sulfur-oxidizing bacteria as plant growth promoting rhizobacteria for canola
TRANSCRIPT
Sulfur-oxidizing bacteria as plant growth promoting rhizobacteria for canola'
SUSAN J. GRAYSTON AND JAMES J. GERMIDA~ Department of Soil Science, University of Saskatchewan, Saskutoon, Sask., Canada S7N OW0
Received September 1 1, 1990 Accepted December 1 8, 1990
GRAYSTON, S. J., and GERMIDA, J. J. 1991. Sulfur-oxidizing bacteria as plant growth promoting rhizobacteria for canola. Can. J. Microbiol. 37: 521-529.
Canola (Brassica napus) has a high sulfur requirement during vegetative growth and exhibits symptoms of sulfur deficiency when cropped on Saskatchewan soils low in plant available sulfur. Elemental sulfur (So) is frequently used as a fertilizer to alleviate this deficiency. The potential of sulfur-oxidizing microorganisms to enhance the growth of canola in So fertilized soils was assessed. Sulfur-oxidizing bacteria and fungi were isolated from the rhizosphere and rhizoplane of canola grown in four different Saskatchewan soils under growth chamber conditions. Of 273 bacterial isolates, 245 (89.7%) oxidized So to thiosulfate or tetrathionate in vitro, and 133 (48.7%) oxidized So to sulfate; 70 fungal isolates oxidized So to sulfate. Eighteen bacterial isolates demonstrating the highest in vitro sulfur oxidation were tested as seed inoculants under growth chamber conditions, with So as sulfur source. Fourteen isolates increased canola leaf size measured at the bud stage of growth, and seven isolates increased root and pod dry weights at maturity. Three of the 14 isolates were also able to stimulate canola leaf area in the presence of plant available sulfate. The shoot material from canola inoculated with two of these isolates contained more iron, sulfur, and magnesium than uninoculated canola. Three of the 14 isolates inhibited the growth of the canola fungal pathogens, Rhizoctoniu soluni AG2- I , R. solani AG4, and Leptosphaeriu rnaculans "Leroy." Another isolate was antagonistic towards both R. soluni strains and another inhibited the growth of R. solani AG2- 1 and L. rnuculans "Leroy." Thus some sulfur-oxidizing isolates appear to stimulate canola growth due to the enhancement of mineral nutrient uptake, whereas in other cases antibiosis towards canola pathogens may also be involved.
Key words: elemental sulfur, oxidation, canola, rhizosphere, plant growth promoting rhizobacteria.
GRAYSTON, S. J., et GERMIDA, J. J. 1991. Sulfur-oxidizing bacteria as plant growth promoting rhizobacteria for canola. Can. J. Microbiol. 37 : 521-529.
Le canola (Brassica napus) a des exigences ClevCes en soufre pour sa croissance vegetative et, lorsqu'il est cultive sur les sols pauvres en soufre disponible de la Saskatchewan, la deficience en cet element est manifeste. Pour rCmCdier a cette situation, du soufre Clementaire (So) est frkquement utilise comme amendement. Pour rehausser la croissance du canola, le potentiel de microorganismes qui oxydent le soufre a Cte Cvalue dans des sols amendis avec du So. Des batteries et des champignons oxydant le So ont CtC isolCs de rhizosphkres et de rhizoplans de canola croissant sur quatre sols differents de Saskatchewan, et ce dans des conditions phytotroniques. Sur 273 isolates bacteriens, 245 (89,7%) ont oxyde in vitro le So en thiosulfate ou en tetrathionate et 133 (48,7%) ont oxydC le So en sulfate; 70 isolats fongiques ont oxydC le So en sulfate. Les isolats bacteriens, au nombre de 18, qui ont le plus oxydC le soufre in vitro, ont semi a inoculer des graines et ont ete testes en chambre de croissance avec du So comme seule source de soufre. De ces isolats, 14 on accnj la dimension des feuilles de canola, mesurees au stade de bourgeon en dkveloppement, et sept 6nt accnj le poids sec des racines et des gousses. Trois de ces 14 isolats ont aussi stimule la surface des feuilles, meme en presence de sulfate disponible pour les plants. La tige de plantes inoculkes avec deux de ces isolats s'est aver6 contenir plus de fer, de soufre et de magnesium que les plantes non inoculCes. Des 14 isolats, trois ont inhibe la croissance des pathogknes fongiques du canola, soit les souches AG2-1 et AG-4 de Rhizoctonia soluni et le Leptosphaeria rnaculans << Leroy D. Un autre isolat s'est rCvClC antagoniste pour les deux souches de R. soluni et un autre a inhibe la croissance de R. solani AG2- 1 et de L. rnaculans << Leroy D. Ainsi, certains isolats qui oxydent le soufre se sont revC1Cs des stimulants de la croissance du canola en rehaussant l'absorption du nutriment mineral, alors que dans certains cas une antibiose envers les pathogknes du canola a CtC manifeste.
Mots clks : soufre ClCmentaire, oxydation, canola, rhizosphkre, rhizobacteries promotrices de croissance vkgetale. [Traduit par la redaction]
Introduction dominant oxidizers in Saskatchewan soils (Lawrence and Canola has the highest sulfur requirement of any crop grown
in western Canada. As a consequence the yield of canola is severely affected in sulfur-deficient soils. Four million of the 36 million ha of cultivated arable land in the Canadian prairies are currently sulfur deficient (Bettany et al. 1983). It is in these areas that the effects of sulfur deficiency on canola growth can be clearly demonstrated. Elemental sulfur (SO) is frequently used as a fertilizer to alleviate sulfur deficiency in these soils. This reduced sulfur fertilizer must be oxidized to sulfate before it becomes plant available. Sulfur oxidation in soils is primarily a microbial process (Germida 1985; Grayston et al. 1986; Wain- wright 1978, 1984). Heterotrophic microorganisms are the
Germida 1988a; Lawrence et al. 1988). The use of bacteria from the rhizosphere (rhizobacteria) to
enhance plant growth is receiving increasing attention and is reviewed elsewhere (Gaskins et al. 1985; Kloepper et al. 1989; Okon and Hadar 1987). Inoculation of canola with rhizobacteria increased shoot and root elongation and phosphorus uptake (Lifshitz et al. 1987). Similarly, seedling emergence, leaf area, and yield of canola were increased by inoculation of the canola seeds with rhizobacteria (Kloepper et al. 1988). There are a number of postulated mechanisms by which rhizobacteria may promote crop growth, including nitrogen fixation (Kapulnik et al. 1981; Schank et al. 1981; Okon 1985), suppression of plant pathogens (Howie and Suslow 1986; Schroth and Hancock 1982;
'Contribution No. R635, Saskatchewan Institute of Pedology. Suslow 1982), mineralization of organic phosphorus or solubili- 2Author to whom all correspondence should be addressed. zation of inorganic phosphorus compounds (Brown 1974),
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TABLE 1. Characteristics of the four Saskatchewan soils
Soil % organic SO4-S association Soil type Texture PH matter (kg .g- ' )
Haverhill Brown Loam 7.6 2.2 4.0 Chernozemic
Carrot River Dark Gray Loamy sand 5.5 1.7 3.5 Luvisolic
Asquith Dark Brown Loamy sand 7.7 2.3 9.0 Chernozemic
Laird Black Clay loam 6.5 6.9 > 12.0 Chernozemic
production of plant growth regulators (Brown 1974), production of siderophores (Kloepper et al. 1980), and enhanced mineral uptake (Lin et al. 1983). Some of the heterotrophic sulfur- oxidizing microorganisms found in the rhizosphere of crops could be postulated to enhance sulfur availability to these crops. The aim of this research was to determine the potential of heterotrophic sulfur-oxidizing microorganisms isolated from the canola rhizosphere and rhizoplane to enhance canola growth through increased sulfur uptake.
Materials and methods lsolation of sulfur-oxidizing microorganisms
Canola (Brassica napus L. var. Regent) was grown in four different Saskatchewan soils (Table 1) under growth chamber conditions to obtain rhizosphere soils. Two canola seedlings were grown in 15-cm pots fertilized with Long Ashton nutrient solution (Hewitt 1966). After 6 weeks the canola was harvested and the roots shaken to remove adhering rhizosphere soil. The roots were then washed in sterile deionized water to remove any remaining soil prior to maceration to obtain rhizoplane microorganisms. Presumptive SO oxidizing bacteria were isolated from the canola rhizophere soils by plating serial dilutions of the soils on trypticase soy agar (TSA, Difco; 1/10 strength) amended with 0.2% (wlv) flowable sulfur (So) (Stoller Chemical Co., Inc., Houston). Presumptive So oxidizing fungi were isolated using heterotrophic sulfur medium (HSM, Germida 1985) amended with 0.2% (wlv) flowable sulfur and bromothymol blue indicator (0.0025%). Presumptive So oxidizing microorganisms were isolated from the rhizoplane of canola by plating serial dilutions of the macerated canola roots on the above media.
Sulfur oxidation in vitro The ability of the bacterial isolates to oxidize So in vitro was
measured qualitatively. For these determinations 0.1 mL of overnight cultures (organisms grown in 1110th strength trypticase soy broth (TSB)) was inoculated into wells of microtiter plates (24 wells; 3.5 mL capacity per well; Linbro Plastics Ltd) containing TSB ( 1110th strength) and flowable sulfur (1% wlv) (Lawrence and Germida 1988b). Control microtiter plates containing the medium and no flowable sulfur, and uninoculated plates containing the medium and flowable sulfur, were included to detect false positive results. The plates were incubated in polyethylene bags at 25°C for 10 days. A colorimetric ferric thiocyanate method was used to score the wells positive for thiosulfateltetrathionate production (Nor and Tabatabai 1976). The intensity of the orange color produced was scored on a scale of 1-5 (5 being the most intense). Sulfate producers were enumerated turbidimetrically (Hesse 197 1). This involved adding a spatula of barium chloride to each well. Formation of a white color indicated the presence of sulfate. The ability of the fungal isolates to oxidize SO in vitro was assessed qualitatively by measuring the zone of acid production by these fungi when growing on HSM agar plates containing flowable sulfur and bromothymol blue
indicator. Control plates containing the indicator but no flowable sulfur were included to detect any acid production by fungi in the absence of sulfur.
Effect of sulfur-oxidizing rhizobacteria on canola growth
Study 1 The best in vitro sulfur oxidizers were chosen to be tested as
potential plant growth promoting rhizobacteria (PGPR) for canola. These were the 18 isolates producing the largest concentrations of sulfur oxy-anions (thiosulfate, tetrathionate, and sulfate) from the oxidation of So in vitro. These bacteria were tentatively identified as pseudomonads, as described previously (deFreitas and Germida 1990). The bacteria to be assessed were grown for 3 days on King's B medium (King et al. 1954), and then aseptically scraped into 2 mL of 0.1 M MgSO,. Canola seeds (0.2 g) were gently shaken in the appropriate bacterial suspension for 2 h. The seeds were aseptically sown (four seedslpot) in quintupli- cate pots of Haverhill soil (2 kg) containing flowable sulfur (50 as a sulfur source. This study was conducted in two trials to facilitate data collection. Controls consisting of canola seeds soaked in auto- claved bacterial suspensions were sown into soil containing no added sulfur source, flowable sulfur, or Na2S04 (50 SO4-S) as the added sulfur source. The controls with no added sulfur were included to determine the growth of canola in sulfur-deficient conditions. At the other extreme, the controls with Na2S04 as the sulfur source were included to determine the growth of canola in the presence of readily available sulfate. The studies were conducted in soil to which flowable sulfur was added to determine the potential of the isolates to stimulate canola growth by increasing plant available sulfate in the rhizosphere by sulfur oxidation. The pots were placed in a growth chamber photosynthetic irradiance of -450-500 p,~.m-~.s- ' ; 22°C 16 h day; 16"C, 8 h night; for 9 weeks. After 7 days the canola was thinned to two plants per pot, and this was reduced to one per pot after 14 days. After 30 days (bud stage of growth) the width and length of the two largest leaves on each plant were measured. At the end of the incubation period the plants were harvested and the root, shoot, and pod dry weights determined. The shoot and pod material from the plants that showed the largest growth promotion were analyzed for nutrient composition (N, P, K, S, Ca, Mg, Cu, Fe, Mn, Zn, B). All nutrients were analyzed by ICP after nitriclperchloric digestion, with the exception of nitrogen, which was measured using a Leco model 600 CHN analyzer.
Study 2 Leaf size was seen to be a good indicator of growth promotion in the
first growth chamber experimefits. Therefore, leaf area was used in Study 2 to determine if the isolates also stimulated growth in the absence of sulfur. Three isolates that had shown growth promotion of canola in the presence of sulfur were used in this experiment. Isolates 6, 13, and 14 were used as seed inoculants for canola, and were prepared and used to coat the seeds as described in Study 1. Four seeds were sown into each of five pots containing either fertilizer sulfur beads (prilled sulfur; 50 kg-g-') or Na2S04 (50 SO4-S) as the sulfur
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TABLE 2. Numbers of sulfur-oxidizing bacteria isolated from the rhizosphere and rhizoplane of canola grown in a growth chamber
No. of isolates producing:
Area of Total Szo3/s406
Soil isolation isolates S2O3/S4o6 SO4 and so4
Haverhill Rhizosphere Rhizoplane
Carrot River Rhizosphere Rhizoplane
Asquith Rhizosphere Rhizoplane
Laird Rhizosphere Rhizoplane
Total bacteria
"~ulfur oxidizers as percentage of total isolates.
source. Prilled sulfur was used in this study because it is the major form TABLE 3. Canola leaf width and length at 30 days after seed inoculation of sulfur applied to the soil by farmers. Controls consisting of canola with sulfur-oxidizing rhizobacteria seeds soaked in autoclaved bacterial suspensions were also sown into pots containing either fertilizer So beads or Na,SO, as the sulfur source. Treatment Leaf width (cm) Leaf length (cm) The pots were placed in the growth chamber as above, and thinned to one plant per pot after 14 days. The leaf area of the canola in each pot Trial 1 was measured after 30 days (before bolting) using a leaf area meter (Licor Ltd., Lincoln, Nebraska). Control
Control + SO
Inhibition of canola pathogens by sulfur-oxidizing rhizobacteria The 18 isolates screened for their plant growth promoting abil-
ity were assessed for antagonism towards the canola pathogens Rhizoctonia solani AG2- 1, R. solani AG4, and Leptosphaeria maculans "Leroy." A 6-mm core of one of the fungal pathogens previously grown on potato dextrose agar was inoculated on one half of a potato dextrose agar plate. Bacterial inoculum was streaked parallel to the fungal pathogen. The plates were incubated at 25OC for 4 days in the case of R. solani AG 4, 6 days for R. solani AG 2- 1, and 18 days for L. maculans "Leroy," after which the plates were observed for any inhibition of fungal growth.
Control + SO4 Isolate 1 + SO
Isolate 2 + SO
Isolate 3 + SO
Isolate 4 + SO
Isolate 5 + SO
Isolate 6 + SO
Isolate 7 + SO
Isolate 8 + SO
Isolate 9 + SO
Trial 2 Statistical analyses
The growth chamber experiments were carried out in a completely randomized design. Probability levels for statistical significance were made using Fisher's protected LSD.
Control Control + SO
Control + SO4 Isolate 10 + SO
Results Isolate 11 + SO 13.0k 1 .0 Isolate 12 + SO 13.6k0.6
Isolation of sulfur-oxidizing microorganisms from the canola Isolate 13 + SO 12.8k0.8 rhizosphere Isolate 14 + SO 13.7k0.8
A total of 273 bacteria and 70 fungi were isolated from the l,,late 15 + SO 13.3k0.8 rhizosphere and rhizoplane of canola. Of the bacteria, 245 lsolate 16 + SO 14.020.7 (89.7%) were able to oxidize So to thiosulfate or tetrathionate sola ate 17 + SO 13.3k0.9 and 133 (48.7%) oxidized SO to sulfate in vitro (Table 2). In all Isolate 18 + SO 13.7k0.7 soils, except the Asquith soil, a higher percentage of the rhizo-
NOTE: Plants grown in 2 kg of soil amended with appropriate sulfur source ( i . e . , 50 p g plane than the rhizosphere population oxidize 70 flowable So or SO,-S per gram of soil). Controls were inoculated with an autoclaved culture fungal isolates oxidized So to sulfate, producing acid on plates of isolates I and 10 for trials I and 2 , respectively. The two largest leaves were
containing SO and a pH indicator, but not on the same plates m~~~~ ~ P , " e ~ ~ g ~ ~ ~ ~ ~ , ! ; a t e S standard deviation,
lacking So.
Effect of sulfir-oxidizing rhizobacteria on canola growth of growth (before bolting), whereas the other isolates produced Study I nonsignificant increases in these parameters (Table 3). These Eighteen of the best in vitro sulfur-oxidizing isolates were increases were above those for canola inoculated with autoclaved
tested as potential PGPR for canola. Isolates 7,9, 10, and 1 1 had isolates and grown in soil containing flowable sulfur as the sulfur no effect on the canola leaf width and (or) length at the bud stage source (control + So).
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FIG. 1. Stimulation of canola growth by inoculation with sulfur-oxidizing bacteria.
The visual increase in growth of inoculated canola can be seen in Fig. 1. The figure shows the growth of canola at 4 weeks, inoculated with isolates 1 and 8, compared to canola inoculated with autoclaved bacteria (i.e., control). The growth of inoculated canola was compared to the control that had the flowable sulfur addition (control + So). The increased growth of canola by the addition of sulfur to the soil can be seen by comparing the growth of uninoculated control plants under different sulfur treatments (Fig. 1). Sulfur applied to soil in either a reduced
form as flowable sulfur (control + So) or an oxidized form as Na,SO, (control + SO,) stimulated the growth of canola above that of plants of grown in soil to which no sulfur was added (con- trol). The symptoms of sulfur deficiency in canola can be seen in the control plant.
Seven of the isolates (3,4,5,8, 13, 15, and 17) increased root and pod dry weight of canola at maturity above that of the controls to which flowable sulfur had been added (C + S) (Fig. 2). Isolates 1, 6, 7, 9, 12, 14, and 18 increased the root dry
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6000 pod
ar, a ROO^ E .- 5000 shoot u r: .? 4000 aJ 3 A 3000
2000
1000
0
Treatment
C C+SC+S04 10 11 12 13 14 15 16 17 18
Treatment
FIG. 2. Effect of inoculation with sulfur-oxidizing rhizobacteria on the dry weight of canola. (Means of five replicates; bars represent + standard deviation; C, control; S, sulfur; SO,, sulfate.)
weight of canola at maturity, and inoculation with isolate 11 increased the pod dry weight of canola (Fig. 2). Due to the large standard deviation between treatments (as shown in Fig. 2), these increases were not significant at the 5% level. The variations between the dry weights of the control plants in the two trials were probably due to variability in conditions in the growth chamber (e.g., light, humidity). The controls to which no sulfur had been added yielded very few pods due to the sulfur-deficient conditions.
Analysis of some of the plant tissues from these experiments showed that the canola shoots and pods from plants inoculated with some of the isolates contained more sulfur, iron, and magnesium than canola inoculated with autoclaved bacteria (Table 4). The values shown in Table 4 are total element contents of the tissues (normalized against dry weight). Inocula- tion of canola with isolate 14 significantly increased the sulfur, iron, and magnesium content of the canola shoots (Table 4). Similarly, inoculation of canola with isolate 13 resulted in significantly higher iron content of the canola shoots (Table 4). Isolate 17 had no effect on total element contents of canola tissues. Concentrations of all the other elements analyzed (N, P,
K, Ca, Cu, Mn, Zn, B) were similar in the shoot and pod tissue of both the inoculated and control plants. This suggests that the increased growth of canola in the presence of these isolates may be due to enhanced iron and possibly sulfur and magnesium uptake.
Study 2 The effect of the three sulfur-oxidizing isolates 6, 13, and 14
on the growth of canola in the presence of a reduced or oxidized sulfur source is shown in Fig. 3. Inoculation of canola with all three isolates significantly increased the leaf area of canola after 30 days growth. This growth promotion occurred in both the presence and absence of So as the sulfur source.
Inhibition of canola pathogens by sulfur-oxidizing rhizobacteria Five of the 18 sulfur-oxidizing rhizobacteria inhibited the
growth of at least one of the canola pathogens. These five isolates belonged to the group of 14 that had increased leaf size of canola in Study 1. Three of the isolates (4, 12, and 13) inhibited the growth of the fungal pathogens R. solani AG2- 1, R. solani AG4, and L. muculans "Leroy." The inhibitory effect of isolate 13 (HWRS2) against the two strains of R. solani can be
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TABLE 4. Sulfur, iron, and magnesium content of canola shoots and pods after seed inoculation with sulfur- oxidizing rhizobacteria
Total element content of plant tissue
Treatment Laboratory code Plant tissue Mg (mg) s (mg) Fe (pg)
Control Shoots 9 .3 (1.6) 21.6 (2.8) 438.5 (64.3) Isolate 13 HWRS2 Shoots 1 1 . 1 (1.1) 23.2 (2.4) 656.6* (155.0) Isolate 14 HWRS4 Shoots 11.9 (1.2)* 28.1* (1.2) 727.1* (118.1)
Control Pods 9.7 (1 .2) 9 .2 (1.2) 216.5 (44.1) Isolate 13 HWRS2 Pods 10.5 (0.8) 10.2 (1.6) 217.7 (25.0) Isolate 14 HWRS4 Pods 8 .3 (1.6) 8.5 (1.8) 248.1 (65.5)
NOTE: Plants grown in 2 kg of soil amended with prilled So fertilizer (50 k g . g - ' soil) in growth chamber. The control was inoculated with an autoclaved culture of isolate 10. Means of five replicates (SD).
*Significant increase above control (P < 0.05).
Conuol 6 13 14
Isolate
Conuol 6 13 14
Isolate
FIG. 3. Effect of inoculation with sulfur-oxidizing rhizobacteria on the leaf area of canola, when grown in soil containing (a) NaSO, as the sulfur source and (b ) sulfur pellets as the sulfur source. Controls were inoculated with autoclaved bacteria. (Means of five replicates; bars represent + standard deviation; bars followed by different letters are significantly different at P < 0. 05.)
seen in Fig. 4 (CRT4 and HH3 are negative controls that had no effect on the growth of the pathogens). Isolate 14 (HWRS4) was antagonistic towards both R. solani strains (Fig. 5). Isolate 1 inhibited the growth of R. solani AG2-1 and L. maculans "Leroy." None of the other 13 isolates inhibited the growth of the three fungal pathogens.
Discussion Research on PGPR is increasing as the potential of these
inoculants is realized (Kloepper et al. 1989). In previous studies looking for PGPR for canola, large numbers of rhizobacteria were screened for their ability to increase canola root length (Lifshitz et al. 1987) or leaf area (Kloepper et al. 1988). In this study we took a different approach: we looked for PGPR with a specific mechanism of action, namely, those that would enhance sulfur uptake in canola. Canola has a very high requirement for sulfur. In the Canadian prairies, where 850 000 ha of land are sown to canola each year (Anon 1983), the soil is sulfur deficient (Bettany et al. 1983). Reduced sulfur fertilizers are added to the soil to overcome these deficiencies. However, the sulfur is poorly oxidized to plant available sulfate in these soils (Germida 1985; Germida et al. 1985; Janzen and Bettany 1986). The aim, therefore, was to find rhizobacteria that were good sulfur oxidizers and postulate that they could increase sulfur oxidation in the rhizosphere of canola, thereby increasing sulfur uptake in the crop.
Previously, we found that numbers and activity of sulfur- oxidizing bacteria were stimulated in the rhizosphere soil of different crops, including canola (Grayston and Germida 1990). In this study, a high proportion of isolates from the rhizosphere and rhizoplane of canola were able to oxidize So in vitro. From 73 to 98% of the heterotrophic bacteria isolated from the rhizosphere of canola grown in different soils were able to oxidize So to thiosulfate or tetrathionate, and 37-70% of the heterotrophs could fully oxidize So to sulfate. Nearly 100% of the heterotrophs that oxidized So to sulfate also produced thio- sulfate or tetrathionate during sulfur oxidation. Many heterotrophic microorganisms produce thiosulfate during ,the oxidation of So (Wainwright 1984; Germida et al. 1985; Gray- ston et al. 1986). However, the pathway of heterotrophic sulfur oxidation remains unclear, and it is not known whether thio- sulfate is an intermediate or by-product in the oxidation of SO to sulfate.
Eighteen of the best So oxidizing bacterial isolates were tested as PGPR for canola. Inoculation of canola with 14 of these isolates produced an increase in leaf size of canola, in the growth chamber. Seven of these isolates also increased the root and pod dry weight of canola at maturity. The plant material from these inoculation studies was analyzed to determine if increased sulfur uptake by canola could be postulated to be the mechanism of action of these PGPR. Sulfur uptake did increase in the shoots of canola inoculated with some these isolates, suggesting that
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FIG. 4. Inhibitory effect of isolate 13 (HWRS2) on the growth of Rhizoctonia solani AG 2- 1 and R. solani AG4. (CRT4 and HH3 are negative controls with no inhibitory effect on the pathogens.)
increased sulfur uptake may be one growth-promoting mecha- nism. However, the isolates also increased canola growth in the absence of a reduced sulfur source, suggesting that sulfur uptake may not be the only mechanism of growth promotion.
It has been reported that some PGPR achieve their growth promotion through siderophore-mediated biocontrol (Kloepper et al. 1980), or by producing, or stimulating the plant to produce, more growth hormones (Brown 1974). For example, Kloepper et al. (1980) proposed that pseudomonad PGPR that excrete siderophores may make iron more directly available to plants or, alternatively, deprive native, phytopathogenic microflora of iron, thus limiting their growth. It may be that some of our sulfur- oxidizing PGPR stimulated the growth of canola in such a manner. Isolates 4, 12, 13, and 14 inhibited the in vitro growth of several phytopathogenic fungi, and thus biocontrol might be an important mechanism of growth promotion. However, we did not assess canola growth in soil infested with fungal pathogens, and the indigenous level of such organisms in our studies was not known. Furthermore, some of our isolates produce phytohor-
mones (de Freitas and Germida 1990), and stimulation of root growth could lead to an increase in mineral nutrient uptake by canola. Most So oxidizing isolates increased the root dry weight of canola, and isolates 13 and 14 also increased nutrient (i.e., Mg, S, Fe) uptake by canola. Thus some of these PGPR may enhance canola growth via two (or more) mechanisms simulta- neously.
Canola shoot tissue from plants inoculated with sulfur- oxidizing PGPR contained a significantly higher iron content than uninoculated plants, demonstrating that inoculation with those PGPR increased iron uptake in the plants. In addition to increasing sulfur and iron uptake by canola, inoculation with some of these isolates also increased magnesium uptake by canola. This might be explained by sulfur-oxidizing bacteria acidifying the microenvironment around canola roots and solubilizing iron and (or) other elements, which are then taken up by the plants. Previous studies on PGPR for canola (Lifshitz et al. 1987) showed that inoculation of canola with PGPR increased phosphorus uptake by canola. This suggests that rather than
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FIG. 5. Inhibitory effect of isolate 14 (HWRS4) on the growth of Rhizoctonia solani AG 2- 1 and R. solani AG4. (CRT4 and HH3 are negative controls with no inhibitory effect on the pathogens.)
enhancement of uptake of a specific nutrient, PGPR may stimulate uptake of nutrients in general.
The activity of heterotrophic sulfur-oxidizing bacteria in crop rhizospheres is affected by both crop and soil characteristics (Grayston and Gennida 1990). Thus So fertilizer use efficiency may be affected by selective manipulation of sulfur-oxidizing bacteria in the rhizosphere of canola. This study demonstrates that some sulfur-oxidizing bacteria may be effective PGPR for canola. Enhanced uptake of sulfur may be one, but not the only, mechanism by which these PGPR exert their effect. Additional studies are required to identify other sulfur-oxidizing bacteria as PGPR to specifically enhance sulfur nutrition of plants.
Acknowledgements This study was supported by grants from the Western Grains
Research Foundation and the Natural Sciences and Engineering Research Council of Canada.
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