Effects of gamma-irradiated Acinetobacter calcoaceticus on ... Text/vol23/V23_12.pdfMalaysian...

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ISSN: 1394-7990 Malaysian Journal of Soil Science Vol. 23: 149-166 (2019) Malaysian Society of Soil Science Effects of gamma-irradiated Acinetobacter calcoaceticus on nitrogen and phosphorus uptake of green mustard (Brassica chinensis) Phua Choo Kwai Hoe 1 , Halimi Mohd Saud 1 , Khairuddin Abdul Rahim 2 ,Che Fauziah Binti Ishak 3 and Puteri E. Megat Wahab 4 1 Universiti Putra Malaysia, Department of Agriculture Technology, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia 2 Malaysian Nuclear Agency, Agrotechnology and Bioscience Division, Bangi, 43000 Kajang, Selangor, Malaysia 3 Universiti Putra Malaysia, Department of Land Management, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia 4 Universiti Putra Malaysia, Department of Crop Science, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia ABSTRACT Several biofertiliser microorganisms are subjected to gamma-irradiation for mutagenesis to improve capabilities of N 2 fixation and P solubilisation, and also to satisfy biofertiliser market demands. The effects of gamma-irradiated Acinetobacter calcoaceticus on N and P uptake of choy sum or green mustard (Brassica chinensis) were investigated in a greenhouse experiment. Eight bacteria isolated from compost, soil and plants which were to be used as biofertilisers were gamma irradiated at 50–400 Gy and screened for the best N-fixing and P-solubilising mutants. The gamma-irradiated A. calcoaceticus M100/200 mutant showed higher N 2 fixation and phosphate solubilisation than those of the wild-type in vitro. The selected mutant (M100/200) and wild-type (M100) A. calcoaceticus were then tested on green mustard in a greenhouse experiment. N, P and K contents in soil, as well as pH, were determined using a soil nutrient analyser. Urea and rock phosphate were used as nitrogen and phosphate sources, respectively. Two-week- old seedlings were treated with biofertiliser (mutant or wild type) with either N or P source alone or in combination. The control treatments comprised of biofertiliser or N or P source alone (positive control) and without treatment (negative control). Crops were harvested after 2 months. Fresh and dry weight, height, chlorophyll content, leaf area and total N and P in the tissue of the crops were measured. Mutant M100/200 with N and P source treatment or with P source only showed superior growth and nutrient uptake in comparison to those of the samples given other treatments in the greenhouse experiment. Nitrogen input did not enhance growth, whereas phosphorus input and administration of phosphate solubiliser increased plant growth indirectly through a better rooting system. Green mustard inoculated with mutant strain demonstrated better nutrient uptake in a greenhouse experiment than those inoculated with the wild type. ___________________ *Corresponding author :

Transcript of Effects of gamma-irradiated Acinetobacter calcoaceticus on ... Text/vol23/V23_12.pdfMalaysian...

Page 1: Effects of gamma-irradiated Acinetobacter calcoaceticus on ... Text/vol23/V23_12.pdfMalaysian Journal of Soil Science Vol. 23 2019 151 2015; Halim et al. 2016; Andhika and Augustini

ISSN: 1394-7990Malaysian Journal of Soil Science Vol. 23: 149-166 (2019) Malaysian Society of Soil Science

Effects of gamma-irradiated Acinetobacter calcoaceticus on nitrogen and phosphorus uptake of green mustard (Brassica

chinensis)

Phua Choo Kwai Hoe1, Halimi Mohd Saud1, Khairuddin Abdul Rahim2,Che Fauziah Binti Ishak3 and Puteri E. Megat Wahab4

1Universiti Putra Malaysia, Department of Agriculture Technology, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia

2Malaysian Nuclear Agency, Agrotechnology and Bioscience Division, Bangi,43000 Kajang, Selangor, Malaysia

3Universiti Putra Malaysia, Department of Land Management, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia

4Universiti Putra Malaysia, Department of Crop Science, Faculty of Agriculture, 43300 UPM Serdang, Selangor, Malaysia

ABSTRACTSeveral biofertiliser microorganisms are subjected to gamma-irradiation for mutagenesis to improve capabilities of N2 fixation and P solubilisation, and also to satisfy biofertiliser market demands. The effects of gamma-irradiated Acinetobacter calcoaceticus on N and P uptake of choy sum or green mustard (Brassica chinensis) were investigated in a greenhouse experiment. Eight bacteria isolated from compost, soil and plants which were to be used as biofertilisers were gamma irradiated at 50–400 Gy and screened for the best N-fixing and P-solubilising mutants. The gamma-irradiated A. calcoaceticus M100/200 mutant showed higher N2 fixation and phosphate solubilisation than those of the wild-type in vitro. The selected mutant (M100/200) and wild-type (M100) A. calcoaceticus were then tested on green mustard in a greenhouse experiment. N, P and K contents in soil, as well as pH, were determined using a soil nutrient analyser. Urea and rock phosphate were used as nitrogen and phosphate sources, respectively. Two-week-old seedlings were treated with biofertiliser (mutant or wild type) with either N or P source alone or in combination. The control treatments comprised of biofertiliser or N or P source alone (positive control) and without treatment (negative control). Crops were harvested after 2 months. Fresh and dry weight, height, chlorophyll content, leaf area and total N and P in the tissue of the crops were measured. Mutant M100/200 with N and P source treatment or with P source only showed superior growth and nutrient uptake in comparison to those of the samples given other treatments in the greenhouse experiment. Nitrogen input did not enhance growth, whereas phosphorus input and administration of phosphate solubiliser increased plant growth indirectly through a better rooting system. Green mustard inoculated with mutant strain demonstrated better nutrient uptake in a greenhouse experiment than those inoculated with the wild type.

___________________*Corresponding author :

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INTRODUCTIONA biofertiliser is a substance composed of living microorganisms that colonise the rhizosphere or the interior parts of plants and promote growth by increasing the supply or availability of primary nutrients to the host plant (Vessey 2003). Biofertilisers with arbuscular mycorrhiza fungi and nitrogen fixing bacteria (NFB) were developed in Malaysia in the late 1980s and early 1990s. Since then, phosphate solubilising microorganisms (PSM) and plant growth promoting microorganisms (PGPR) have been used as biofertiliser inoculants. In 2012, bioinoculants mainly nitrogen fixing bacteria (NFB) such as rhizobia, which constitute 79% of the global demand of biofertiliser application (Transparency Market Research, 2014). This shows that biofertiliser products target limited activities. Thus, it is prudent for multifunctional biofertilisers to be developed. Multifunctional activities include N2 fixation, phosphate solubilisation, potassium solubilisation and plant growth promotion in a single application. Current practices to produce multifunctional biofertilisers involve mixing of microorganisms. As a consequence, this approach may cause other problems such as contamination, antagonistic effects among microorganisms, and decreased biofertiliser shelf life. Mutagenesis or genetic recombination of biofertiliser microorganisms may improve their multifunctional activities to satisfy market demands. Gamma-irradiation is widely used for plant mutagenesis (Anna et al. 2008; Ibrahim et al. 2013 and Sikder et al. 2013). The microbial mutation method by gamma irradiation has been applied in the fermentation industry (Huma et al. 2012 and Mehdikhani et al. 2011), whereas disease-control mutants have been utilised in the agriculture industry (Haggag 2002; Haggag and Mohamed 2002). To date, mutagenesis of biofertiliser microbes has not been conducted in Malaysia. In this study, A. calcoaceticus was gamma-irradiated to enhance N2 fixation and phosphate solubilisation relative to those of the wild-type and ultimately satisfy biofertiliser market demands. Nitrogen and phosphorus are two important plant nutrients. Biological nitrogen fixation (BNF) by N2-fixing bacteria is considered to be important in determining the nitrogen balance in soil systems (Lugtenberg et al. 2013). Meanwhile, phosphate solubilisers comprise a group of heterotrophic microorganisms known to solubilise inorganic phosphorus from insoluble sources. Applying phosphate-solubilising microorganisms substantially increases the abundance of active and effective microorganisms at the root rhizosperic zone and leads to an increases in plant nutrient uptake (Mehrvarz et al. 2008; Shen et al. 2011). Vegetable and cash crop statistics in Malaysia have shown that green mustard has the highest production (15,522.6 ha of harvested area and 275,732.4 Mt) of vegetables in Malaysia (DOA 2014). Green mustard (Brassica chinensis) has been inoculated with biofertilisers, such as mycorrhiza, Rhizobium sp., Azotobacter sp., Azospirillum . Aspergillus niger and Bacillus sp. (Kalay et al.

Keywords: Acinetobacter baumannii, Acinetobacter calcoaceticus, gamma- irradiation, multifunctional biofertiliser

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2015; Halim et al. 2016; Andhika and Augustini 2017; Widnyana et al., 2018), and positive results in terms of growth and yield have been obtained. The effects of gamma-irradiated A. calcoaceticus on the nitrogen and phosphorus uptake of green mustard were evaluated in a greenhouse experiment.

MATERIALS AND METHODS

Mutagenesis of A. calcoaceticus through gamma-irradiationA. calcoaceticus was isolated from paddy soil in MADA (Muda Agricultural Development Authorities) field (Kedah). The bacterial strains were identified by using the 16S rRNA method (Tahir et al. 2013). The radiation mutagenesis procedure was performed with gamma irradiation (50–400 Gy) from the gamma cell in Malaysian Nuclear Agency as described by Rugthaworn et al. (2007) but with slight modifications. Serial dilution and plate count methods were employed to determine the lethal dosage (LD50) and to obtain the survival curve. N2 fixing activity was screened by culturing the isolates on yeast extract–mannitol agar (YMA) containing 25 µg/ml bromothymol blue (BTB) (Swelim et al. 2010). N2-fixing bacteria produced a blue zone on this agar plate. The colonies that produced larger blue zones than those of the non-irradiated colonies were selected. Phosphate-solubilising activities were screened on phosphate agar plates (PDYA) (Freitas et al. 1997). Phosphate-solubilising bacteria produced halo zones on this agar plate. The colonies that produced larger halo zones than those of the non-irradiated colonies were selected. Stable mutants (stable N2-fixing and phosphate solubilisation activities) were selected after five screening times. Data were analysed by ANOVA, with the means separated by Duncan’s test (P ≤ 0.05).

Nitrogen and Phosphorus Uptake of Choy Sum (B. chinensis) in a Greenhouse ExperimentA. calcoaceticus M100 (wild-type) and M100/200 (mutant) were prepared by culturing on nutrient broth for 24 h. Then, 2-wk-old green mustard seedlings were transplanted into pots containing 5 kg soil mixture of soil, peat and sand at a ratio of 2:1:1. The soil was then gamma sterilised at 50 kGy. N, P, K and pH were determined by using a soil nutrient analyser. Urea (120 kg ha-1 of N) and phosphate rock (80 kg ha-1 of P) were applied as N and phosphate sources, respectively. Treatments are listed in Table 1. Plants were harvested after two months. Fresh and dry weights, height, root length, SPAD, chlorophyll content, leaf area, and total N and P were measured. Total chlorophyll (chlorophyll a and b) were extracted and measured as described by Coombs et al. (1985). The total N in the samples was determined by Kjeldahl digestion and titration (Jones, 2001). The total P in the samples was extracted by the wet digestion method (Jones , 2001) and measured by inductively coupled plasma (ICP) optical emission spectrometry. Data were analysed by ANOVA, with means separated by Duncan’s Multiple Range test (P ≤ 0.05).

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RESULTS AND DISCUSSION

Mutagenesis of A. calcoaceticus Through Gamma IrradiationThe wild-type (M100) and mutant (M100/200) strains were identified as A. calcoaceticus by the 16S rRNA method. Table 2 shows that the LD50 for M100 was 488.50 Gy (50% of log cfu/ml bacteria were killed at the dose of 488.50 Gy, where cfu stands for colony-forming units). On N2 fixing agar media, M100/200 formed a 2.20 cm zone, which was larger than those of the other irradiated isolates (Figure 1). The diameters of the clear zones after 0, 50, 100, 300 and 400 Gy irradiations were 2.05, 1.95, 1.88, 2.02 and 2.00 cm, respectively. On phosphate-solubilising agar media, M100/200 showed the clear zone with the largest diameter (2.38 cm). Other irradiated isolates exhibited clear zones with diameters of 1.98, 2.025, 1.88,

TABLE 1Treatments of green mustard in greenhouse experiment

6

Nitrogen and Phosphorus Uptake of Choy Sum (B. chinensis) in a Greenhouse Experiment A. calcoaceticus M100 (wild-type) and M100/200 (mutant) were prepared by

culturing on nutrient broth for 24 h. Then, 2-wk-old green mustard seedlings were

transplanted into pots containing 5 kg soil mixture of soil, peat and sand at a ratio

of 2:1:1. The soil was then gamma sterilised at 50 kGy. N, P, K and pH were

determined by using a soil nutrient analyser. Urea (120 kg/ha of N) and phosphate

rock (80 kg/ha of P) were applied as N and phosphate sources, respectively.

Treatments are listed in Table 1. Plants were harvested after two months. Fresh

and dry weights, height, root length, SPAD, chlorophyll content, leaf area, and

total N and P were measured. Total chlorophyll (chlorophyll a and b) were

extracted and measured as described by Coombs et al. (1985). The total N in the

samples was determined by Kjeldahl digestion and titration (Jones, 2001). The

total P in the samples was extracted by the wet digestion method (Jones , 2001)

and measured by inductively coupled plasma (ICP) optical emission spectrometry.

Data were analysed by ANOVA, with means separated by Duncan’s Multiple

Range test (P ≤ 0.05).

TABLE 1

Treatments of green mustard in greenhouse experiment

Treatments N (4.3 g/pot)

P (3.85 g/pot)

N (2.15 g/pot) and P (1.93 g/pot)

Without N and P

M100 (50 mL/pot)

T1 T4 T7 T10

M100/200 (50 mL/pot)

T2 T5 T8 T11

Control T3 T6 T9 T12

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Figure 1. Determination of the N2-fixing activity of the gamma-irradiated and

non-irradiated (A) A. calcoaceticus (M100) on yeast extract mannitol

Agar (YMA) with bromothymol blue (BTB)

Notes: All values are expressed as means of four replications with one plate per

replication. Means followed by the same letter are insignificantly different

from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test .

0

0.5

1

1.5

2

2.5

0 50 100 200 300 400Zone

Siz

e (c

m) f

or N

itrog

en fi

xing

activ

ity

for A

cine

toba

cter

cal

coac

etic

us

Gamma irradiated dose (Gy)

bc ab a

c bc bc

0

0.5

1

1.5

2

2.5

3

0 50 100 200 300 400

Zone

Size

(cm

) for

Pho

spha

te

solu

bilis

ing

activ

ity fo

r Acin

etob

acte

r ca

lcoac

eticu

s

Gamma irradiated dose (Gy)

ab abc a

dc bc

Fig. 1: Determination of the N2-fixing activity of the gamma-irradiated and non-irradiated (A) A. calcoaceticus (M100) on yeast extract mannitol Agar (YMA) with

bromothymol blue (BTB) Notes: All values are expressed as means of four replications with one plate per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test .

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2.15 and 2.05 cm after irradiation with 0, 50, 100, 300 and 400 Gy, respectively (Figure 2). These findings reveal that Acinetobacter sp. could be improved as multifunctional biofertiliser inoculants through gamma irradiation. The plant growth-promoting activity of Acinetobacter sp. has been widely reported recently. The Acinetobacter rhizosphaerae strain BIHB 723 from the cold deserts of the Himalayas exhibited plant-growth-promoting attributes, namely, inorganic and organic phosphate solubilisation, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, ammonia generation and siderophore production. This isolate significantly increased the growth of pea, chickpea, maize and barley. In the field tests, the growth and yield of the pea plants significantly increased (Gulati et al. 2009). Sarode et al. (2009) reported that the siderophore-producing A. calcoaceticus SCW 1, which was isolated from wheat rhizospheres of black cotton soils of the North Maharashtra region, improved wheat plant growth in pot and field studies. The PGPR activities of this isolate were phosphate solubilisation and IAA production. Endophytic A. calcoaceticus from coffee plant tissue produced phosphatase and IAA in vitro (Silva et al. 2012). Acinetobacter schindleri nbri 05 tolerates arsenic-contaminated environments by producing the plant growth-promoting substance IAA and siderophores (Srivastava and Singh 2014). A. calcoaceticus has also been reported as a gibberellin-producing species that promotes cucumber plant growth (Kang et al. 2012). These reports demonstrate that Acinetobacter sp. exhibits various plant-growth-promoting activities, such as plant growth hormone production, biological control effect, phosphate solubilisation, siderophore production or phytoremediation. 9

Figure 2. Determination of the phosphate-solubilising activity of gamma-

irradiated and non-irradiated (A) A. calcoaceticus (M100) on phosphate-

solubilising yeast extract agar (PDYA)

Notes: All values are expressed as means of four replications with one plate per

replication. Means followed by the same letter are insignificantly different

from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test .

The plant growth-promoting activity of Acinetobacter sp. has been widely

reported recently. The Acinetobacter rhizosphaerae strain BIHB 723 from the

cold deserts of the Himalayas exhibited plant-growth-promoting attributes,

namely, inorganic and organic phosphate solubilisation, auxin production, 1-

aminocyclopropane-1-carboxylate deaminase activity, ammonia generation and

siderophore production. This isolate significantly increased the growth of pea,

chickpea, maize and barley. In the field tests, the growth and yield of the pea

plants significantly increased (Gulati et al. 2009). Sarode et al. (2009) reported

that the siderophore-producing A. calcoaceticus SCW 1, which was isolated from

wheat rhizospheres of black cotton soils of the North Maharashtra region,

0

0.5

1

1.5

2

2.5

3

0 50 100 200 300 400

Zone

Size

(cm

) for

Pho

spha

te

solu

bilis

ing

activ

ity fo

r Acin

etob

acte

r ca

lcoac

eticu

s

Gamma irradiated dose (Gy)

ab abc a

dc bc

Fig. 2: Determination of the phosphate-solubilising activity of gamma-irradiated and non-irradiated (A) A. calcoaceticus (M100) on phosphate-solubilising

yeast extract agar (PDYA)Notes: All values are expressed as means of four replications with one plate per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test .

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Nitrogen and Phosphorus Uptake of Choy Sum (B. chinensis) in a Greenhouse ExperimentSoil N, P and K elements were 18.27, 27.51 and 190 mg kg-1 respectively while soil pH was approximately 7.7. Highest plant heights were obtained from plants inoculated with T5 (mutant M100/200 with N source) and T8 (mutant M100/200 with a combination of N and P sources) (Figure 3). The T5 and T8 significantly differed from other treatments (P ≤ 0.05). The plant height for T5 was 29.5 cm and for T8, it was 28.75 cm. The plant heights for other treatments were in the range of 18.25–25.5 cm.

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improved wheat plant growth in pot and field studies. The PGPR activities of this

isolate were phosphate solubilisation and IAA production. Endophytic A.

calcoaceticus from coffee plant tissue produced phosphatase and IAA in vitro

(Silva et al. 2012). Acinetobacter schindleri nbri 05 tolerates arsenic-

contaminated environments by producing the plant growth-promoting substance

IAA and siderophores (Srivastava and Singh 2014). A. calcoaceticus has also been

reported as a gibberellin-producing species that promotes cucumber plant growth

(Kang et al. 2012). These reports demonstrate that Acinetobacter sp. exhibits

various plant-growth-promoting activities, such as plant growth hormone

production, biological control effect, phosphate solubilisation, siderophore

production or phytoremediation.

TABLE 2

Biofertiliser identification by 16S RNA, isolation sources and LD50

Isolates Isolation source Organism identification

Gen Bank accession no.

Similarity (%)

LD50 value (Gy)

M100 Soil from paddy field: MADA, Kedah.

Acinetobacter calcoaceticus

JF681282 97 448.5

M100/200 Radiated mutant at dose 200 Gy. Nuclear Malaysia

Acinetobacter calcoaceticus

JF681282 94

Nitrogen and Phosphorus Uptake of Choy Sum (B. chinensis) in a Greenhouse Experiment Soil N, P and K elements were 18.27, 27.51 and 190 mg kg-1 respectively while

soil pH was approximately 7.7. Highest plant heights were obtained from plants

inoculated with T5 (mutant M100/200 with N source) and T8 (mutant M100/200

TABLE 2Biofertiliser identification by 16S RNA, isolation sources and LD50

11

with a combination of N and P sources) (Figure 3). The T5 and T8 significantly

differed from other treatments (P ≤ 0.05). The plant height for T5 was 29.5 cm

and for T8, it was 28.75 cm. The plant heights for other treatments were in the

range of 18.25–25.5 cm.

Figure 3. Effects of A. calcoaceticus on green mustard height in a greenhouse

experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figures 4 and 5 show the top fresh and dry weights of the green mustard.

Only T8 showed the highest (P ≤ 0.05) fresh (66.33 g) and dry (3.83 g) weights

among treatments, followed by T5 with fresh and dry weights of 53.25 and 3.52 g,

respectively. The lowest fresh weight was observed in T12 (no treatment),

whereas the lowest dry weight was observed in T3 (only N treatment).

0

5

10

15

20

25

30

35

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

Heig

ht (c

m)

Treatments

bc c

bc

a

bc ab

a

ab bc bc

c bc

Fig. 3: Effects of A. calcoaceticus on green mustard height in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

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Figures 4 and 5 show the top fresh and dry weights of the green mustard. Only T8 showed the highest (P ≤ 0.05) fresh (66.33 g) and dry (3.83 g) weights among treatments, followed by T5 with fresh and dry weights of 53.25 and 3.52 g, respectively. The lowest fresh weight was observed in T12 (no treatment), whereas the lowest dry weight was observed in T3 (only N treatment).

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Figure 4. Fresh weights of top part of green mustard in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Fig. 4: Fresh weights of top part of green mustard in a greenhouse experiment.

Fig. 5: Dry weights of the top parts of green mustard plants in a greenhouse experiment

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Figure 4. Fresh weights of top part of green mustard in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

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Similar results were obtained for root length. The longest root lengths were achieved by T5 and T8 (Figures 6). The T5 (13.13 cm) and T8 (13.38 cm) achieved significantly longer (P ≤ 0.05) root lengths than those of the other treatments (7.13 cm to 10.25 cm). Figure 7 shows the highest (P ≤ 0.05) fresh weight of 4.9 g. Dry weight results (Figure 8) of roots showed insignificant difference (P ≤ 0.05) between T5 and T8. The dry weights for T5 and T8 were 0.75 and 0.97 g, respectively.

13

Figure 5. Dry weights of the top parts of green mustard plants in a greenhouse

experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Similar results were obtained for root length. The longest root lengths

were achieved by T5 and T8 (Figures 6). The T5 (13.13 cm) and T8 (13.38 cm)

achieved significantly longer (P ≤ 0.05) root lengths than those of the other

treatments (7.13 cm to 10.25 cm). Figure 7 shows the highest (P ≤ 0.05) fresh

weight of 4.9 g. Dry weight results (Figure 8) of roots showed insignificant

difference (P ≤ 0.05) between T5 and T8. The dry weights for T5 and T8 were

0.75 and 0.97 g, respectively.

Figure 6. Root length of green mustard plants in a greenhouse experiment

0

2

4

6

8

10

12

14

16

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

Root

leng

th (c

m)

Treatments

bcd ab

cd

bc

a

cd

bc

a

cd bcd

cd d

Fig. 6: Root length of green mustard plants in a greenhouse experiment

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Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 7. Fresh weights of the roots of green mustard plants in a greenhouse experiment

Fig. 7: Fresh weights of the roots of green mustard plants in a greenhouse experiment.Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

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Figure 9 shows the largest leaf area of 938.62 cm2 for T8. The second and third largest leaf areas were observed for T5 and T7 (M100 with N and P), accounting for 808.21 and 761.99 cm2, respectively. The smallest leaf area was observed for T12 (335.29 cm2).

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Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 8. Dry weights of the roots of green mustard plants in a greenhouse

experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 9 shows the largest leaf area of 938.62 cm2 for T8. The second and

third largest leaf areas were observed for T5 and T7 (M100 with N and P),

accounting for 808.21 and 761.99 cm2, respectively. The smallest leaf area was

observed for T12 (335.29 cm2).

Fig. 8: Dry weights of the roots of green mustard plants in a greenhouse experiment.Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

16

Figure 9. Leaf areas of green mustard plants in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 10 shows that an insignificant difference (P ≤ 0.05) in SPAD

reading was detected among T5 (38.30), T7 (38.33) and T8 (40.48). These

treatments showed a higher SPAD reading than in other treatments. The lowest

SPAD reading was observed for T12 (27.38) followed by T3 (30.13). Total

chlorophyll obtained followed almost the same trend as the SPAD reading results.

The chlorophyll contents for T7 (1.93 mg cm−2) and T8 (1.97 mg cm−2) did not

significantly differ (Figure 11). The lowest total chlorophyll was observed for

T12 (0.76 mg cm−2), followed by T3 (1.01 mg cm−2).

Fig. 9: Leaf areas of green mustard plants in a greenhouse experiment.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

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Figure 10 shows that an insignificant difference (P ≤ 0.05) in SPAD reading was detected among T5 (38.30), T7 (38.33) and T8 (40.48). These treatments showed a higher SPAD reading than in other treatments. The lowest SPAD reading was observed for T12 (27.38) followed by T3 (30.13). Total chlorophyll obtained followed almost the same trend as the SPAD reading results. The chlorophyll contents for T7 (1.93 mg cm-2) and T8 (1.97 mg cm-2) did not significantly differ (Figure 11). The lowest total chlorophyll was observed for T12 (0.76 mg cm-2), followed by T3 (1.01 mg cm−2).

17

Figure 10. SPAD reading of green mustard plants in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 11. Total chlorophyll contents of green mustard plants in a greenhouse

experiment

0

0.5

1

1.5

2

2.5

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

Tota

l chl

orop

hyll

(mg

cm-2

)

Treatments

ab

cde ef

cd

ab

de

a a

bc ab

a

f

Fig. 11: Total chlorophyll contents of green mustard plants in a greenhouse experiment

17

Figure 10. SPAD reading of green mustard plants in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 11. Total chlorophyll contents of green mustard plants in a greenhouse

experiment

0

0.5

1

1.5

2

2.5

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

Tota

l chl

orop

hyll

(mg

cm-2

)

Treatments

ab

cde ef

cd

ab

de

a a

bc ab

a

f

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

Fig. 10: SPAD reading of green mustard plants in a greenhouse experiment

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An insignificant difference (P ≤ 0.05) in the total N content of the oven-dried samples for T4 (4.17 %), T5 (4.69 %), T6 (4.20%) and T7 (4.30 %) was observed. The difference in the total P of T5 (10.10 mg/L), T6 (9.72 mg/L), T7 (11.29 mg/L), T8 (9.82 mg/L) and T11 (9.59 mg/L) was insignificant (P ≤ 0.05). Figure 12 shows that T5 attained the highest total N content, whilst Figure 13 shows that T7 achieved the highest total P content of 11.29 mg/L.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

Notes: All values are expressed as the means of four replications with one seedling per replication. Means followed by the same letter are insignificantly different from each other (P ≤ 0.05) as determined by Duncan’s Multiple Range test.

18

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

An insignificant difference (P ≤ 0.05) in the total N content of the oven-

dried samples for T4 (4.17 %), T5 (4.69 %), T6 (4.20%) and T7 (4.30 %) was

observed. The difference in the total P of T5 (10.10 mg/L), T6 (9.72 mg/L), T7

(11.29 mg/L), T8 (9.82 mg/L) and T11 (9.59 mg/L) was insignificant (P ≤ 0.05).

Figure 12 shows that T5 attained the highest total N content, whilst Figure 13

shows that T7 achieved the highest total P content of 11.29 mg/L.

Figure 12. Total N of green mustard plants in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

Fig. 12: Total N of green mustard plants in a greenhouse experiment.

19

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Figure 13: Total P contents of green mustard plants in a greenhouse experiment

Notes: All values are expressed as the means of four replications with one seedling

per replication. Means followed by the same letter are insignificantly

different from each other (P ≤ 0.05) as determined by Duncan’s Multiple

Range test .

Overall, T8 (mutant A. calcoaceticus M100/200 with N and P sources) and

T5 (mutant A. calcoaceticus M100/200 with P source only) exhibited superior

growth and nutrient uptake compared with those of the other treatments.

Amongst the functions of biofertilisers, N2 fixation and phosphate

solubilisation play important roles in plant growth. Studies on the effect of

phosphate solubilisation on N2 fixation (Afzal and Bano 2008; Linu et al. 2009;

Fig. 13: Total P contents of green mustard plants in a greenhouse experiment.

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Overall, T8 (mutant A. calcoaceticus M100/200 with N and P sources) and T5 (mutant A. calcoaceticus M100/200 with P source only) exhibited superior growth and nutrient uptake compared with those of the other treatments. Amongst the functions of biofertilisers, N2 fixation and phosphate solubilisation play important roles in plant growth. Studies on the effect of phosphate solubilisation on N2 fixation (Afzal and Bano 2008; Linu et al. 2009; Argaw 2012 and Wahid et al. 2016) have demonstrated that phosphate solubilising microorganisms influence nutrient uptake and root zone biodiversity. The effects of wild-type and mutant A. calcoaceticus strains on N2 and phosphorus uptake of green mustard were tested in a greenhouse experiment in this study. A similar report observes that a combination of N2 fixing bacteria and phosphate solubiliser increases the chlorophyll content, leaf area index, total root length and root volume of rice grown under aerobic conditions than those of the control (Nur’ain et al. 2017). A. calcoaceticus used in this study has both N2 fixing and phosphate-solubilising activities. Overall, treatment with T8 (mutant M100/200 with the combination of N and P sources) and T5 (mutant M100/200 with P sources) was found to lead to better plant heights, top fresh and dry weights, root and leaf growth and chlorophyll contents compared to other treatments. Chlorophyll content and leaf area were correlated with the nutrient uptake of N. The majority of leaf N accumulated in the chloroplast where photosynthesis occurs, resulting in a strong association between plant photosynthesis and leaf N status. Leaf N can be rapidly estimated by using an SPAD chlorophyll meter (Zakeri et al. 2015), a non-destructive indirect method for evaluating plant N status in real time. However, the relationship between chlorophyll concentration and SPAD values is not always linear (Ferreira et al. 2015). Conversely, other studies have demonstrated that the relationship amongst SPAD reading, chlorophyll content and leaf N content per leaf area is linear. In some cases, this relationship is affected by environmental factors and leaf features of crop species (Xiong et al. 2015). In our study, total chlorophyll content was linearly related to SPAD reading. Leaf area was also linearly associated with chlorophyll content and SPAD reading. This linear result could be due to the growth of green mustard in a greenhouse which was a controlled environment. Both wild-type (M100) and mutant (M100/200) showed significant chlorophyll content, SPAD reading and leaf area compared with those in the other treatments, indicating that wild-type and mutant treatment had N2 fixation activities that enhanced the growth of leafy parts. The root systems of the wild-type and the mutant supplied with P had significant growth. However, these treatments provided additional N but promoted insignificant growth. The results revealed that phosphate solubilisation with P source played an important role. Phosphate is essential for root growth. Good root systems enhance leaf growth, thereby increasing chlorophyll and N uptake. Afzal and Bano (2008) also reported that a single inoculation of Rhizobium (symbiotic N) cannot increase grain yield, but the dual inoculation of Rhizobium and phosphate solubilising bacteria increases the yield by 29% and 25% with

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and without fertiliser, respectively. Seed inoculation of cowpea by phosphate solubilisers improves its nodulation, root and shoot biomasses, straw and grain yields and P and N uptakes (Linu et al. 2009). A. calcoaceticus enhanced the growth of hazelnut seedlings and increased N, P, K, Ca, Mg, Fe, Cu, Mn, Zn, B and Al concentrations in plant tissues (Erturk et al. 2011). In our study, A. calcoaceticus increased N and P uptake. Overall, the total N and P of the plant and soil samples treated with the mutant strain and phosphate rock (T5) or the wild-type strain with urea and phosphate rock (T7) were significantly high. Thus, the combination of biofertiliser with additional nutrient sources could enhance plant nutrient uptake. The treatment with the mutant strain and urea mixed with phosphate rock (T8) was the best combination treatment as shown by physical measurements.

CONCLUSIONSIn conclusion, Acinetobacter sp. could be improved as a multifunctional biofertiliser inoculant through gamma irradiation. The mutant from gamma-irradiation, A. calcoaceticus M100/200, showed superior N2-fixation and phosphate solubilisation in comparison to the wild-type in vitro and under a greenhouse experiment on green mustard plants. For future investigations, 15N isotope could be considered for quantitative N determination to elucidate the influence of P on N utilisation and plant development.

ACKNOWLEDGEMENTSThe authors wish to express their gratitude to Universiti Putra Malaysia, Malaysian Nuclear Agency (Nuclear Malaysia), Ministry of Energy, Science, Technology, Environment and Climate Change (MESTECC) and the Public Service Department (JPA) for technical and financial support. Technical assistance from Dr. Ahmad Zainuri B. Mohd Dzomir, Latiffah Norddin, Abdul Razak Ruslan, Hazlina Abdullah, Shuhaimi B. Shamsudin, Sharpudin Md Nor and Nur Fariz Bt. Hj Sulaiman is greatly appreciated.

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