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Effects of soil microorganisms on mycorrhizal contribution to growth of big bluestem grass in non-sterile soil
Soil Biol. B&hem. Vol. 20, No. 4, pp. 501-507,1988 Britain. All rights reserved
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Printed in Great
0038-0717/88 s3.00 + 0.00 1988 Pergamon Press plc
EFFECTS OF SOIL MICROORGANISMS ON MYCORRHIZAL CONTRIBUTION TO GROWTH OF BIG BLUESTEM GRASS IN NON-STERILE SOIL B. A. DANIELSHETRICK,’ G. THOMPSON WILSON,’ D. GERSCHEFSKE Kin’ and A. P. SCHWAB~ Departments
of ‘Plant Pathology and 2Agronomy, Kansas State University, Manhattan, KS 66506, U.S.A. (Accepted 25 November 1987)
Summary-Plant dry weight and mycorrhizal root colonization of big bluestem (Andropogon gerurdii Vitman) inoculated with Glomus etunicurum Becker and Gerd. were suppressed if non-sterile prairie soil sievings or filtrate were added to pasteurized soil. Addition of prairie soil microorganisms, isolated onto peptone yeast extract, King’s B, or starch casein agar media, to pasteurized soil also reduced dry weight and mycorrhizal root colonization of big bluestem insulate with G. etunicatum. In contrast, addition of non-sterile soil sievings or filtrate or organisms isolated onto potato dextrose or p~udomonas isolation agar to pasteurized soil improved growth of non-myco~hi~l big bluestem. These effects of soil microorganisms on plant growth were further quantified by comparing 32P uptake by fungicide treated and untreated mycorrhizal plants and by non-mycorrhizal plants in pasteurized and non-sterile soil. Mycorrhizal plants grown in pasteurized soil absorbed ‘c IO times more “P than mycorrhizai plants grown in non-sterile soil. Application of propiconazole (Tilt) or fenarimol (Rub&an), fungicides which inhibit )*P uptake by mycorrhizas. resulted in approx. 40- and S-fold reductions in 32Puptake in pasteurized and non-sterile soil, respectively. Thus, more 32P is absorbed in pasteurized than in non-sterile soil, probably because soil microorganisms limit mycorrhizal activity in non-sterile soil. Assessments of mycorrhizal contribution to plant growth conducted in sterilized soils may significantly overestimate the effects of
VAM fungi because other soil microorganisms are not considered.
A wide array of interactions between vesiculararbuscular mycorrhizal (VAM) fungi and soil microorganisms have been studied, including interactions with plant pathogens (Schenck and Kellam, 1978; Dehne, 1982), nitrogen-fixing organisms (Bagyaraj and Menge, 1978; Bagyaraj et al., 1979; Manjunath et al., 1981; Mosse er al., 1976; Bethlenfalvay et al., 1982), phosphate-solubilizing microorganisms (Barea et al., 1975; Manjunath et al., 1981) and hyperparasites (Ross and Daniels, 1982). It has been demonstrated that the microflora population was different in the rhizosphere of mycorrhiza1 plants as compared with non-rny~o~hi~l plants (Ames et al., 1984). The influence of the rhizosphere microflora on mycorrhizas is still poorly understood, although positive effects of the microflora on mycorrhizal root colonization (Azcon-Aguilar and Barea, 1985) and plant growth (Meyer and Linderman, 1986) have been reported. However, the influence of the soil microflora on mycorrhizal contribution to plant growth has not been assessed. VAM fungi can dramatically improve growth of many crop plants. However, the ubiquitous nature of VAM fungi in soils necessitates that they be first eliminated from soil and then reinoculated under controlled conditions before their effects on plant growth can be assessed by comparing mycorrhizai and non-myco~hiza1 plants. The effect of other soil microorganisms, eliminated during soil sterilization, on plant growth or mycorrhizal growth response 501
is generally not considered. The effects of these organisms is probabfy si~ificant, however, since additions of non-sterile soil to pasteurized soil can suppress mycorrhizal growth response and inhibit mycorrhizal root colonization in big bluestem (Hetrick et al., 1986). The contribution of VAM fungi to plant growth in non-sterile soil may differ from that demonstrated in sterilized soil. However, it is extremely difficult to assess the contribution of VAM fungi to plant growth in non-sterile soil. We examined the restoration of soil microorganisms including mycorrhizal fungi to pasteurized soit. Uptake of j2P by mycorrhizal plants in nonsterile and pasteurized soil was compared using a modi~cation of the technique developed by Gray and Gerdemann (1969). We also assessed the effects of soil microorganisms on mycorrhizal symbiosis in big bluestem, a native plant of tallgrass prairies. MATERIALSAND METHODS Plant and inoculum preparation
For each experiment, seeds of big bluestem (Androwere germinated in vermiculite. Two weeks after emergence, one seedling was transplanted to each pot containing freshly-collected tallgrass prairie soil (T&y silty clay loam, fine, mixed mesic Pachic Argiustoll) containing 5 pgg-’ plant available P (Bray test I). Experiments were conducted in 6 x 25 cm plastic pots except those involving 32P uptake which had 9 x 12 cm plastic sleeves inside a
pogon gerardii Vitman)
502
B. A.
DANIELS HETRXK et
15 x 16 cm pot. Spores of Glomus etunicat~m Becker and Gerd. were collected from Sudan grass (~o~g~~ uu!gare var. sirdanense [piper] Hitch) pot cultures by wet sieving, decanting and sucrose centrifugation (Daniels and Skipper, 1982). Inoculation was accomplished by pipeting approx 400 spores onto roots of each seedling at transplanting.
Injluence of non-sterile soil sievings and filtrate growth of big bluestem
on
Freshly-collected prairie soil (400g dry wt) was blended with 4000 ml sterile water and sieved ( < 38 pm). The sieved soil suspension was added in 125-ml aliquots to 14 pots containing 400 g (dry wt) pasteurized prairie soil. Sterile water (125 ml) was added to 14 control pots. The remaining suspension was then further filtered through 615 grade filter paper (Whatman 1) and 125 ml of this filtrate was added to 14 pots containing pasteurized soil. Half of the 14 pots of each treatment were inoculated with G. etunicatum spores. To determine whether fungi or bacteria were present in the sieved suspension or filtrate, 0.1 ml of suspension or filtrate was plated onto Petri dishes containing peptone yeast extract agar, tryptic soy or potato dextrose agar, and plates were maintained at 22°C for 7days. Z~~~e~ce offingi and bacteria from non-sterjle soil on mycorrhj~aI growth response Three 25-g samples of freshly-collected tallgrass prairie soil were each blended with 250ml sterile water and 0.1 ml of the suspension was plated onto two Petri dishes containing 0.6% Difco potato dextrose agar (PDA) (amended with 0.1 gl-’ streptomycin and chloramphenical), peptone yeast extract (PYE), King’s B (KB) or tryptic soy agar (TSA) media (Difco Laboratories, Detroit, Mich.). Plated agar dishes were maintained at 22°C for 7 days, then flooded with 10 ml sterile distilled water and scraped with a glass rod to remove microbial growth. For each of the four media, organisms from all replicate plates were combined. Sterile distilled water was added to produce a finat volume of 100 ml. For each of the four media, 5 ml of microbial suspension were pipetted into I2 pots containing 430g (dry wt) of pasteurized prairie soil. For controls, 5 ml of water acquired from flooding non-inoculated plates were added to 12 pots containing pasteurized and nonsterile soil. The use of a water control ensured that nutrient contained in culture media did not cause the observed effects. Half of the 12 pots of each treatment were inoculated with G. etunicatum spores. In a subsequent experiment these collections were repeated but starch casein agar (10 g starch, 0.3 g casein, 2.0 g KNO,, 2.0 g NaCl, 2.Og K~HPO~,5OmgMgSO~~7H~O,2Omg~a~O~, 1Omg FeSO,.H?O, 18 g agar, 50 pg g-l cycloheximide) (Hirsch and Christenson, 1983) and pseudomonas isolation (PI) agar (Difco Laboratories, Detroit, Mich.) were included for isolation of actinomycetes and pseudomonads, respectively. Rhizosphere fungi were separated from soil fungi in this subsequent experiment by removing three 25-g (fresh wt) root samples from soil. Roots were shaken to remove adherent soil, blended in 250 ml water, and 0.1 ml plated onto 0.6% PDA (amended with 0.1 g 1-l strep-
al.
tomycin and chloramphenicol). Soil fungi were isolated by dry-sieving soil through a 355~grn sieve to remove roots. Three 25-g samples of sieved soil were blended in 250 ml water, and 0.1 ml plated onto PDA as above. For comparison. soil samples containing roots were also plated as in the previous experiment. After 7days, microorganisms were removed from the media and diluted with distilled water as previously described. Solution turbidities of 30, 20. 85 and 100 Klett units were determined for organisms removed from TSA, PYE. KB and PI media, respectively (Klett-Summerson Photoelectric Colorimeter, New York). Fungi grown on PDA were washed from the culture medium and added to sterile distilled water to a total volume of 100 ml. Hemacytometer readings from roots contained approx. 1.4 x IO’ fungal spores ml-‘, soil without roots contained 1.3 x 10’ fungal spores ml-’ and soil with roots contained 4.4 x 10’ spores ml-‘. influence of non-sterile soil on uptake of 3’P bJ mycorrhizae
Seven fungicides were screened for inhibition of nutrient uptake by mycorrhizal fungi. Initially, 9 x 12 cm plastic sleeves were placed inside 15 x 16 cm pots. Each sleeve was filled with 600 g (dry wt) pasteurized prairie soil, into which one seedling was transplanted; 75 such sleeves were inoculated with G. etun~catam and 5 were not. Seedings were grown in the greenhouse for 7 weeks. Thereafter, 25 ml of a fungicide were applied to five inoculated plants as a soil drench and to five inoculated plants as a foliar spray in the following concentrations (pg 8-l): 6.15 triadimefon (Bayleton); 4.65 pentachloronitrobenzene (PCNB); 47.7 imazilil (Imazilil); 21.8 carboxin/thiram (Vitavax); 24.6 benomy1 (Benlate); 15.4 fenarimol (Rubigan); and 4.05 propiconazole (Tilt). As a control, 25 ml sterile distilled water were applied as a soil drench and a foliar spray to the remaining five inoculated and five noninoculated plants. After 72 h, the plastic sleeves were removed without disturbing the soil mass containing the plant. Additional soil (325 g) was placed around the original soil mass in three layers. The bottom and top layers consisted of 75 g (dry wt) each of pasteurized prairie soil while the middle layer consisted of 175 g pasteurized prairie soil amended with 10 FCi ‘*P tracer solution. This ensured that roots at the bottom of the pot were not placed in direct contact with tracer. The upper non-amended soil layer blocked radiation sufficiently to allow the use of a 6-m tube survey meter to determine when 3zP had been translocated into plant shoots. Seven days after “P addition, plant shoots were harvested, dried, weighed, wet-ashed (in 15 mf 5:2: 1 nitric-perchloric-sulfuric acid), and diluted to 50 ml with distilled water. A 2.5-ml aliquot of this solution was mixed with 15 ml Aquasol II cocktail (DuPont NEN Research Products, Boston, Mass.) and radioassayed using a United Technologies Packard scintillation counter. Roots from each pot were carefully removed from soil with tweezers and washed free of soil, stained in trypan blue, and examined microscopically to determine percentage root colonization and colonization intensity (Kormanik and McGraw, 1982).
503
Soil microbes and mycorrhizae A simple analysis of variance (P = 0.05) was performed on log values of scintillation counts g-’ shoot dry wt, and on root colonization data using Duncan’s multiple range test for mean separation. The influence of non-sterile soil on mycorrhizal uptake of 32P was studied using Tilt and Rubigan, the fungicides which proved effective in this initial experiment. Sixty plastic sleeves were filled with 600 g (dry wt) soil: 30 with pasteurized prairie soil and 30 with freshly-collected non-sterile prairie soil. Half of the pots containing each soil treatment were inoculated with G. etunicatum at the time of seedling transplanting. In addition, 10 sleeves were filled with 600g pasteurized soil amended with lOpgg_‘P as KH2P04, and not inoculated. This produced growth of non-mycorrhizal plants comparable to mycorrhizal plants, allowing comparison of equal-sized plants. Seedlings were grown in the greenhouse for 6 weeks, after which 25ml aliquots of 31.2pgg-’ Rubigan or 8.37 pgg-’ Tilt or sterile water were applied as soil drenches to five replicate inoculated and non-inoculated plants in pasteurized and nonsterile soil. Higher fungicide dosages were used in this experiment to ensure maximum effectiveness and to assess whether these fungicides could be fungitoxic. A 25ml aliquot of each fungicide was applied to five of the P-amended plants to evaluate whether or not the fungicides were phytotoxic. Sterile water was applied to the remaining replications of inoculated, non-inoculated and P-amended plants. Three days after fungicide application, the sleeves were removed and 20 pCi 32P tracer were added. Ten days later plant shoots were harvested, dried, wet-digested and radio-assayed. Roots were also harvested, stained and examined for root colonization. Experimental design and maintenance
For each experiment, the containers were arranged in a completely-randomized design and plants grown in a 15-25’C greenhouse. Plants were fertilized every other week with 368 mg Peter’s No-Phos Special fertilizer solution (Peter’s Fertilizer Products, Fogelsville, Pa) delivered in 10ml H20. For all microbial isolation studies, top and root dry weights were determined at harvest after 13 weeks. The dried roots were subsampled, stained in trypan blue (Phillips and Hayman, 1970), and examined microscopically to assess percentage root colonization and colonization
Table
I. The
influence
of non-sterile
soil sievings
and
bluestem
treatment
Pasteurized
with
non-sterile
soil sievings
Amended
with
non-sterile
soil filtrate
with
filtrate
on growth
plant
dry
and
mycorrhizal
root
colonizatmn
of big
soil WI (g)’
Mean
Non-inoculated
root
Inoculated’
colonizatmn’ Non-inoculated
the same superscript
letters
4.08’
0.2Y
413”
0’
2.9gb
2.23’
313’
0
I .09
3.43b are not significantly
(P = 0.05)
212y
different
as determined
0’ by Duncan‘s
multiple
test.
‘Percentage
colomzatron
5 = 76-100% scattered multiplied
(first
colonization.
sites; 2 = larger by intensity
number): Root
but separate
rating
0 = no
colonization
with
400 spores
colonization; intensity
sites; 3 = feeder
of G. efunicorum.
I = l-S%:
(second roots
and these scores were subjected
test. ‘Inoculated
DISCUSSION
soil
Amended
range
AND
Big bluestem plants inoculated with G. etunicatum grew significantly larger than non-inoculated plants, regardless of non-sterile soil amendment (Table 1). Adding soil microbes to mycorrhizal plants significantly reduced big bluestem growth. whereas adding soil microbes to non-inoculated plants significantly improved plant growth. Non-inoculated plants amended with non-sterile soil sievings were significantly larger than those amended with filtrate. When sievings and filtrate were plated onto various media it was evident that sievings contained high populations of fungi (280cfumll’) and bacteria (3.0 x lo4 cfu ml-‘). In contrast, bacteria (2.5 x lo4 cfu ml-‘), but not fungal colonies, grew from the filtrate. Therefore, the lesser growth of plants amended with filtrate rather than sievings may reflect the lack of soil fungi in the filtrate. Mycorrhizal root colonization decreased significantly when non-sterile soil sievings were added to pasteurized soil (Table 1). Root colonization was further reduced in plants amended with filtrate. No mycorrhizal root colonization was evident in noninoculated plants, even those amended with nonsterile soil sievings or filtrate. This suggests that sieving or filtration eliminated or reduced mycorrhizal fungus inoculum to a level unable to colonize or be detected in 3-month-old plants. Since no root colonization was evident in non-inoculated plants, growth improvements following sieving or filtrate amendment is most likely not attributable to indigenous VAM fungi, i.e. those present in non-sterile soil. Addition of sievings or filtrate to pasteurized soil could increase soil organic matter and, therefore, soil fertility explaining the stimulation of non-inoculated plant growth. More likely, however, soil microorganisms in the amendments directly affected plant growth (perhaps by mineralization of nutrients) since addition of the amendments to VAM fungusinoculated plants decreased plant growth. Fertilizer amendment can reduce mycorrhizal root colonization while maintaining or improving plant growth
Inoculated’
Control
‘Means
RESULTS
in pasteurized
Mean Soil
intensity (Kormanik and McGraw, 1982). A simple analysis of variance (P = 0.05) was performed on dry weights and root colonization using Duncan’s multiple range test for mean separation.
number):
almost
2 = &25%;
entirely
to analysis
3 = 2650%;
0 = no colonization; colonized.
of variance
4 = 5l-75%; I = small.
Colonization
and Duncan’s
widely
ratmg multiple
was range
504
B.
Table
2. Dry
weight
or pasteurized
A.
of big bluestem
soil amended
with
DANIEL~
plants after
organisms TSA Mean
Medium
I2 weeks
et al.
growth
dilution-plated or KB
plant
Inoculated’
Non-sterile
HETRICK
aaar
dry
from
in non-sterile non-sterile
or pasteurized
soil onto
PDA.
media
WI (g)’
Mean
Non-inoculated
root
Inoculated’
colonization’ Non-inoculated
I ,I”
2.42d
2.06d
2’1’
Non-amended
3.97’b
0.10’
513”
w
PDA-fungi
3.94.b
1.49E
312’
w
PYE-bacteria
3.17’
0.40’
3.2’
0’
TSA-bacteria
3.81b
0.54’
5.‘3”
0’
KB-bacteria
3.20’
0.38’
413”
0
Bacteria
4.44”
2.4Sd
2,‘2”
0’
Pasteurized
‘-‘See 4All
soil
+ fungi’
footnotes cultured
to Table
organisms
I. were
mixed
together
in equal
(Hayman, 1983). Since the reduction in root colonization in this case accompanied reduced plant growth, the action of soil microorganisms, not fertility changes, is again suggested. Hetrick et af. (1986) demonstrated that additions of 1 or 10% non-sterile soil to pasteurized soil resulted in significant suppression of mycorrhizal plant growth and stimulation of non-VAM fungus-inoculated plant growth. Because it is unlikely that addition of 1% non-sterile soil would dramatically alter soil fertility, they concluded that soil microorganisms were probably responsible for the observed growth responses. Addition of specific groups of soil microorganisms to pasteurized soil significantly altered growth of big bluestem (Table 2). Non-mycorrhizal plant growth was not affected by amendment with bacteria which grew from PYE, TSA or KB; i.e. there was no difference in growth of these compared with non-amended plants. However, non-inoculated plants amended with PDA-fungi or fungi and bacteria together grew significantly larger than non-amended plants. In fact, growth of non-inoculated plants amended with bacteria and fungi together was equal to inoculated plants grown in non-sterile soil. This suggests that plant growth similar to that obtained in natural conditions may be achieved without mycorrhizal fungi. Furthermore, these results suggest that fungi, more than bacteria, are responsible for the observed stimulation of non-mycorrhizal plant growth by nonsterile soil. In plants inoculated with G. erunica~m, plant
Table
3. The influence
of mycorrhizal media
Inoculated’
Non-sterile
soil
Pasteurized
soil
Unamended
volumes
in this treatment.
and soil microorgamsms
of bin bluestem plant
of water
growth in pasteurized soil was significantly greater than that occurring in non-sterile soil. This is consistent with the data of Hetrick et al. (1986) and with the results of the sievings and filtrate experiment. Addition of PDA-fungi or fungi and bacteria together did not alter growth when compared with the non-amended. mycorrhizal plants grown in pasteurized soil. However. addition of PYE- and KBbacteria significantly decreased plant growth but not to the extent observed in non-sterile soil. Apparently, it is the mycorrhizal growth response which is affected by these soil bacteria since they had no effect on non-mycorrhizal plant growth. Fungi grown from plated, non-sterile soil and roots (but not from soil or roots alone) significantly improved growth of non-mycorrhizal plants when compared with non-amended, non-mycorrhizal plants (Table 3). TSA-, PYE- and KB-bacteria did not alter growth when compared with the non-amended, nonmycorrhizal control plants. Bacteria from PI plates, however, significantly improved plant growth while SCA actinomycetes had no effect. Plant growth in non-sterile soil was similar whether or not G. etunicatum inoculum was added. Plants grown in non-sterile soil, as before, were significantly smaller than those grown with mycorrhizal fungus inoculum in pasteurized soil. Growth of mycorrhizal plants was not altered significantly by additions of fungi from roots alone, but was somewhat reduced by addition of fungi from non-sterile soil and roots together and significantly reduced by addition of
inoculation
on growth Mean
Medium
dry
WI (g)’
Non-inoculated
in pasteurized
Isolated
on various
Soil
and
Soil
alone
Mean Inoculated’
root
colomzatlon’ Non-inoculated
2.73’
3.04’
211’
?,‘I‘
5.91’
0.07h
513”
w
5.4C’b
2.06”
2/2”
0’
4.77”
0.10”
3/2”
0’
6.04”
0.25h
5/3”
0’
PI-bacteria
5.64’b
0.938
4i3’
0’
TSA-bacteria
5.45’b
0.05h
413’
0’
KB-bacteria
5.05”
O.OSh
3/2”’
0
PYE-bacteria
5.16bc
0.25h
212””
0’
SCA
3.38’
0.03h
0’
4.25d
0.32gh
312” I/2’?
Roots
“See
roots
plated
plated
alone
plated
actinomycetes bacteria footnotes
+ fungi to Table
I.
culture
soil
PDA-fungi
All
so11 PYE.
0’
Soil
microbes and mycorrhizae
505
Table 4. The effect of fungicide applicatiofts on uptake of 3zP by mycotrhizas Mean ‘*P counts gg’ dry shoot wt’ Fungicide fUR
ai
R-’
SOii)
inoculated’ Bayleton (6.15) PCNB (4.65) Imazalil(46.7) Vitavax (21.8) Benomyl(24.6) Tilt (4.05) Rubigan (I 5.4) No fungicide Non-inoculated No fungicide
Mean root colonization’
--Plant application
Soil application
1802” I I .764’b 10.404=b 2275”& 297 I rbcd 249”’ 847=+”
18,083’ 405vm Il.814~b 821” 1475&d 512c+ 392’ !5.844~b
Plant application
3/Z” 2/2X’ 3/Z” 2/l” 212” 312’ 312”
Z/Z”’ 3i2” 2/2X’ 2/l”? 2;Jl” 3,‘2’! 3!2’ 3,‘2”
0’
0’
‘Means with the same superscript letters are not significantly (P = 0.05) different as dete~~n~ rangeteston log values of counts gg’ shoot dry wt. *.>See footnotes to Table I.
fungi from non-sterile
soil alone. Root colonization was also significantly reduced in the treatments containing non-sterile soil fungi. The inconsistent effect of root and soil fungi on big bluestem growth is difficult to explain. Apparently, fungi from soil alone are deleterious to mycorrhizal plant growth while fungi from roots are not. Further research will be necessary to explain why soil and root fungi stimulate non-mycorrhizal plant growth only when combined but not separately. Growth of mycorrhizal plants was not affected by bacteria from Pf or TSA plates even though significantly less mycorrhizal root colonization was evident in these treatments. Mycorrhizal plant growth was significantly reduced by addition of bacteria from KB or PYE and by bacteria and fungi together. Similarly, the SCA actinomycetes significantly suppressed plant growth and their inclusion in this experiment may account for the high degree of growth suppression observed in the combined fungal and bacterial treatment. Krishna et al. (1982) also observed suppression of mycorrhizal growth response and root colonization by actinomycetes but in their experiments the actinomycetes were beneficial to plant growth in the absence of mycorrhizal fungi. In our experiments no benefit from actinomycetes was evident in nonmycorrhizal plants but the large variety of actinomycete species in that treatment may mask positive and negative growth effects. The fact that all treatments which suppressed plant growth also suppressed mycorrhizal root colonization suggests that these are related phenomena. Hetrick et af. (1986) observed that P amendment to pasteurized soil significantly improved plant growth overcoming the stunting which occurs in the absence of mycorrhizas in pasteurized soil. Therefore, if the fungi which stimulate non-mycorrhizal plant growth are decomposers of organic matter, more P or other nutrients may be available for plant growth explaining the observed increase in plant growth. The presence of such organisms would result in increased plant growth, although perhaps not to the same extent as mycorrhizal fungi, because of nutrient immobilization by soil microbes. Further research will be necessary to determine whether this proposed mechanism is correct and to elucidate the mechanism for growth simulation by PI-bacteria. Similarly, the mechanism for suppression of mycorrhizal plant
----
Soil application
by Duncan’s multiple
growth by certain fungi, actinomycetes and KB- or PYE-bacteria will require further research. However, hyperparasitism, inhibition of spore germination, competition for root colonization sites or competition for nutrients are possible explanations. From these data it appeared that soil microorganisms limit the need for or establishmnent of mycorrhizae. Since these data were obtained under somewhat artificial conditions [i.e. by adding nonsterile soil sievings, filtrate or microorganisms to pasteurized soil), the suppressive effect of soil microorganisms was also tested in non-sterile soil using fungicides to eliminate mycorrhizal nutrient uptake. Of the seven fungicides tested for activity against mycorrhizas, both Tilt and Rubigan significantly reduced (almost 20-fold) mycorrhizal ‘*P uptake, whether they were applied as a soil drench or foliar spray (Table 4). No uptake of 32P was apparent in non-inoculated plants, probably reflecting the stunted growth pattern of these plants which limited penetration of roots into soil containing “P. Root colonization by the mycorrhizal fungus was not affected by fungicide applications, although Vitavax appeared to slightly reduce colonization. The time between fungicide application and experiment termination may not have been sufficient for roots colonized before inoculation to be sloughed off and significant differences in root colonization realized. Altematively, the fungicides may have primarily halted 32P uptake without affecting root colonization. Subsequently, when Tilt and Rubigan were added to pasteurized soil, similar levels of inhibition of 3ZP uptake were evident in VAM fungus-inoculated plants (Table S), while again no uptake occurred in non-mycorrhizal plants. Plants grown in non-sterile soil without additional VAM fungus in~ulum had significantly greater “P uptake than did nonmycorrhizal plants in pasteurized soil. However, “P uptake in non-sterile soil was approx. 10 times lower than that occurring in ptants grown in steamed, mycorrhizal fungus-inoculated soil. Application of Tilt or Rubigan resulted in approx. 4O- and S-fold reductions in 32P uptake by plants grown in pasteurized-inoculated and non-sterile soil, respectively. 32P uptake concentrations in fungicide-treated plants were similar whether soil was pasteurized and inoculated or remained non-sterile (with or without
B. A.
506 Table
5. The
influence
DANIELS HETRICK et al.
of pasteurized
and “P
Soil
treatment
Pasteurized Tilt
Non-sterile Tilt
P-amended
“See
soil
(8.1)
g-’
soil on uptake dry shoot
(30.8pg
558”” ai gg’)
soil
;:
of ‘*P
by mycorrhizas
wt’
Non-inoculated
22,553’
Root Inoculated”
colonization’ Non-inoculated
513’
(Y
5!3’
w
545d
0’
S/3’
w
2159&
5313b
3/2?
3i2’
4046’
93l’d
3 ‘2’
3i2y
(30.8 cg ai gg’)
6735d
106206
312’
312y
pasteurized
ND
300&
ND
w
ND
2316’
ND
w
(8.1 pg ai g-l)
Rubigan Tilt
Inoculated’
(8.1 pg ai g-‘)
Rubigan
non-sterile
counts
and
footnotes
soil
Rubigan
(30.8 pg ai g-‘)
to Table
1.
additional VAM fungus inoculum). Mycorrhizal 32P uptake was inhibited by fungicide application, since 32P uptake by fungicide-treated mycorrhizal plants was similar to uptake by non-mycorrhizal plants growing in fertilized soil. The fact that Tilt and Rubigan application to P-fertilized plants did not alter 32P uptake as compared with non-fungicidetreated, fertilized non-mycorrhizal plants suggests that decreased 32P uptake in fungicide-treated plants is not the result of phytotoxicity. Although mycorrhizal root colonization was significantly lower in non-sterilized soil than in pasteurized, inoculated soil, the fungicides had no detectable influence on root colonization. The effect of fungicides on mycorrhizal fungi is usually evaluated by comparing sporulation or mycorrhizal root colonization in fungicide-treated and nontreated plants (Trappe et al., 1984). Our results support the suggestion by Rhodes and Larsen (1981) that root colonization levels may not accurately gauge the influence of a given fungicide on mycorrhizas because root colonization ratings cannot distinguish between active and inactive mycorrhizas. Studying fungicide effects on mycorrhizal nutrient uptake may provide a more useful approach to fungicide screening. This 32P assay measures the ability of plant roots to explore new soil for P. Even though plants with low growth rate or adequate fertility may be less likely to explore new soil, the present technique has considerable advantages over similar techniques. The technique of Gray and Gerdemann (1969), required 32P to be poured onto plants in small beakers. The relative immobility of P in soil restricted their technique to use in sand. Injection of 12P into soil also proved unacceptable because roots were severed. Distribution of hyphae and roots relative to the injection point was unpredictable and yielded erratic “P uptake. Thus, the present technique is advantageous because it can be used in natural soils and 32P can be uniformly distributed in the outer soil zone, independent of the root or mycelium distribution in the original soil mass. The two fungicides, Tilt and Rubigan, may be more versatile than the PCNB used by Gray and Gerdemann (1969) since either soil or plant application is effective. The approx. IO-fold difference between 32P uptake in steamed-inoculated and non-sterile soil is presumably attributable to microbial suppression of mycorrhizal activity. As in the previous experiments, mycorrhizal root colonization was less in non-sterile soil and 32P uptake is apparently affected as well. Thus, estimates of mycorrhizal benefit to plants, obtained in sterilized soils may significantly over-
estimate mycorrhizal contribution to plant growth. Estimates of mycorrhizal contribution to plant growth obtained in sterilized soil must be reevaluated and viewed as the potential contribution of VAM fungi to plant growth if those factors (presumably soil microbes) in non-sterile soil which limit that potential can be controlled. The microorganisms which stimulate plant growth in pasteurized soil apparently contribute significantly to plant growth in non-sterile soil. Whether such microorganisms can be manipulated to further benefit plant growth in nonsterile soil is yet to be determined. The suppression phenomena studied are obviously related to soil microorganisms other than those associated with mycorrhizal fungus spores, since plant growth is suppressed in pasteurized soil without mycorrhizal fungi but not suppressed in pasteurized soil in the presence of mycorrhizal fungi. It is only when soil microorganisms are present with the mycorrhizal fungus that suppression is observed. Mychorrhizal fungus spore-associated bacteria appear to benefit plant growth, since Azcon-Aguilar and Barea (1985) demonstrated that surface-sterilization of spores inhibited mycorrhiza formation. Similarly, Mayo et al. (1986) demonstrated that mycosphere microorganisms positively influence germination of mycorrhizal fungus spores. Thus, we did not surfacesterilize spores because such treatment would confound results and was not the phenomenon we wished to study. Whether the microbial effects on mycorrhizas, reported here, are widespread and occur for other soils, plant or VAM fungus species remains to be determined. However, they may indeed be widespread since other researchers have observed reduced growth response and root colonization in non-sterile soil (Mosse et al., 1969; Berthelin and Leyval, 1982). That such growth reductions are attributable to soil microorganism suppression of mycorrhizal activity is conceivable since Bowen and Theodorou (1978) observed suppression of ectomycorrhizal root colonization by bacteria. Similarly, Ross (1980) demonstrated that the soil microflora could inhibit VAM fungus sporulation. In contrast, Azcon-Aguilar and Barea (1985) observed increased incidence of mycorrhizas and infectivity of mycorrhizal fungi when non-sterile soil filtrate was added to a sterilized soil-sand mix. These effects were attributed to stimulation of saprophytic growth of mycorrhizal fungi by soil microorganisms. The results presented here suggest more diverse effects of soil microorganisms on mycorrhizal symbiosis, since both positive and negative effects were observed.
Soil microbes and mycorrhizae REFERENCES
Ames R. N., Reid C. P. P. and Ingham E. R. (1984) Rhizosphere bacterial population responses to root colonization by a vesicular-arbuscular mycorrhizal fungus. New, Phytologist %, 555-563. Azcon-Aguilar C. and Barea J. M. (1985) Effect of soil micro-organisms on formation of vesicular arbuscular mycorrhizas. Transactions of the British Mycological Society 84, 536537.
Bagyaraj D. J. and Menge J. A. (1978) Interaction between a VA mycorrhiza and Azorobacter and their effects on rhizosphere microflora and plant growth. New Phytologist 80, 567-573.
Bagyaraj D. J., Manjunath A. and Patil R. B. (1979) Interaction between a VA mycorrhiza and Rhizobium and their effects on soybean in the field. New Phytologist 82, 141-145.
Barea J. M., Azcon R. and Hayman D. S. (1975) Possible synergistic interactions between Endogone and phosphate-solubilizing bacteria in low-phosphate soils. In Endomycorrhizas (F. E. Sanders, B. Mosse and P. B. Tinker, Eds), pp. 409-418. Academic Press, London. Berthelin J. and Leyval C. (1982) Ability of symbiotic and nonsymbiotic rhizospheric microflora of maize (Zea mays) to weather micas and to promote plant growth and plant nutrition. Plant and Soil 68, 369-377. Bethlenfalvay G. J., Pacovoky R. S., Bayne H. G. and Stafford A. E. (1982) Interactions between nitrogen fixation, mycorrhizal colonization, and host-plant growth in the Phaseolus-RhizobiumClomus symbiosis. Plant Physiology 70, 446450.
Bowen G. D. and Theodorou C. (1978) Interactions between bacteria and ecto-mycorrhizal fungi. Soil Biology & Biochemistry 11, 119-126.
Daniels B. A. and Skipper H. D. (1982) Methods for the recovery and quantitative estimation of propagules from soil. In Methods and Principles of Mycorrhizal Research (N. C. Schenck, Ed.), pp. 29-37. American Phytopathological Society, St Paul, Minn. Dehne H. W. (1982) Interaction between vesiculararbuscular mycorrhizal fungi and plant pathogens. Phytopathology 72, 1115-l 118. Gray L. E. and Gerdemann J. W. (1969) Uptake of phosphorus-32 by vesicular-arbuscular mycorrhizae. Plant Soil 30, 415422.
Hayman D. S. (1983) The physiology of vesiculararbuscular endomycorrhizal symbiosis. Canadian Journal of Botany 61, 944963.
Hetrick B. A. D., Kitt D. G. and Wilson G. T. (1986) The influence of phosphorus fertilization, drought, fungal species and soil microorganisms on mycorrhizal growth response in tallgrass prairie plants. Canadian Journal of Botany. 64, 11991203. Hirsch C. F. and Christenson D. L. (1983) Novel method for selective isolation of actinomycetes. Applied and Environmental Microbiology 46, 925-929.
Kormanik P. P. and McGraw A.-C. (1982) Quantification of vesicular-arbuscular mycorrhizae in plant roots. In
507
Methods and Principles of Mycorrhizal Research (N. C.
Schenck, Ed.), pp. 3747. American Phytopathological Society, St Paul, Minn. Krishna K. R., Balakrishna A. N. and Bagyaraj D. J. (1982) Interaction between a vesicular-arbuscular mycorrhizal fungus and Streptomyces cinnamomeous and their effects on finger millet. New Phytologist 92, 401-405. Manjunath A., Mohan R. and Bagyaraj D. J. (1981) Interaction between Beijerinckia mobilis. Aspergillus niger and Glomus fasciculatus and their effects on growth of onion. New: Phytologist 87, 723-727. Mayo K., Davis R. E. and Motta J. (1986) Stimulation of germination of spores of Glomus verstforme by spore associated bacteria. Mycologia 78, 42a3 I. Menge J. A. (1983) Utilization of vesicular-arbuscular mycorrhizal fungi in agriculture. Canadian Journal of Botany 61, 1015-1024.
Meyer J. R. and Linderman R. G. (1986) Response of subterranean clover to dual inoculation with vesiculararbuscular mycorrhizal fungi and a plant growthpromoting bacterium, Pseudomonas putida. Soil Biology & Biochemistry 18, 185-190. Mosse B., Hayman D. S. and Ide G. J. (1969) Growth responses of plants in unsterilized soil to inoculation with vesicular-arbuscular mycorrhiza. ‘Vature, London 224, 1031-1032.
Mosse B., Powell C. L. and Hayman D. S. (1976) Plant growth responses to vesicular-arbuscular mycorrhiza. XI. Interactions between VA mycorrhiza, rock phosphate and symbiotic nitrogen fixation. Nen’ Phytologist 76, 331-342.
Phillips J. M. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 1588160. Rhodes L. H. and Larsen P. 0. (1981) Effects of fungicides on mycorrhizal development of creeping bentgrass. Plant Disease 65, 145-147.
Ross J. P. (1980) Effect of nontreated field soil on sporulation of vesicular-arbuscular mycorrhizal fungi associated with soybean. Plant Pathologist, USDA, Science and Education Administration, Agricultural Research, and Department of Plant Pathology, North Carolina State University, Raleigh, N.C. Ross J. P. and Daniels B. A. (1982) Hyperparasitism of endomycorrhizal fungi. In Methods and -Principles of Mvcorrhizal Research (N. C. Schenck. Ed.). DD. 5-59. American Phytopathological Society, St Pa;i, ‘Minn. Schenck N. C. and Kellam M. K. (1978) The influence of vesicular arbuscular mycorrhizae on disease development. Agricultural Experiment Station, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Fla, Bulletin 799. Trappe J. M., Molina R. and Castellano M. (1984) Reactions of mycorrhizal fungi and mycorrhiza formation to pesticides. Annual Review of Phytopathology 22, 331-359.
Copyright 0
Printed in Great
0038-0717/88 s3.00 + 0.00 1988 Pergamon Press plc
EFFECTS OF SOIL MICROORGANISMS ON MYCORRHIZAL CONTRIBUTION TO GROWTH OF BIG BLUESTEM GRASS IN NON-STERILE SOIL B. A. DANIELSHETRICK,’ G. THOMPSON WILSON,’ D. GERSCHEFSKE Kin’ and A. P. SCHWAB~ Departments
of ‘Plant Pathology and 2Agronomy, Kansas State University, Manhattan, KS 66506, U.S.A. (Accepted 25 November 1987)
Summary-Plant dry weight and mycorrhizal root colonization of big bluestem (Andropogon gerurdii Vitman) inoculated with Glomus etunicurum Becker and Gerd. were suppressed if non-sterile prairie soil sievings or filtrate were added to pasteurized soil. Addition of prairie soil microorganisms, isolated onto peptone yeast extract, King’s B, or starch casein agar media, to pasteurized soil also reduced dry weight and mycorrhizal root colonization of big bluestem insulate with G. etunicatum. In contrast, addition of non-sterile soil sievings or filtrate or organisms isolated onto potato dextrose or p~udomonas isolation agar to pasteurized soil improved growth of non-myco~hi~l big bluestem. These effects of soil microorganisms on plant growth were further quantified by comparing 32P uptake by fungicide treated and untreated mycorrhizal plants and by non-mycorrhizal plants in pasteurized and non-sterile soil. Mycorrhizal plants grown in pasteurized soil absorbed ‘c IO times more “P than mycorrhizai plants grown in non-sterile soil. Application of propiconazole (Tilt) or fenarimol (Rub&an), fungicides which inhibit )*P uptake by mycorrhizas. resulted in approx. 40- and S-fold reductions in 32Puptake in pasteurized and non-sterile soil, respectively. Thus, more 32P is absorbed in pasteurized than in non-sterile soil, probably because soil microorganisms limit mycorrhizal activity in non-sterile soil. Assessments of mycorrhizal contribution to plant growth conducted in sterilized soils may significantly overestimate the effects of
VAM fungi because other soil microorganisms are not considered.
A wide array of interactions between vesiculararbuscular mycorrhizal (VAM) fungi and soil microorganisms have been studied, including interactions with plant pathogens (Schenck and Kellam, 1978; Dehne, 1982), nitrogen-fixing organisms (Bagyaraj and Menge, 1978; Bagyaraj et al., 1979; Manjunath et al., 1981; Mosse er al., 1976; Bethlenfalvay et al., 1982), phosphate-solubilizing microorganisms (Barea et al., 1975; Manjunath et al., 1981) and hyperparasites (Ross and Daniels, 1982). It has been demonstrated that the microflora population was different in the rhizosphere of mycorrhiza1 plants as compared with non-rny~o~hi~l plants (Ames et al., 1984). The influence of the rhizosphere microflora on mycorrhizas is still poorly understood, although positive effects of the microflora on mycorrhizal root colonization (Azcon-Aguilar and Barea, 1985) and plant growth (Meyer and Linderman, 1986) have been reported. However, the influence of the soil microflora on mycorrhizal contribution to plant growth has not been assessed. VAM fungi can dramatically improve growth of many crop plants. However, the ubiquitous nature of VAM fungi in soils necessitates that they be first eliminated from soil and then reinoculated under controlled conditions before their effects on plant growth can be assessed by comparing mycorrhizai and non-myco~hiza1 plants. The effect of other soil microorganisms, eliminated during soil sterilization, on plant growth or mycorrhizal growth response 501
is generally not considered. The effects of these organisms is probabfy si~ificant, however, since additions of non-sterile soil to pasteurized soil can suppress mycorrhizal growth response and inhibit mycorrhizal root colonization in big bluestem (Hetrick et al., 1986). The contribution of VAM fungi to plant growth in non-sterile soil may differ from that demonstrated in sterilized soil. However, it is extremely difficult to assess the contribution of VAM fungi to plant growth in non-sterile soil. We examined the restoration of soil microorganisms including mycorrhizal fungi to pasteurized soit. Uptake of j2P by mycorrhizal plants in nonsterile and pasteurized soil was compared using a modi~cation of the technique developed by Gray and Gerdemann (1969). We also assessed the effects of soil microorganisms on mycorrhizal symbiosis in big bluestem, a native plant of tallgrass prairies. MATERIALSAND METHODS Plant and inoculum preparation
For each experiment, seeds of big bluestem (Androwere germinated in vermiculite. Two weeks after emergence, one seedling was transplanted to each pot containing freshly-collected tallgrass prairie soil (T&y silty clay loam, fine, mixed mesic Pachic Argiustoll) containing 5 pgg-’ plant available P (Bray test I). Experiments were conducted in 6 x 25 cm plastic pots except those involving 32P uptake which had 9 x 12 cm plastic sleeves inside a
pogon gerardii Vitman)
502
B. A.
DANIELS HETRXK et
15 x 16 cm pot. Spores of Glomus etunicat~m Becker and Gerd. were collected from Sudan grass (~o~g~~ uu!gare var. sirdanense [piper] Hitch) pot cultures by wet sieving, decanting and sucrose centrifugation (Daniels and Skipper, 1982). Inoculation was accomplished by pipeting approx 400 spores onto roots of each seedling at transplanting.
Injluence of non-sterile soil sievings and filtrate growth of big bluestem
on
Freshly-collected prairie soil (400g dry wt) was blended with 4000 ml sterile water and sieved ( < 38 pm). The sieved soil suspension was added in 125-ml aliquots to 14 pots containing 400 g (dry wt) pasteurized prairie soil. Sterile water (125 ml) was added to 14 control pots. The remaining suspension was then further filtered through 615 grade filter paper (Whatman 1) and 125 ml of this filtrate was added to 14 pots containing pasteurized soil. Half of the 14 pots of each treatment were inoculated with G. etunicatum spores. To determine whether fungi or bacteria were present in the sieved suspension or filtrate, 0.1 ml of suspension or filtrate was plated onto Petri dishes containing peptone yeast extract agar, tryptic soy or potato dextrose agar, and plates were maintained at 22°C for 7days. Z~~~e~ce offingi and bacteria from non-sterjle soil on mycorrhj~aI growth response Three 25-g samples of freshly-collected tallgrass prairie soil were each blended with 250ml sterile water and 0.1 ml of the suspension was plated onto two Petri dishes containing 0.6% Difco potato dextrose agar (PDA) (amended with 0.1 gl-’ streptomycin and chloramphenical), peptone yeast extract (PYE), King’s B (KB) or tryptic soy agar (TSA) media (Difco Laboratories, Detroit, Mich.). Plated agar dishes were maintained at 22°C for 7 days, then flooded with 10 ml sterile distilled water and scraped with a glass rod to remove microbial growth. For each of the four media, organisms from all replicate plates were combined. Sterile distilled water was added to produce a finat volume of 100 ml. For each of the four media, 5 ml of microbial suspension were pipetted into I2 pots containing 430g (dry wt) of pasteurized prairie soil. For controls, 5 ml of water acquired from flooding non-inoculated plates were added to 12 pots containing pasteurized and nonsterile soil. The use of a water control ensured that nutrient contained in culture media did not cause the observed effects. Half of the 12 pots of each treatment were inoculated with G. etunicatum spores. In a subsequent experiment these collections were repeated but starch casein agar (10 g starch, 0.3 g casein, 2.0 g KNO,, 2.0 g NaCl, 2.Og K~HPO~,5OmgMgSO~~7H~O,2Omg~a~O~, 1Omg FeSO,.H?O, 18 g agar, 50 pg g-l cycloheximide) (Hirsch and Christenson, 1983) and pseudomonas isolation (PI) agar (Difco Laboratories, Detroit, Mich.) were included for isolation of actinomycetes and pseudomonads, respectively. Rhizosphere fungi were separated from soil fungi in this subsequent experiment by removing three 25-g (fresh wt) root samples from soil. Roots were shaken to remove adherent soil, blended in 250 ml water, and 0.1 ml plated onto 0.6% PDA (amended with 0.1 g 1-l strep-
al.
tomycin and chloramphenicol). Soil fungi were isolated by dry-sieving soil through a 355~grn sieve to remove roots. Three 25-g samples of sieved soil were blended in 250 ml water, and 0.1 ml plated onto PDA as above. For comparison. soil samples containing roots were also plated as in the previous experiment. After 7days, microorganisms were removed from the media and diluted with distilled water as previously described. Solution turbidities of 30, 20. 85 and 100 Klett units were determined for organisms removed from TSA, PYE. KB and PI media, respectively (Klett-Summerson Photoelectric Colorimeter, New York). Fungi grown on PDA were washed from the culture medium and added to sterile distilled water to a total volume of 100 ml. Hemacytometer readings from roots contained approx. 1.4 x IO’ fungal spores ml-‘, soil without roots contained 1.3 x 10’ fungal spores ml-’ and soil with roots contained 4.4 x 10’ spores ml-‘. influence of non-sterile soil on uptake of 3’P bJ mycorrhizae
Seven fungicides were screened for inhibition of nutrient uptake by mycorrhizal fungi. Initially, 9 x 12 cm plastic sleeves were placed inside 15 x 16 cm pots. Each sleeve was filled with 600 g (dry wt) pasteurized prairie soil, into which one seedling was transplanted; 75 such sleeves were inoculated with G. etun~catam and 5 were not. Seedings were grown in the greenhouse for 7 weeks. Thereafter, 25 ml of a fungicide were applied to five inoculated plants as a soil drench and to five inoculated plants as a foliar spray in the following concentrations (pg 8-l): 6.15 triadimefon (Bayleton); 4.65 pentachloronitrobenzene (PCNB); 47.7 imazilil (Imazilil); 21.8 carboxin/thiram (Vitavax); 24.6 benomy1 (Benlate); 15.4 fenarimol (Rubigan); and 4.05 propiconazole (Tilt). As a control, 25 ml sterile distilled water were applied as a soil drench and a foliar spray to the remaining five inoculated and five noninoculated plants. After 72 h, the plastic sleeves were removed without disturbing the soil mass containing the plant. Additional soil (325 g) was placed around the original soil mass in three layers. The bottom and top layers consisted of 75 g (dry wt) each of pasteurized prairie soil while the middle layer consisted of 175 g pasteurized prairie soil amended with 10 FCi ‘*P tracer solution. This ensured that roots at the bottom of the pot were not placed in direct contact with tracer. The upper non-amended soil layer blocked radiation sufficiently to allow the use of a 6-m tube survey meter to determine when 3zP had been translocated into plant shoots. Seven days after “P addition, plant shoots were harvested, dried, weighed, wet-ashed (in 15 mf 5:2: 1 nitric-perchloric-sulfuric acid), and diluted to 50 ml with distilled water. A 2.5-ml aliquot of this solution was mixed with 15 ml Aquasol II cocktail (DuPont NEN Research Products, Boston, Mass.) and radioassayed using a United Technologies Packard scintillation counter. Roots from each pot were carefully removed from soil with tweezers and washed free of soil, stained in trypan blue, and examined microscopically to determine percentage root colonization and colonization intensity (Kormanik and McGraw, 1982).
503
Soil microbes and mycorrhizae A simple analysis of variance (P = 0.05) was performed on log values of scintillation counts g-’ shoot dry wt, and on root colonization data using Duncan’s multiple range test for mean separation. The influence of non-sterile soil on mycorrhizal uptake of 32P was studied using Tilt and Rubigan, the fungicides which proved effective in this initial experiment. Sixty plastic sleeves were filled with 600 g (dry wt) soil: 30 with pasteurized prairie soil and 30 with freshly-collected non-sterile prairie soil. Half of the pots containing each soil treatment were inoculated with G. etunicatum at the time of seedling transplanting. In addition, 10 sleeves were filled with 600g pasteurized soil amended with lOpgg_‘P as KH2P04, and not inoculated. This produced growth of non-mycorrhizal plants comparable to mycorrhizal plants, allowing comparison of equal-sized plants. Seedlings were grown in the greenhouse for 6 weeks, after which 25ml aliquots of 31.2pgg-’ Rubigan or 8.37 pgg-’ Tilt or sterile water were applied as soil drenches to five replicate inoculated and non-inoculated plants in pasteurized and nonsterile soil. Higher fungicide dosages were used in this experiment to ensure maximum effectiveness and to assess whether these fungicides could be fungitoxic. A 25ml aliquot of each fungicide was applied to five of the P-amended plants to evaluate whether or not the fungicides were phytotoxic. Sterile water was applied to the remaining replications of inoculated, non-inoculated and P-amended plants. Three days after fungicide application, the sleeves were removed and 20 pCi 32P tracer were added. Ten days later plant shoots were harvested, dried, wet-digested and radio-assayed. Roots were also harvested, stained and examined for root colonization. Experimental design and maintenance
For each experiment, the containers were arranged in a completely-randomized design and plants grown in a 15-25’C greenhouse. Plants were fertilized every other week with 368 mg Peter’s No-Phos Special fertilizer solution (Peter’s Fertilizer Products, Fogelsville, Pa) delivered in 10ml H20. For all microbial isolation studies, top and root dry weights were determined at harvest after 13 weeks. The dried roots were subsampled, stained in trypan blue (Phillips and Hayman, 1970), and examined microscopically to assess percentage root colonization and colonization
Table
I. The
influence
of non-sterile
soil sievings
and
bluestem
treatment
Pasteurized
with
non-sterile
soil sievings
Amended
with
non-sterile
soil filtrate
with
filtrate
on growth
plant
dry
and
mycorrhizal
root
colonizatmn
of big
soil WI (g)’
Mean
Non-inoculated
root
Inoculated’
colonizatmn’ Non-inoculated
the same superscript
letters
4.08’
0.2Y
413”
0’
2.9gb
2.23’
313’
0
I .09
3.43b are not significantly
(P = 0.05)
212y
different
as determined
0’ by Duncan‘s
multiple
test.
‘Percentage
colomzatron
5 = 76-100% scattered multiplied
(first
colonization.
sites; 2 = larger by intensity
number): Root
but separate
rating
0 = no
colonization
with
400 spores
colonization; intensity
sites; 3 = feeder
of G. efunicorum.
I = l-S%:
(second roots
and these scores were subjected
test. ‘Inoculated
DISCUSSION
soil
Amended
range
AND
Big bluestem plants inoculated with G. etunicatum grew significantly larger than non-inoculated plants, regardless of non-sterile soil amendment (Table 1). Adding soil microbes to mycorrhizal plants significantly reduced big bluestem growth. whereas adding soil microbes to non-inoculated plants significantly improved plant growth. Non-inoculated plants amended with non-sterile soil sievings were significantly larger than those amended with filtrate. When sievings and filtrate were plated onto various media it was evident that sievings contained high populations of fungi (280cfumll’) and bacteria (3.0 x lo4 cfu ml-‘). In contrast, bacteria (2.5 x lo4 cfu ml-‘), but not fungal colonies, grew from the filtrate. Therefore, the lesser growth of plants amended with filtrate rather than sievings may reflect the lack of soil fungi in the filtrate. Mycorrhizal root colonization decreased significantly when non-sterile soil sievings were added to pasteurized soil (Table 1). Root colonization was further reduced in plants amended with filtrate. No mycorrhizal root colonization was evident in noninoculated plants, even those amended with nonsterile soil sievings or filtrate. This suggests that sieving or filtration eliminated or reduced mycorrhizal fungus inoculum to a level unable to colonize or be detected in 3-month-old plants. Since no root colonization was evident in non-inoculated plants, growth improvements following sieving or filtrate amendment is most likely not attributable to indigenous VAM fungi, i.e. those present in non-sterile soil. Addition of sievings or filtrate to pasteurized soil could increase soil organic matter and, therefore, soil fertility explaining the stimulation of non-inoculated plant growth. More likely, however, soil microorganisms in the amendments directly affected plant growth (perhaps by mineralization of nutrients) since addition of the amendments to VAM fungusinoculated plants decreased plant growth. Fertilizer amendment can reduce mycorrhizal root colonization while maintaining or improving plant growth
Inoculated’
Control
‘Means
RESULTS
in pasteurized
Mean Soil
intensity (Kormanik and McGraw, 1982). A simple analysis of variance (P = 0.05) was performed on dry weights and root colonization using Duncan’s multiple range test for mean separation.
number):
almost
2 = &25%;
entirely
to analysis
3 = 2650%;
0 = no colonization; colonized.
of variance
4 = 5l-75%; I = small.
Colonization
and Duncan’s
widely
ratmg multiple
was range
504
B.
Table
2. Dry
weight
or pasteurized
A.
of big bluestem
soil amended
with
DANIEL~
plants after
organisms TSA Mean
Medium
I2 weeks
et al.
growth
dilution-plated or KB
plant
Inoculated’
Non-sterile
HETRICK
aaar
dry
from
in non-sterile non-sterile
or pasteurized
soil onto
PDA.
media
WI (g)’
Mean
Non-inoculated
root
Inoculated’
colonization’ Non-inoculated
I ,I”
2.42d
2.06d
2’1’
Non-amended
3.97’b
0.10’
513”
w
PDA-fungi
3.94.b
1.49E
312’
w
PYE-bacteria
3.17’
0.40’
3.2’
0’
TSA-bacteria
3.81b
0.54’
5.‘3”
0’
KB-bacteria
3.20’
0.38’
413”
0
Bacteria
4.44”
2.4Sd
2,‘2”
0’
Pasteurized
‘-‘See 4All
soil
+ fungi’
footnotes cultured
to Table
organisms
I. were
mixed
together
in equal
(Hayman, 1983). Since the reduction in root colonization in this case accompanied reduced plant growth, the action of soil microorganisms, not fertility changes, is again suggested. Hetrick et af. (1986) demonstrated that additions of 1 or 10% non-sterile soil to pasteurized soil resulted in significant suppression of mycorrhizal plant growth and stimulation of non-VAM fungus-inoculated plant growth. Because it is unlikely that addition of 1% non-sterile soil would dramatically alter soil fertility, they concluded that soil microorganisms were probably responsible for the observed growth responses. Addition of specific groups of soil microorganisms to pasteurized soil significantly altered growth of big bluestem (Table 2). Non-mycorrhizal plant growth was not affected by amendment with bacteria which grew from PYE, TSA or KB; i.e. there was no difference in growth of these compared with non-amended plants. However, non-inoculated plants amended with PDA-fungi or fungi and bacteria together grew significantly larger than non-amended plants. In fact, growth of non-inoculated plants amended with bacteria and fungi together was equal to inoculated plants grown in non-sterile soil. This suggests that plant growth similar to that obtained in natural conditions may be achieved without mycorrhizal fungi. Furthermore, these results suggest that fungi, more than bacteria, are responsible for the observed stimulation of non-mycorrhizal plant growth by nonsterile soil. In plants inoculated with G. erunica~m, plant
Table
3. The influence
of mycorrhizal media
Inoculated’
Non-sterile
soil
Pasteurized
soil
Unamended
volumes
in this treatment.
and soil microorgamsms
of bin bluestem plant
of water
growth in pasteurized soil was significantly greater than that occurring in non-sterile soil. This is consistent with the data of Hetrick et al. (1986) and with the results of the sievings and filtrate experiment. Addition of PDA-fungi or fungi and bacteria together did not alter growth when compared with the non-amended. mycorrhizal plants grown in pasteurized soil. However. addition of PYE- and KBbacteria significantly decreased plant growth but not to the extent observed in non-sterile soil. Apparently, it is the mycorrhizal growth response which is affected by these soil bacteria since they had no effect on non-mycorrhizal plant growth. Fungi grown from plated, non-sterile soil and roots (but not from soil or roots alone) significantly improved growth of non-mycorrhizal plants when compared with non-amended, non-mycorrhizal plants (Table 3). TSA-, PYE- and KB-bacteria did not alter growth when compared with the non-amended, nonmycorrhizal control plants. Bacteria from PI plates, however, significantly improved plant growth while SCA actinomycetes had no effect. Plant growth in non-sterile soil was similar whether or not G. etunicatum inoculum was added. Plants grown in non-sterile soil, as before, were significantly smaller than those grown with mycorrhizal fungus inoculum in pasteurized soil. Growth of mycorrhizal plants was not altered significantly by additions of fungi from roots alone, but was somewhat reduced by addition of fungi from non-sterile soil and roots together and significantly reduced by addition of
inoculation
on growth Mean
Medium
dry
WI (g)’
Non-inoculated
in pasteurized
Isolated
on various
Soil
and
Soil
alone
Mean Inoculated’
root
colomzatlon’ Non-inoculated
2.73’
3.04’
211’
?,‘I‘
5.91’
0.07h
513”
w
5.4C’b
2.06”
2/2”
0’
4.77”
0.10”
3/2”
0’
6.04”
0.25h
5/3”
0’
PI-bacteria
5.64’b
0.938
4i3’
0’
TSA-bacteria
5.45’b
0.05h
413’
0’
KB-bacteria
5.05”
O.OSh
3/2”’
0
PYE-bacteria
5.16bc
0.25h
212””
0’
SCA
3.38’
0.03h
0’
4.25d
0.32gh
312” I/2’?
Roots
“See
roots
plated
plated
alone
plated
actinomycetes bacteria footnotes
+ fungi to Table
I.
culture
soil
PDA-fungi
All
so11 PYE.
0’
Soil
microbes and mycorrhizae
505
Table 4. The effect of fungicide applicatiofts on uptake of 3zP by mycotrhizas Mean ‘*P counts gg’ dry shoot wt’ Fungicide fUR
ai
R-’
SOii)
inoculated’ Bayleton (6.15) PCNB (4.65) Imazalil(46.7) Vitavax (21.8) Benomyl(24.6) Tilt (4.05) Rubigan (I 5.4) No fungicide Non-inoculated No fungicide
Mean root colonization’
--Plant application
Soil application
1802” I I .764’b 10.404=b 2275”& 297 I rbcd 249”’ 847=+”
18,083’ 405vm Il.814~b 821” 1475&d 512c+ 392’ !5.844~b
Plant application
3/Z” 2/2X’ 3/Z” 2/l” 212” 312’ 312”
Z/Z”’ 3i2” 2/2X’ 2/l”? 2;Jl” 3,‘2’! 3!2’ 3,‘2”
0’
0’
‘Means with the same superscript letters are not significantly (P = 0.05) different as dete~~n~ rangeteston log values of counts gg’ shoot dry wt. *.>See footnotes to Table I.
fungi from non-sterile
soil alone. Root colonization was also significantly reduced in the treatments containing non-sterile soil fungi. The inconsistent effect of root and soil fungi on big bluestem growth is difficult to explain. Apparently, fungi from soil alone are deleterious to mycorrhizal plant growth while fungi from roots are not. Further research will be necessary to explain why soil and root fungi stimulate non-mycorrhizal plant growth only when combined but not separately. Growth of mycorrhizal plants was not affected by bacteria from Pf or TSA plates even though significantly less mycorrhizal root colonization was evident in these treatments. Mycorrhizal plant growth was significantly reduced by addition of bacteria from KB or PYE and by bacteria and fungi together. Similarly, the SCA actinomycetes significantly suppressed plant growth and their inclusion in this experiment may account for the high degree of growth suppression observed in the combined fungal and bacterial treatment. Krishna et al. (1982) also observed suppression of mycorrhizal growth response and root colonization by actinomycetes but in their experiments the actinomycetes were beneficial to plant growth in the absence of mycorrhizal fungi. In our experiments no benefit from actinomycetes was evident in nonmycorrhizal plants but the large variety of actinomycete species in that treatment may mask positive and negative growth effects. The fact that all treatments which suppressed plant growth also suppressed mycorrhizal root colonization suggests that these are related phenomena. Hetrick et af. (1986) observed that P amendment to pasteurized soil significantly improved plant growth overcoming the stunting which occurs in the absence of mycorrhizas in pasteurized soil. Therefore, if the fungi which stimulate non-mycorrhizal plant growth are decomposers of organic matter, more P or other nutrients may be available for plant growth explaining the observed increase in plant growth. The presence of such organisms would result in increased plant growth, although perhaps not to the same extent as mycorrhizal fungi, because of nutrient immobilization by soil microbes. Further research will be necessary to determine whether this proposed mechanism is correct and to elucidate the mechanism for growth simulation by PI-bacteria. Similarly, the mechanism for suppression of mycorrhizal plant
----
Soil application
by Duncan’s multiple
growth by certain fungi, actinomycetes and KB- or PYE-bacteria will require further research. However, hyperparasitism, inhibition of spore germination, competition for root colonization sites or competition for nutrients are possible explanations. From these data it appeared that soil microorganisms limit the need for or establishmnent of mycorrhizae. Since these data were obtained under somewhat artificial conditions [i.e. by adding nonsterile soil sievings, filtrate or microorganisms to pasteurized soil), the suppressive effect of soil microorganisms was also tested in non-sterile soil using fungicides to eliminate mycorrhizal nutrient uptake. Of the seven fungicides tested for activity against mycorrhizas, both Tilt and Rubigan significantly reduced (almost 20-fold) mycorrhizal ‘*P uptake, whether they were applied as a soil drench or foliar spray (Table 4). No uptake of 32P was apparent in non-inoculated plants, probably reflecting the stunted growth pattern of these plants which limited penetration of roots into soil containing “P. Root colonization by the mycorrhizal fungus was not affected by fungicide applications, although Vitavax appeared to slightly reduce colonization. The time between fungicide application and experiment termination may not have been sufficient for roots colonized before inoculation to be sloughed off and significant differences in root colonization realized. Altematively, the fungicides may have primarily halted 32P uptake without affecting root colonization. Subsequently, when Tilt and Rubigan were added to pasteurized soil, similar levels of inhibition of 3ZP uptake were evident in VAM fungus-inoculated plants (Table S), while again no uptake occurred in non-mycorrhizal plants. Plants grown in non-sterile soil without additional VAM fungus in~ulum had significantly greater “P uptake than did nonmycorrhizal plants in pasteurized soil. However, “P uptake in non-sterile soil was approx. 10 times lower than that occurring in ptants grown in steamed, mycorrhizal fungus-inoculated soil. Application of Tilt or Rubigan resulted in approx. 4O- and S-fold reductions in 32P uptake by plants grown in pasteurized-inoculated and non-sterile soil, respectively. 32P uptake concentrations in fungicide-treated plants were similar whether soil was pasteurized and inoculated or remained non-sterile (with or without
B. A.
506 Table
5. The
influence
DANIELS HETRICK et al.
of pasteurized
and “P
Soil
treatment
Pasteurized Tilt
Non-sterile Tilt
P-amended
“See
soil
(8.1)
g-’
soil on uptake dry shoot
(30.8pg
558”” ai gg’)
soil
;:
of ‘*P
by mycorrhizas
wt’
Non-inoculated
22,553’
Root Inoculated”
colonization’ Non-inoculated
513’
(Y
5!3’
w
545d
0’
S/3’
w
2159&
5313b
3/2?
3i2’
4046’
93l’d
3 ‘2’
3i2y
(30.8 cg ai gg’)
6735d
106206
312’
312y
pasteurized
ND
300&
ND
w
ND
2316’
ND
w
(8.1 pg ai g-l)
Rubigan Tilt
Inoculated’
(8.1 pg ai g-‘)
Rubigan
non-sterile
counts
and
footnotes
soil
Rubigan
(30.8 pg ai g-‘)
to Table
1.
additional VAM fungus inoculum). Mycorrhizal 32P uptake was inhibited by fungicide application, since 32P uptake by fungicide-treated mycorrhizal plants was similar to uptake by non-mycorrhizal plants growing in fertilized soil. The fact that Tilt and Rubigan application to P-fertilized plants did not alter 32P uptake as compared with non-fungicidetreated, fertilized non-mycorrhizal plants suggests that decreased 32P uptake in fungicide-treated plants is not the result of phytotoxicity. Although mycorrhizal root colonization was significantly lower in non-sterilized soil than in pasteurized, inoculated soil, the fungicides had no detectable influence on root colonization. The effect of fungicides on mycorrhizal fungi is usually evaluated by comparing sporulation or mycorrhizal root colonization in fungicide-treated and nontreated plants (Trappe et al., 1984). Our results support the suggestion by Rhodes and Larsen (1981) that root colonization levels may not accurately gauge the influence of a given fungicide on mycorrhizas because root colonization ratings cannot distinguish between active and inactive mycorrhizas. Studying fungicide effects on mycorrhizal nutrient uptake may provide a more useful approach to fungicide screening. This 32P assay measures the ability of plant roots to explore new soil for P. Even though plants with low growth rate or adequate fertility may be less likely to explore new soil, the present technique has considerable advantages over similar techniques. The technique of Gray and Gerdemann (1969), required 32P to be poured onto plants in small beakers. The relative immobility of P in soil restricted their technique to use in sand. Injection of 12P into soil also proved unacceptable because roots were severed. Distribution of hyphae and roots relative to the injection point was unpredictable and yielded erratic “P uptake. Thus, the present technique is advantageous because it can be used in natural soils and 32P can be uniformly distributed in the outer soil zone, independent of the root or mycelium distribution in the original soil mass. The two fungicides, Tilt and Rubigan, may be more versatile than the PCNB used by Gray and Gerdemann (1969) since either soil or plant application is effective. The approx. IO-fold difference between 32P uptake in steamed-inoculated and non-sterile soil is presumably attributable to microbial suppression of mycorrhizal activity. As in the previous experiments, mycorrhizal root colonization was less in non-sterile soil and 32P uptake is apparently affected as well. Thus, estimates of mycorrhizal benefit to plants, obtained in sterilized soils may significantly over-
estimate mycorrhizal contribution to plant growth. Estimates of mycorrhizal contribution to plant growth obtained in sterilized soil must be reevaluated and viewed as the potential contribution of VAM fungi to plant growth if those factors (presumably soil microbes) in non-sterile soil which limit that potential can be controlled. The microorganisms which stimulate plant growth in pasteurized soil apparently contribute significantly to plant growth in non-sterile soil. Whether such microorganisms can be manipulated to further benefit plant growth in nonsterile soil is yet to be determined. The suppression phenomena studied are obviously related to soil microorganisms other than those associated with mycorrhizal fungus spores, since plant growth is suppressed in pasteurized soil without mycorrhizal fungi but not suppressed in pasteurized soil in the presence of mycorrhizal fungi. It is only when soil microorganisms are present with the mycorrhizal fungus that suppression is observed. Mychorrhizal fungus spore-associated bacteria appear to benefit plant growth, since Azcon-Aguilar and Barea (1985) demonstrated that surface-sterilization of spores inhibited mycorrhiza formation. Similarly, Mayo et al. (1986) demonstrated that mycosphere microorganisms positively influence germination of mycorrhizal fungus spores. Thus, we did not surfacesterilize spores because such treatment would confound results and was not the phenomenon we wished to study. Whether the microbial effects on mycorrhizas, reported here, are widespread and occur for other soils, plant or VAM fungus species remains to be determined. However, they may indeed be widespread since other researchers have observed reduced growth response and root colonization in non-sterile soil (Mosse et al., 1969; Berthelin and Leyval, 1982). That such growth reductions are attributable to soil microorganism suppression of mycorrhizal activity is conceivable since Bowen and Theodorou (1978) observed suppression of ectomycorrhizal root colonization by bacteria. Similarly, Ross (1980) demonstrated that the soil microflora could inhibit VAM fungus sporulation. In contrast, Azcon-Aguilar and Barea (1985) observed increased incidence of mycorrhizas and infectivity of mycorrhizal fungi when non-sterile soil filtrate was added to a sterilized soil-sand mix. These effects were attributed to stimulation of saprophytic growth of mycorrhizal fungi by soil microorganisms. The results presented here suggest more diverse effects of soil microorganisms on mycorrhizal symbiosis, since both positive and negative effects were observed.
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