob Ob Ob Ob ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND
oc oc oc oc
r Ca(NO& or (NHJ2S04, both with N-Serve 24, were applied at the rate of O.lOg N kg-’ soil. ’ Averages of 4 replicates. Averages within paired rows of isolation pH 5.5 and 7.0 that are followed by the same letter are not significantly different at the 0.05 level of Duncan’s Multiple Range Test. 3 gets 33, 3.5, and 57 for Wimmera-NH,; sets 33, 59, and 62 for Wimmera-NOJ; sets 57, 60, 62 and 64 for Rosedale-NH,; and sets 47, 49 and 64 for Rosedale-NO,. 4 Set 17 for Wimmera-Nap; set 13 for Wimmera-NON; set 13 for Rosedale-NH,; and sets 13, 29 and 32 for Rosedale-NO,. ’ Antagonism Rating = decimal percentage of antagonists x zone of inhibition distance in mm. ND = no determination.
P. fiuorescens, other sets & biotypes
P. fluorescens,
P. jhorescens
P. putida
Fluorescent p~udomonads
Antagonism Rating5 Nonfluorescent pseudomonads
set 30, biotype G
P. fluorescens,
P. jluorescens
P. putida
Fluorescent pseudomonads
Antagonists (70 Nan~uores~nt
374
RICHARD V?. SMILEY
Inaculum, incubation, and sampling
Smiley (1978a) described techniques used in these investigations. Inoculum of G. graminis-colonized crushed oats was blended into the soil at a rate of 0.05% (w/w), or the soil remained noninoculated. TWO “Halberd” wheat plants were grown in pots filled with treated soils that were packed into 0.6-I plastic pots and placed in a controlled environment chamber. Treatments were replicated four times and sampling was 3 weeks after seedling emergence. Roots were gently shaken from the soil and the remaining soil adhering to roots was retained for measurement of pH and N concentrations. Roots were then washed gently in sterile water to remove remaining soil particles, transferred to 100 ml of sterile water containing glass beads, and vigorously shaken on a wrist-action shaker for 10min to dislodge the majority of rhizoplane-inhabiting microorganisms. Roots were dried at 25°C and then weighed. The rhizoplane suspension was diluted in a 5-fold dilution series with 10 mM Tris [(2-amino, 2-hydroxymethyl, 1,3-propanediol)-HCl] buffer; pH 7.2. One drop (18 ~1) of each suspension per half plate was spread on NPCC medium (Simon et af., 1973) prepared at pH 5.5 or 7.0. Incubation was at 30°C. Pseudomonads were counted after 2 days, with ~uorescent species counted under U.V.radiation. Twelve pseudomonad isolates (6 fluorescent and 6 nonfluorescent) from each of four replicates of each treatment for each soil were selected 7-10 days after plating on NPCC at pH 5.5 or 7.0 (e.g. 960 isolates), and transferred to potato-dextrose-agar (PDA) medium for tests of antagonism toward G. graminis (Smiley, 1978a). The basis for selection of antagonists was inhibition of growth; the mechanism (e.g. antibiotic production, medium acidi~catio~ nutrient competition, etc.) was not evaluated. All fluorescent pseudomonads from two of the replicates of nonfumigated G. graminis-inoculated Wimmera and Rosedale soils were further identified to species (P. jluorescens or P. putida), biotype, and set (Stanier et al., 1966), following the diagnostic testing procedure outlined by Sands and Rovira (1970). The tests included gelatin hydrolysis, levan production, denitrificatio~ ethanol utilization trehalose utilization, and sorbitol utilization. Fluorescent isolates from noninoculated soils were identified only to species. Most of the study was repeated, and the results were similar to comparable treatments of the first study. All data were evaluated (P = 0.05) according to Duncan’s Multiple Range Test. RESULTS ln~uen~e of Gaeumannomy~s
graminis Numbers of pseudomonads on rhizoplanes differed for healthy and G. graminis-infected roots (Table 1). Pseudomonads were as much as four times more numerous on infected roots than on healthy roots in the Rosedale soil, and the opposite relationship was found in the alkaline Wimmera soil. The number of rhizoplane pseudomonads on roots parasitized by G. gruminis was nearly equal in the two soils, but the numbers on healthy roots differed greatly. This presumably resulted from nutritional and pH similarities at the root:soil interface for infected roots, and to
dissimilar rhizoplane properties in the two soils when roots were intact. Although the proportions of the detected fluorescent and nonfluorescent pseudomonads were not changed by the presence or absence of the pathogen, much higher proportions of the Wimmera-soil pseudomonads were antagonistic to G. graminis if recovered from the inoculated soil rather than the noninoculated treatments. This inoculum effect was small or nonexistent in the Rosedale soil. Influence of soil type
Pseudomonads were equally numerous on the rhizoplanes of diseased wheat in the inoculated Wimmera and Rosedale soils, but in the absence of the pathogen, the numbers in Wimmera soil were significantly higher than in the Rosedale soil (Table 1). The pseudomonad component of the population of rhizoplane bacteria in inoculated Wimmera and Rosedale and noninoculated Rosedale soils was similar, but the population was about three times higher in noninoculated Wimmera soil. The proportions of pseudomonads that produced fluorescent pigments were two-fold higher in the Rosedale than in the Wimmera soil. Pseudomonas putida isolates comprised less than half of the fluorescent pseudomonads in the rhizoplanes, with the lowest proportions being in the Rosedale soil; a reciprocal relationship occurred for P. jhorescens since isolates of P. aeruginosu were not detected in either soil. The NPCC pseudomonad isolation medium of Simon et al. (1973), which is designed for the selective isolation of fluorescent species from soil, did not effectively prevent the isolation of nonfluorescing species. The medium satisfied its purpose best with the acidic Rosedale soil that characterizes the South Australian soils for which the medium was prepared (Sands and Rovira, 1970). Wheat rhizoplanes in the two soils generally harbored different physiological sets of P. jiuorescens and P. putida, although P. jhorescens set 30 was clearly the dominant set in both soils. Set 17 of P. fluorescens was found only in Wimmera soil, sets 29 and 32 were only found in Rosedale soil, and set 13 was common to both soils. For P. putida, the sets found only in Wimmera soil included sets 33, 45 and 59 whereas only Rosedale soil yielded sets 47, 49, 60 and 64. Sets 57 and 62 were common to rhizoplanes in both soils, The proportion of the Pseudomonas isolates that were antagonistic in oitro toward G. graminis were greater for Wimmera than for Rosedale soil (Table 1). The percentage differences in most inoculated soil treatments were significant for each pseudomonad group except P. putida, where few antagonists were recovered from either soil. Most of the antagonistic fluorescent pseudomonads in both soils were P. jhorescens: however, higher proportions of both P. jluorescens and P. putida were antagonistic when isolated from Wimmera soil rather than from the Rosedale soil. In inoculated soils, a large proportion of the antagonists were identified as set 30 of P. jhorescens. Although several other sets also contained a high percentage of antagonists, their magnitude of antagonism on PDA was generally weak, resulting in low antagonism ratings. The tendency for different sets to occur in these soils, therefore,
Pseudomonads
had little or no apparent gonism ratings, except for of set 17 which occurred in NH:-treated Wimmera
and Gaeumannompces
importance to the antahighly antagonistc isolates at a low percentage only soil.
The pseudomonad ~mponents of bacterial populations estimated from wheat rhizoplanes in the Wimmera and Rosedale soils were not significantly altered when N was supplied primarily as NH:-N or as NOT-N (Table 1). Generally, different sources of N also did not alter proportions of the composite fluorescent and nonfluorescent groups that were antagonistic toward G. graminis or their antagonistic ability. Two exceptions included the occurrence of highly antagonistic isolates of set 17 of P. Jluorescens only in NH:-treated Wimmera soil, and the generally higher antagonism ratings of set 30 from NHftreated, than from NO;-treated, Wimmera soils. Fumigation of soil with methyl bromide caused significant reductions in the populations of all pseudomonads, and eliminated the detectable fluorescent component for at least 6 weeks. No antagonistic pseudomonads were detected in the fumigated soils. InJluence of isolation
medium pH
The isolation of pseudomonads on PDA adjusted to pH 5.5 or 7.0 had little or no effect on the apparent numbers or proportions of pseudomonad groups, species, or physiological sets found in wheat rhizospheres. The isolation medium pH also did not affect the apparent proportions of antagonistic nonfluorescent pseudomonads, but it had a very large effect on the activity of fluorescent species. Greater proportions of fluorescent species were ~tagonisti~ and antagonism ratings were much higher among pseudomonads isolated at pH 5.5 than at 7.0, even though the antagonism tests were all conducted on media adjusted to pH 7.0. The antagonism of G. gram~nis by fluorescent pseudomonads was, therefore, very responsive to changes in pH, but not to the prevalence of a specific form of N. There also appears to be variable antagonistic capabilities within specific fluorescent pseudomonad sets that separates them further than the biochemical and nutritional tests used in this study. The severity of take-all on 7-week-old plants (Smiley, 1978a) was inversely correlated with the antagonism ratings for pseudomonads in the inoculated Wimmera soil. Regression coefficients and their significance for isolations at pH 5.5 and 7.0 were -0.904 (P = 0.01) and -0.554 (NS), respectively. There were no correlations between disease and pseudomonads in the Rosedale soil. DISCUSSION
Colonization of wheat roots by Gaeumannomyces var. tritici is accomplished by production of infection hyphae which arise from ectotrophie “runner” hyphae. Penetration of roots by G. graminis and the enlargement of infection sites is dependent upon the ability of the pathogen’s runner hyphae to endure biotic and abiotic adversities in the rhizoplane. An example is the pathogen’s sensitivity to rhizoplane acidity that is caused by absorption of NH:-N by graminis
375
roots (Smiley and Cook, 1973; Smiley, 1974). Most of the NH:-induced inhibition of G. graminis growth on wheat roots is eliminated by soil fumigation (methyl bromide), and is reinstated by small additions (ly/, w/w) of certain nonsterile soils to the fumigated soil (Smiley, 1978b). This effect apparently results from pH-mediated interactions between the microorganisms and the pathogen. Pseudo~~s species appear well adapted as potential antagonists of this root parasite. They are highly competitive colonizers of the ecological niche in which G. graminis must compete (Marshall and A!exander, 1960; Vasantharajan, 1969) because they are selectively stimulated near wheat roots (Sands and Rovira, 1970), are tolerant of antibiotics (Simon et al., 1973), and have high growth rates (Rouatt and Katznelson, 1961). They are also very tolerant of acidic environments (Chan and Katznelson, 1961; Jensen, 1963) and are highly antagonistic toward other root parasites (Broadbent et al., 1971). Cook and Rovira (1976) and Smiley (1978b) have suggested that pseudomonads could have a role in the suppression of take-all in some soils. These hypotheses resulted from observations that treatments of soil with aerated steam (Baker, 1970) at 4@-6o’C or with methyl bromide increased take-all severity and the pathogen’s growth rate on roots (Shipton, 1972; Smiley and Cook, 1973), and also nearly eliminated the pseudomonad component of the rhizosphere microflora (Broadbent et al., 1971; Rovira and Ridge, 1973; Smiley, unpublished). Each of these effects was absent in soils treated with chloropicrin (Rovira and Ridge, 1973). When selected microbial species or groups were reintroduced into fumigated soils (Smifey, 1978b) the p~udomonads were more consistently associated with decreased pathogen activity than were the other soil microorganisms. Results of my study support the hypothesis that pseudomonads in some soils are antagonistic toward G. graminis, and it further identifies the pseudomonads which are primarily responsible for this disease-suppressive effect. Antagonism of G. graminis by Pseudomonas species appears to be very dependent upon environmental conditions in the rhizoplane. The conditions studied here included soil type, and pH and nutrition. The antagonism was most effective where isolations were made at low pH, and at this pH, a negative correlation existed between the antagonism ratings and disease severity. Isolates from healthy roots were antagonistic only if they came from NH:-treated Wimmera soil, but where roots were infected by G. graminis, there were no antagonism differences among These isolates from the NH:- and NO;-treatments. results suggest that a pH-nutritional interaction may occur in the rhizoplane of healthy roots, and that the nutritional effects alone are unimportant, at least after roots have become infected. I have demonstrated (Smiley, 1978b) that pseudomonads can reduce the rate of this pathogen’s ectotrophic growth. The acidifying influence of NH: -N may therefore depend more upon a slowing of the increase in infection site size than upon prevention of infections. This supports the observation (Smiley and Cook, 1973) that the numbers of infection sites on roots in NH:- and NOT-N treated soils are equal, and that they enlarge rapidly in the NOT-treatments, but not in the NH:-
376
RICHARDW. SMILEY
treatments. Rhizoplane conditions that are more favorable to the pathogen than to the antagonistic potential of Pseudomonas species may therefore lead to greater losses from take-all. The Wimmera soil has been considered suppressive of take-all and the Rosedale soil as nonsuppressive (Cook and Rovira, 1976). In these growth chamber studies, however, the disease was more severe in Wimmera than in Rosedale soil (Smiley, 1978a). Factors that were uniformly favorable to disease in each soil were the temperature, moisture, immaturity of seedlings, and an abundance of G. gruminis inoculum (Smiley, 1978a). The alkalinity of Wimmera soil was also very favorable, whereas the acidity of Rosedale soil was not. The pseudomonads in Wimmera soil were very inhibitory toward G. graminis, but they were incapable of countering the combined diseasefavoring factors in this growth cabinet study. Treatment of Wimmera soil with NH:-N apparently assisted in disease control by increasing the antagonistic capability of the pseudomonads and by discouraging growth by this acid-sensitive pathogen. The greater acidity of Rosedale soil than Wimmera soil appears to be largely responsible for the lower level of disease in Rosedale soil. However, the presence of pathogen-suppressing influence of NH;treated Rosedale soil, which could be eliminated by fumigation (Smiley, 1978aj, could not be attributable to antagonism by pseudomonads. This could have resulted from a reduction of antagonism by other members of the microflora (Smiley, 1978a,b), or to a factor that was not studied. This uncertainty in Rosedale soil indicates that a full interpretation of the negative correlation between antagonistic pseudomonads and disease severity in the Wimmera soil must await further investigation. Differences in take-all suppression among the two soils and their three treatments may have resulted from differences in the composition of pseudomonads in the rhizoplane, but this was not conclusively demonstrated. One highly antagonistic set (No. 17) was isolated only from the NH:-N treatment of Wimmera soil, where the growth of G. graminis was also inhibited most. Highly antagonistic pseudomonads were never isofated primarily from treatments where G. gramin~s growth was favored. Most of the set 30, biotype G isolates of P. Jfuorescens were antagonistic if isolated on acidified agar medium from Wimmera soil, and were not antagonistic if isolated from this soil on agar at pH 7, or if isolated from Rosendale soil. Antagonistic capabilities within a given set probably cannot be irreversibly altered by isolation medium pH, but differences in the medium’s acidity could selectively favor the active growth and therefore the isolation of a particular set or “sub-set”. These results are interpreted to mean that additional divisions existed within P. Juorescens set 30, and that these divisions were detectable only by their differences in antagonism toward G. graminis. Acknowledgements-This work was conducted while I was a Visiting Research Scientist, CSIRO, Division of Soils, Glen Osmond, South Australia, 5064. I thank A. D. Rovira, J. K. Martin, E. H. Ridge, G. D. Bowen, A. Simon, and MS J. Price, all of CSIRO, for expert advice and assistance; and the Departments of Agriculture of Victoria and
South Australia for supplying the soils used in these investigations. REFERENCES BAKERK. F. (1970) Selective killing of soil microorganisms by aerated steam. In Root Diseases and Soil-Borne PUQIOgens (T. A. Toussoun, R. V. Bega and P. E. Nelson. Eds), pp. 234-239. University of California Press, Berkeley. BROADRENT P., BAKERK. F. and WATERWORTH Y. (1971) Bacteria and actinomycetes antagonistic to fungal root nathogens in Australian soils. Australian Journal of Bioiogicai Science 24, 925-944. CHANE. C. S. and KATZNEL~N H. (1961) Growth interac. tions of Arthrobacter globiformis and Pseudomonas sp. in relation to the rhizosphere effect. Canadian Journal of ‘~~robiolog~ 7, 759-767. COOKR. J. and ROVIRAA. D. (1976) The role of bacteria in the biological control of G~eu~~~~yces graminis by suppressive soils. Soil Biology & Biochemistry 8, 269-273. GERLAGHM. (1968) Introduction of 0. graminis into new polders and its decline. Netherlands Journal of Plant Pathology 74, (Suppl. 2) l-97. JEN!ENV. (1963) Studies on the microflora of Danish beech forest soils. 3. Properties and composition of the bacterial flora. Zenrra~b~arr ftir Bak~er~o~ogie, AhteiIungen 116, 593611. MARSHALLK. S. and ALEXANDER M. (1960) Competition between soil bacteria and Fusurium. Pianr and Soil 12, 143.-153.
ROUATTJ. W. and KATZNEL~VN H. (1961) A study of the bacteria on the root surface and in the rhizosphere of crop plants. Journal of Applied Bacteriology 24, 164-171. ROVIRAA. D. and RIDGEE. H. (1973) The use of a selective medium to study the cology of Pseudomonas spp. in soil. Brflleiin Ecofoqical Research Communications, Sfockholm 17, 329-335. I SANDS D. C. and ROVIRAA. D. (1970) Pseudo~nas jfuorescens Biotype G., the dominant fluorescent pseudomonad in South Australia soils and wheat rhizospheres. Journal of Applied Bacteriology 34, 261-275.
SHIPTONP. J. (1972) Take-all in spring sown cereals under continuous cultivation; Disease progress and decline in relation to crop succession and nitrogen. Annaks of Applied Biology 71, 3346. SIMON A.,
ROVIRAA. D. & SANDSD. C. (1973) An improved selective medium for the isolation of fluorescent pseudomonads. journal qf Applied Bacrerjo~0g.v 36, 141-145.
SMILEYR. W. (1974) Rhizosphere pH as influenced by plants, soils, and nitrogen fertilizers. Proceedings of the Soil Science Society of America 38, 795-799. SMILEYR. W. (1978a) Antagonists of Gaeumannomyces graminis from the rhizoplane of wheat in soils fertilized with ammonium- vs. nitrate-nitrogen. Soil Biology & Biochemistry 10, 169-174. SMILEYR. W. (1978b) Colonization of wheat roots by Gaet~mQ~no~~~~ces graminjs inhibited by specific soils, microorganisms, and ammonium-nitrogen. Soil Binlog!: & Biochemistry to, 175-179. SMILF.Y R. W. and Coon R. 1. (1973) Relationship between take-all of wheat and rhizosphere pH in soils fertilized with ammonium- vs. nitrate-nitrogen. Phytopatholoa\ 1. _ 63, 882-890. STANER R. Y.. PALLERONI N. J. and DOUDCIROFF M. (1966) The aerobic pseudomonads: a taxonomic study. Journal of Generaf Microbiology 43, 159271. V A~NTWARAJAN V. N. (1969) Investigations into the pattern of microbial colonization of early rhizosphere with model systems. Journal of the Indian institute of Science 52, 148-154.