Effect of co-inoculation with phosphate and potassium

solubilizing bacteria on mineral uptake and growth of pepper

and cucumber

H.S. Han1, Supanjani2, K.D. Lee2

1Department of Biology, Suncheon National University, Jeonnam, Korea

2Department of Plant Science, McGill University, MacDonald Campus, Quebec, Canada

ABSTRACT

Biofertilizers have been used as sources to improve plant nutrients in sustainable agriculture. Experiments were
conducted to evaluate the potential of phosphate solubilizing bacteria (PSB) Bacillus megaterium var. phosphaticum
and potassium solubilizing bacteria (KSB) Bacillus mucilaginosus inoculated in nutrient limited soil planted with
pepper and cucumber. Results showed that rock P and K applied either singly or in combination did not significant-
ly enhance soil availability of P and K, indicating their unsuitability for direct application. PSB was a more potent
P-solubilizer than KSB, and co-inoculation of PSB and KSB resulted in consistently higher P and K availability than
in the control without bacterial inoculum and without rock material fertilizer. Integrated rock P with inoculation
of PSB increased the availability of P and K in soil, the uptake of N, P and K by shoot and root, and the growth of
pepper and cucumber. Similar but less pronounced results were obtained when rock K and KSB were added conco-
mitantly. Combined together, rock materials and both bacterial strains consistently increased further mineral avai-
lability, uptake and plant growth of pepper and cucumber, suggesting its potential use as fertilizer.

Keywords: pepper; cucumber; phosphate solubilizing bacteria; potassium solubilizing bacteria

Significant areas of cultivated soils in Korea 1990). On the other hand, K deficiencies become
and China are deficient of available P and K and problem because K decreases easily in soils due
have a low crop productivity (Xie 1998). The use to crop uptake, runoff, leaching and soil erosion
of plant growth promoting rhizobacteria (PGPR), (Sheng and Huang 2002). Direct application of
including phosphate and potassium solubilizing rock phosphate (rock P) and potassium (rock K)
bacteria (PSB and KSB) as biofertilizers, was sug- materials may be agronomically more useful and
gested as a sustainable solution to improve plant environmentally more feasible than soluble P and K
nutrient and production (Vessey 2003). Phosphate (Rajan et al. 1996). Rock P and K materials are
and potassium are major essential macronutrients cheaper sources of P and K; however, most of
for plant growth and development and soluble them are not readily available to a plant because
P and K fertilizers are commonly applied to re- the minerals are released slowly and their use as
place removed minerals and to optimize yield. fertilizer often causes insignificant yield increases
When phosphate is added into soils as a fertilizer of current crop (Zapata and Roy 2004). PSB have
in relatively soluble and plant available forms, it been used to improve rock P value because they
is easily converted into insoluble complexes with convert insoluble rock P into soluble forms available
calcium carbonate, aluminum and iron oxides, for plant growth (Nahas et al. 1990, Bojinova et
and crystalline and amorphous aluminum silicate al. 1997). This conversion is through acidification,
(Sample et al. 1980). Consequently, to achieve chelation and exchange reactions (Gerke 1992) and
optimum crop yields, soluble phosphate fertiliz- produces, in the periplasm, strong organic acids
ers have to be applied at high rates which cause (Alexander 1977), which have become indicators for
unmanageable excess of phosphate application routine isolation and selection procedures of PSB
and environmental and economic problems (Brady (e.g. Illmer et al. 1995). Bacillus megaterium var.

130 PLANT SOIL ENVIRON., 52, 2006 (3): 130–136

phosphaticum is known for its ability to solubilize incubated on an orbital shaker at 150 rpm for 48 h
rock P material (Schilling et al. 1998). On the other at 27°C. The cells in cultured bacterial broth were
hand, KSB are able to solubilize rock K mineral collected by centrifugation at 2.822 × g for 15 min
powder, such as micas, illite and orthoclases, also at 4°C and washed with sterilized tap water. The
through production and excretion of organic acids pelleted cells were resuspended with sterilized tap
(Friedrich et al. 1991, Ullman et al. 1996). It was water and then the cells were adjusted to about
shown that KSB, such as Bacillus mucilaginosus, 108 cells/ml, based on optical density OD 620 = 0.08
increased K availability in soils and increased (Bhuvaneswari et al. 1980), and 1 ml of inoculum
mineral content in plant (Sheng et al. 2002). An was applied into each seedling.
integrated application of rock P and K materials Experiment for studying the effect of the bac-
with co-inoculation of bacteria that solubilize terial strain on plant growth, P and K uptake of
them might provide faster and continuous supply pepper and cucumber was conducted in pots
of P and K for optimal plant growth. However, lit- (17 cm diameter and 15 cm deep) layered with
tle is known about the combined effects of rock plastic bags and containing 2.0 kg of sterilized
materials and co-inoculation of PSB and KSB on soil. The soil was sterilized by dry autoclaving at
mineral availability in soils, mineral content and 20 psi for 120 min. The experiment was estab-
growth of pepper and cucumber. The aim of the lished with 10 treatments: without rock P and K
study was to evaluate the potential of the direct materials or bacteria inoculation (control), PSB,
application of rock P and K materials and co-in- KSB, (P + K)SB, rock P [rock phosphate powder
oculation with PSB and KSB for the improvement (total P 15%, 106 µm mesh), 3 g/kg soil], rock K
of P and K uptake and growth of pepper and cu- [illite powder (total K 3.9%, 106 µm mesh), 3 g/kg
cumber plants grown under limited P and K soil soil], rock (P + K), rock P + PSB, rock K + KSB,
conditions in a greenhouse. and rock (P + K)+(P + K)SB. Rock materials and
inoculants were mixed thoroughly with the soil
in a plastic pot to ensure the solubilization effect
MATERIAL AND METHODS of rock nutrient by PGPRs. Seeds of pepper and
cucumber were surface-sterilized in 2% sodium
The effects of P- and K-solubilizing bacteria hypochlorite for 3 min and then rinsed 5 times
on pepper (Capsicum annum L.) and cucumber with distilled water (Bhuvaneswari et al. 1980).
(Cucumis sativus L.) growth were examined in The seeds were germinated and grown in sterilized
a greenhouse experiment using Aquepts Series, vermiculite in trays. At 20 days after sowing for
Typic Endoaquepts (USDA, Inceptisols) soil col- pepper and 15 days for cucumber, one seedling
lected from Chinju, Kyungnam province, Korea. was transplanted into each pot. At three days after
The chemical properties of the soil were: pH (1:5 transplanting, depending on the treatment, the
w/v water) 5.4, organic matter 18 g/kg, total ni- healthy seedling was inoculated with 1 ml of inocu-
trogen 2.1 g/kg, available P 9.1 mg/kg, and avail- lum containing around 10 8 cells. The temperature
able K 52.4 mg/kg (1M NH 4-OAc). We used two in the greenhouse was maintained at 30 ± 2°C with
strains of plant growth promoting rhizobacteria a relative humidity of 65% and a 16 hrs photoperiod
(PGPR), i.e. PSB and KSB. Bacillus megaterium created by using supplemental lighting from high-
var. phosphaticum, selected for PSB, was isolated pressure sodium lamps. All plants were harvested
from plastic film house area in Korea by using the 30 days after transplanting. The photosynthesis of
PBY medium (1.0% polypeptone, 0.5% beef extract, plants was measured using a Li-Cor 6400 (Li-Cor
0.2% NaCl, 0.05% yeast extract). This PSB strain Inc., Lincoln, Nebraska, USA) at harvest time, just
was identified by Dr. H. Han (Molecular Biology before harvesting the plants.
Laboratory, Department of Biology, Sunchon To analyze mineral elements, soils were sampled
National University, Korea) and was proven for for chemical analyses before the experiments and
its capability to solubilize many phosphatic com- after harvesting the plants. At harvest, following
pounds, such as aluminum phosphate and rock root removal, soils in the whole pot were mixed
phosphate (Sundara et al. 2002). In the case of and a sample was drawn. Soil samples were air-
KSB, we used Bacillus mucilaginosus (KCTC3870, dried and sieved (2 mm screen) and analyzed
Korean Collection of Type Cultures). for the following: pH (1:5 water extraction), or-
PSB and KSB were cultured in Tryptone Yeast ganic matter content (Wakley and Black method;
(TY) medium (Vincent 1970) and sucrose-minimal Allison 1965), available P content (Lancast; RDA
salts medium (Sheng et al. 2002), respectively, and 1988) and contents of exchangeable or avail-

PLANT SOIL ENVIRON., 52, 2006 (3): 130–136 131

able K + (1M NH 4 -OAc pH 7, AA, Shimazu 660; inoculation of PSB or KSB resulted in a higher
Richards and Bates 1989). Shoot and root tissues mineral availability in which PSB strain was a more
were separated after harvesting and air-dried at potent solubilizer for rock P than KSB strain.
70°C for 5 days. Dried soils were grounded and Co-inoculation with PSB and KSB or combined
then digested in H 2SO 4 for determination of total PSB inoculation and rock P material significantly
nitrogen (Kjeldahl method) or in a ternary solu- increased the availability of P and K. When applied
tion (HNO 3:H 2SO 4:HClO 4 = 10:1:4 with volume) all together, PSB, KSB and rock P and K resulted
for the determination of P and K. in the highest availability of P and K in soils, and
All data were analyzed statistically by analysis of caused an increase of about 36% for P and 31%
variance using CoStat software (CoHort Software, for K as compared to untreated control (without
Monterey, USA). The experiment was structured bacterial inoculum and without rock material
following a randomized complete block design fertilizer). The results also showed that there was
(RCBD) with four replications. Means compari- a relatively higher availability of P and K in soils
sons were conducted using an ANOVA protected planted with pepper than with cucumber.
the least significant difference (LSD) test, with Soil inoculation with PSB or KSB significantly
the ANOVA confidence levels of 0.95. Data were increased N, P and K uptake in pepper and cu-
presented with their standard deviations (SD). cumber plants, especially when the respective rock
P or rock K were added (Tables 2 and 3). Further
significant increases in N, P and K uptakes from
RESULTS AND DISCUSSION the inoculated soils were generally found in the
all-integrated treatment, i.e. soil co-inoculated with
Large areas of cultivated land in Korea and China both bacteria and fertilized with rock P and K. This
are deficient in phosphorus and potassium nutri- combined treatment increased N, P and K uptake
ent; therefore, in our experiment we have included in shoot (21, 31 and 33%, respectively, for pepper
soil with addition of rock P and K and their solu- and 29, 41 and 29% for cucumber) and in root (16,
bilizing bacteria to increase the available P and K 33 and 26% for pepper; 29, 34 and 50% for cucum-
in this soil. Available P and K in the soils 30 days ber). Application of insoluble rock fertilizers in the
following the planting were presented in Table 1. sterilized soil did not increase mineral content in
Addition of rock materials into the soil did not pepper and cucumber. This might occur because
significantly increase available P or K, and single these rock materials are solubilized slowly only by

Table 1. Effects of PSB and KSB strains on available P and K (mg/kg) in soil planted with pepper and cucumber,
30 days after planting; the values are the mean of four replications (± SD)

Pepper Cucumber
Treatments
P K P K
Control 8.0 ± 0.47 41.2 ± 2.08 7.8 ± 0.80 38.3 ± 1.45
R-P 8.5 ± 0.49 42.0 ± 2.14 8.4 ± 0.67 39.2 ± 2.92
R-K 8.3 ± 0.32 43.3 ± 2.27 8.2 ± 0.70 41.6 ± 1.39
R-(P + K) 8.7 ± 0.40 45.1 ± 2.50 8.8 ± 0.28 41.5 ± 1.82
PSB 9.4 ± 1.08 45.5 ± 2.51 9.3 ± 0.40 41.4 ± 2.70
KSB 8.5 ± 0.69 47.3 ± 2.38 8.5 ± 0.30 44.7 ± 2.72
(P + K)SB 9.8 ± 0.48 49.9 ± 5.28 9.7 ± 0.64 46.8 ± 2.21
R-P + PSB 10.1 ± 0.80 47.3 ± 3.82 9.8 ± 0.93 43.8 ± 2.71
R-K + KSB 9.0 ± 0.50 49.5 ± 1.99 8.9 ± 0.50 46.4 ± 2.71
R-(P + K) + (P + K)SB 10.9 ± 1.24 54.2 ± 3.19 10.6 ± 0.58 50.1 ± 1.70
LSD0.05 1.03 4.22 0.91 3.35

R-P = rock phosphate; R-P = rock K (illite); PSP = phosphate solubilizing bacteria; KSB = potassium solubilizing
bacteria; LSD = least significant difference

132 PLANT SOIL ENVIRON., 52, 2006 (3): 130–136

Table 2. Effects of PSB and KSB strains on shoot and root nutrient uptake (mg/plant) of pepper; the values are
the mean of four replications (± SD)

Shoot Root
Treatments
N P K N P K
Control 13.6 ± 0.74 1.61 ± 1.61 19.2 ± 1.69 3.69 ± 0.18 0.46 ± 0.04 5.92 ± 0.25
R-P 14.3 ± 0.80 1.72 ± 0.08 19.9 ± 1.74 3.76 ± 0.23 0.48 ± 0.06 6.19 ± 0.33
R-K 14.1 ± 0.70 1.68 ± 0.09 20.3 ± 2.29 3.71 ± 0.34 0.47 ± 0.05 6.17 ± 0.30
R-(P + K) 14.2 ± 0.67 1.75 ± 0.05 20.9 ± 2.16 3.87 ± 0.16 0.50 ± 0.05 6.68 ± 0.34
PSB 14.5 ± 0.64 1.88 ± 0.12 21.1 ± 1.40 3.90 ± 0.41 0.50 ± 0.05 6.48 ± 0.36
KSB 14.5 ± 0.79 1.75 ± 0.12 21.7 ± 2.19 3.82 ± 0.45 0.49 ± 0.03 6.68 ± 0.29
(P + K)SB 14.7 ± 0.57 1.99 ± 0.23 23.4 ± 2.31 3.92 ± 0.28 0.54 ± 0.06 6.95 ± 0.62
R-P + PSB 15.0 ± 0.56 1.91 ± 0.09 21.7 ± 1.43 3.95 ± 0.26 0.53 ± 0.06 6.70 ± 0.76
R-K + KSB 14.8 ± 0.66 1.84 ± 0.13 22.4 ± 1.59 3.90 ± 0.18 0.51 ± 0.05 6.93 ± 0.32
R-(P + K) + (P + K)SB 16.5 ± 0.83 2.11 ± 0.08 25.7 ± 2.35 4.27 ± 0.31 0.61 ± 0.06 7.46 ± 0.29
LSD 0.05 1.01 0.18 2.81 0.43 0.07 0.60

Explanations see Table 1

plant root system in sterilized soil and therefore and cucumber plants (Table 4). Double inocula-
not generally available to plants (Table 1). It is tion of PSB and KSB increased shoot and root
interesting that jointly bacterial inoculation and dry weight; PSB inoculation increased shoot dry
rock material fertilizer application also increased weight, whereas KSB did not. Although combined
N uptake by plants (Table 3). PSB inoculation with application of rock P con-
Single or double application of rock P and rock K sistently increased shoot and root dry weight as
did not significantly improve plant growth (shoot compared to control, a treatment which joints
and root dry weight) and photosynthesis in pepper together both bacteria and mineral rocks, further

Table 3. Effects of PSB and KSB strains on shoot and root nutrient uptake (mg/plant) of cucumber; the values
are the mean of four replications (± SD)

Shoot Root
Treatments
N P K N P K
Control 16.9 ± 0.94 1.66 ± 0.08 22.8 ± 1.91 3.55 ± 0.12 0.44 ± 0.04 8.67 ± 0.33
R-P 17.3 ± 1.15 1.70 ± 0.08 23.1 ± 2.27 3.60 ± 0.28 0.46 ± 0.02 8.84 ± 0.56
R-K 17.2 ± 0.82 1.70 ± 0.07 24.1 ± 1.55 3.64 ± 0.27 0.46 ± 0.04 9.12 ± 0.93
R-(P + K) 18.9 ± 0.76 1.80 ± 0.07 24.6 ± 1.54 3.87 ± 0.22 0.48 ± 0.06 9.86 ± 0.94
PSB 18.0 ± 1.14 1.85 ± 0.11 24.0 ± 2.69 3.75 ± 0.18 0.49 ± 0.04 9.05 ± 0.26
KSB 17.8 ± 1.06 1.77 ± 0.08 25.5 ± 0.90 3.70 ± 0.31 0.47 ± 0.05 9.97 ± 0.85
(P + K)SB 19.6 ± 1.27 1.93 ± 0.07 26.6 ± 1.63 4.01 ± 0.31 0.51 ± 0.04 10.50 ± 1.11
R-P + PSB 18.5 ± 0.67 1.96 ± 0.11 24.9 ± 0.54 3.95 ± 0.30 0.53 ± 0.05 9.52 ± 0.48
R-K + KSB 18.2 ± 0.62 1.86 ± 0.05 26.4 ± 1.53 3.96 ± 0.30 0.49 ± 0.02 10.48 ± 1.33
R-(P + K) + (P + K)SB 21.8 ± 0.76 2.34 ± 0.11 29.4 ± 0.90 4.59 ± 0.31 0.59 ± 0.07 13.05 ± 1.37
LSD0.05 1.36 0.12 2.41 0.39 0.065 1.30

Explanations see Table 1

PLANT SOIL ENVIRON., 52, 2006 (3): 130–136 133

Table 4. Effects of PSB and KSB strains on dry weight (g/plant) and photosynthetic rate (µmole/cm 2/s) of pepper
and cucumber; the values are the mean of four replications (± SD)

Pepper Cucumber
Treatments dryweight photosynthetic dry weight photosynthetic
shoot root rate shoot root rate

Control 0.670 ± 0.033 0.268 ± 0.023 18.2 ± 0.50 0.757 ± 0.039 0.291 ± 0.016 11.1 ± 0.59
R-P 0.692 ± 0.035 0.288 ± 0.016 18.7 ± 0.67 0.810 ± 0.046 0.307 ± 0.020 11.3 ± 0.61
R-K 0.685 ± 0.037 0.283 ± 0.019 18.5 ± 0.47 0.805 ± 0.042 0.302 ± 0.023 11.5 ± 0.54
R-(P + K) 0.700 ± 0.037 0.289 ± 0.021 18.8 ± 1.13 0.781 ± 0.044 0.317 ± 0.007 11.6 ± 0.49
PSB 0.762 ± 0.035 0.294 ± 0.027 19.4 ± 0.48 0.845 ± 0.043 0.314 ± 0.021 12.1 ± 0.57
KSB 0.723 ± 0.033 0.286 ± 0.013 18.9 ± 0.22 0.819 ± 0.040 0.310 ± 0.023 11.6 ± 0.79
(P + K)SB 0.786 ± 0.055 0.300 ± 0.016 19.7 ± 0.87 0.832 ± 0.069 0.338 ± 0.018 12.1 ± 0.70
R-P + PSB 0.790 ± 0.039 0.310 ± 0.030 20.5 ± 1.02 0.871 ± 0.044 0.339 ± 0.024 12.5 ± 0.32
R-K + KSB 0.765 ± 0.042 0.295 ± 0.024 19.2 ± 0.40 0.828 ± 0.038 0.317 ± 0.014 12.0 ± 12.0
R-(P + K) + (P + K)SB 0.846 ± 0.039 0.345 ± 0.021 21.8 ± 0.54 0.920 ± 0.047 0.369 ± 0.011 12.9 ± 0.74
LSD0.05 0.056 0.031 0.99 0.066 0.027 0.085

Explanations see Table 1

increased plant growth: 26% in shoot and 29% in petition with indigenous less-effective rhizobia
root dry weight for pepper and 22% in shoot and was studied in rhizobial inoculant (Sessitch et al.
27% in root dry weight for cucumber plant over 2002, Lopez-Gracia et al. 2002, Loh and Stacey
the controls during 30 days following planting. 2003), competition in PGPR inoculants was not
Photosynthesis responses of pepper and cucumber intensively explored probably because some PGPR
to the treatments demonstrated similar trends to inoculants are selected for increasing plant sys-
shoot dry weight responses (Table 4). The integrated temic acquired resistance or for suppression of
treatment of co-inoculation with two strains and pathogenic bacteria (Zehnden et al. 2001, Anith et
application of insoluble rock materials significantly al. 2004). Bacteria inoculation, which can improve
increased leaf photosynthesis 20% in pepper and P and K availability in soils by producing organic
16% in cucumber plants over the controls. acids and other chemicals, stimulated growth and
Increasing the bioavailability of P and K in soils mineral uptake of plants (Alexander 1977, Park et
with inoculation of PGPR or with combined in- al. 2003). Combined all together, our results with
oculation and rock materials, which may lead to results from others suggest that integration of
increased P uptake and plant growth, was reported bacteria inoculation with PSB and KSB and rock
by many researchers (e.g. Omer 1998, Wahid and P and K materials amendment can improve crop
Mehana 2000, Lin et al. 2002, Şahin et al. 2004). mineral nutrients in nutrient-deficient soils.
Omer (1998) reported that the application of rock P Our experiment demonstrated the advantage
to nonsterilized soil with bacteria inoculation of co-inoculation of two Bacillus species, which
increased P uptake and shoot and total dry mass have been reported as PSB and KSB strains, on
of wheat plant. Our one-year field experiment mineral uptake and growth of pepper and cucum-
using the same PSB strain demonstrated results ber. Synergistic effects of combined inoculation of
similar to these greenhouse experiments: increased PGPRs have also been reported in various crops,
P availability in the soils and increased dry mater for examples potatoes (Kundu and Gaur 1980),
of lettuce, especially when rock P was added (data rice (Tiwari et al. 1989) and sugar beet and barley
not shown). This indicates that the bacterial strains (Çakmakçi et al. 1999). In our case, co-inoculation
can compete with existing natural bacteria, which of PSB and KSB strains synergistically solubilized
is understandable as they are isolated from similar rock P and K which were added into the soil and
soil conditions in Korea. Although such a com- make them much more available for uptake by plant

134 PLANT SOIL ENVIRON., 52, 2006 (3): 130–136

roots. Higher N, P and K uptake may subsequently Gerke L. (1992): Phosphate, aluminum, and iron in
promote the plant growth. Growth enhancement the soil solution of three different soils in relation to
by Bacillus may be also associated to its ability varying concentrations of citric acid. Z. Pfl.-Ernähr.
to produce hormone, especially IAA (Sheng and Bodenkde, 155: 17–22.
Huang 2001), and siderophore (Ito 1993, Hu and Hu X., Boyer G.L. (1996): Siderophore-mediated alu-
Boyer 1996). Increasing N uptake in our experiment minum uptake by Bacillus megaterium ATCC 19213.
with inoculation with Bacillus may be related to the Appl. Environ. Microbiol., 62: 4044–4048.
fact that Bradyrhizobium sp., a genus which fixes Illmer P., Barbato A., Schinner F. (1995): Solubilization
atmospheric nitrogen in symbiosis with legume, of hardly-soluble AlPO 4 with P-solubilizing micro-
is phyllogenically closer to Bacillus than to other organism. Soil Biol. Biochem., 27: 265–270.
rhizobial genera (Zakhia and Lajudie 2001, Şahin Ito T. (1993): Enzymatic determination of itoic acid,
et al. 2004). Therefore, Bacillus strain used in this a Bacillus subtilis siderophore, and 2,3-dihydroxy-
study might have the capacity to fix atmospheric benzoic acid. Appl. Environ. Microbiol., 59: 2343–
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is important for the uptake of N from soils and Kim T., Jung W., Lee B., Yoneyama T., Kim H., Kim K.
its utilization in plant (Kim et al. 2003). Hence, (2003): P effects on N uptake and remobilization dur-
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N uptake. In short, results from all these and other Kundu B.S., Gaur A.C. (1980): Effect of phosphobacteria
experiments suggest that co-inoculation of PGPR on the yield and phosphate uptake of potato crop.
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Corresponding author:

Dr. Kyung Dong Lee, Department of Plant Science, MacDonald Campus, McGill University, 21111 Lakeshore Road,
Ste-Anne-de-Bellevue, QC Canada H9X 3V9
phone: + 1 514 398 7851, fax: + 1 514 398 7897, e-mail: [email protected]

136 PLANT SOIL ENVIRON., 52, 2006 (3): 130–136