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Potential of plant growth promoting Rhizobacteria to overcome the exposure of pesticide in Trigonella foenum - graecum (fenugreek leaves)
Journal Pre-proof Potential of plant growth promoting Rhizobacteria to overcome the exposure of pesticide in Trigonellafoenum - graecum (fenugreek leaves) S. Nathiya, R. Janani, V. Rajesh Kannan PII:
S1878-8181(19)31263-0
DOI:
https://doi.org/10.1016/j.bcab.2020.101493
Reference:
BCAB 101493
To appear in:
Biocatalysis and Agricultural Biotechnology
Received Date: 12 October 2019 Revised Date:
16 December 2019
Accepted Date: 2 January 2020
Please cite this article as: Nathiya, S., Janani, R., Kannan, V.R., Potential of plant growth promoting Rhizobacteria to overcome the exposure of pesticide in Trigonellafoenum - graecum (fenugreek leaves), Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2020.101493. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Potential of Plant Growth Promoting Rhizobacteria to Overcome the Exposure of Pesticide
2
in Trigonellafoenum - graecum (Fenugreek leaves)
3 4
S. Nathiya, R. Janani, V. Rajesh Kannan*
5 6
Rhizosphere Biology Laboratory, Department of Microbiology, Bharathidasan University,
7
Tiruchirappalli - 620 024, Tamil Nadu, India.
8 9
*Corresponding Author:
10
Dr. V. Rajesh Kannan
11
Email id: [email protected]
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1
1
Abstract
2
Agrochemicals are generally used as additional supplements to the soil for improving soil
3
fertility throughout the world. In this present study was carried out to characterize the pesticide
4
tolerance plant growth promoting rhizobacteria (PGPR) were isolated from fenugreek leaves.
5
Seven bacterial isolates were obtained namely FOW1 to FOW7 and characterized on their
6
colony morphology, size and colour. Among the seven, FOW1 and FOW7 isolates were found to
7
grown well in nutrient medium containing 10 to 20% concentration of pesticide. All the seven
8
isolates were produced indole-3- acetic acid in the range of 56 to 97µg/mL and only two isolates
9
(FOW1 and FOW7) displayed the phosphate solubilising activity in Pikovskaya agar, whereas
10
six isolates were showed positive for production of siderophore. Out of seven isolates, two
11
exhibited in vitro plant growth promotion activities and indicated that these isolates can be
12
exploited as biofertilizers and microbial inoculants for crops as they enhanced plant growth via
13
diverse mechanisms and offered an attractive strategy to replace synthetic fertilizers and
14
pesticides. The morphological features such as germinating ability, shoot length, root length
15
were enhanced in the presence of PGPR and was maximum at 1%.
16
Keywords: Pesticide; PGPR; Fenugreek seed; Rhizosphere; Tolerance.
17 18
1. Introduction
19
Pesticide is a single or combination of toxic chemical or chemical agent that are
20
deliberately let into the environment in order for the control/ destruction of harmful pests.
21
Pesticides are not only toxic to living pests and the ecosystem too and ultimately resulting in
22
their bioaccumulation. Early reports have established the presence of several pesticides such as
23
DDT, lindane and dieldrin in even food products beyond their permissible limits ( Linhart et al.,
24
2019). Few microorganisms on long term exposure show resistance to pesticides and they can be
25
effectively used in the remediation of pesticide contaminated soils (Khan et al., 2009). Microbes
26
perform efficiently in the presence of specific pesticides by using them as nutrients and energy
27
source (Pramanik et al., 2018). Microorganisms degrade these pesticides and use them as a
28
carbon source. Their ability to degrade pesticide is an important phenomenon through which
29
these chemicals are eliminated from the environment and control the environmental pollution
30
(Backer et al., 2018). These bacterial strains can be used in modern agriculture as inoculants
31
under stress conditions such as heavy metal stress (Bhatt and Vyas, 2014), herbicide stress, 2
1
(Ahemad and Khan, 2011a), insecticides stress (Ahemad and Khan, 2011b), fungicides stress,
2
(Ahemad and Khan, 2012) and salinity stress (Tank and Saraf, 2010). Therefore, those microbes
3
are essential component in recycling process and important to maintain soil fertility (Glick,
4
2012).
5
Bioremediation is transformation or degradation of pollutants by microbes into non-
6
hazardous or less toxic substances. It mainly depends on microbial enzymatic attack on the
7
pollutants and converts them to less toxic products. On this strategy which not only proves
8
beneficial in reducing organophosphates accumulation but it’s also enhances plant growth up to
9
in quality aspects. Thus, the course of bioremediation is addressed by plant growth promoting
10
rhizobacteria (PGPR). The PGPR colonises on the roots of the plants and help in promoting plant
11
growth (DalCorso et al., 2019). The PGPR may exert their effects directly or indirectly by
12
several metabolites, singly or in combination with several other factors (Shaheen and Sundari,
13
2013). Generally, PGPR functions by synthesizing particular compounds, inhabiting certain
14
nutrient uptake and guarding the plants from diseases (Castellanos Rozo et al., 2013). Thus it
15
becomes more beneficial to apply such a strategy which remediate the pollutants as well enhance
16
plant growth. The PGPR has been explored on biotechnological applications in various uses such
17
as biofertilizers, biocontrol agents, phytostimulators and bioremediators based on their mode of
18
action in agriculture practices (Jariyal et al., 2018). The PGPR is known for the production of
19
growth hormones, vitamins and other amino acids that influence the growth of the plant by
20
increasing its nutrient availability (Kumar et al., 2011).
21
Addition that the PGPRs like Pseudomonas, Azospirillum, Agrobacterium, Bacillus,
22
Enterobacter and Flavobacterium strains that degrade organic and inorganic contaminants in soil
23
(Myresiotis et al., 2011; Joseph et al., 2012). While the inorganic amendments to agricultural soil
24
improves the efficiency of fertility in certain limits (Htwe et al., 2019) and the excess or
25
unutilized
26
available on biodegradation of soil applied pesticides by PGPR strains and their effects on
27
bacterial growth (Pandey and Gupta, 2019) and the indigenous soil microbial community (Liang
28
et al., 2019). Therefore, the present study was conducted to isolate and characterize the pesticide
29
tolerant rhizobacteria for multiple plant growth promoting traits.
those inorganic nutrients leads to fatal the soil organisms, the limited data are
30 31 3
1
2. Materials and Methods
2
2.1 Sample Source
3
Soil samples were collected from the Trigonellafoenum graecum rhizosphere which was
4
regular cultivation field in Othakadai Village of Tiruchirappalli district in Tamil Nadu, India.
5
Organochlorine pesticide was purchased from certified agrochemical shop in Tiruchirappalli.
6 7
2.2 Isolation and Identification of PGPR
8
Bacterial isolates were obtained from fenugreek plants rhizosphere soils through dilution
9
plate technique (Albdaiwi et al., 2019) in detail, one gram of rhizosphere soil samples were taken
10
and mixed in 10mL of sterile water and then serially diluted. The dilutions of 10-3, 10-4 and 10-5
11
were used for bacterial isolation. Approximately 0.1mL sample was spread over the nutrient agar
12
medium the plates were incubated at 30oC for up to 48h. The colonies were identified based on
13
their morphology and biochemical tests (Aditi et al., 2017; Tirry et al., 2018). The isolates were
14
stored at -4°C for further analyses.
15 16
2.3 Screening of Plant Growth Promoting Rhizobacteria
17
2.3.1 Indole Acetic Acid Production
18
The bacterial strains were cultured in nutrient broth supplement with 500µg tryptophan
19
mL-1 and incubated at 27ºC about two weeks. Approximately 1mL of cultures were withdrawn at
20
different time intervals (2nd - 10th day with 2 days interval) and centrifuged at 6000rpm for
21
30min. Indole acetic acid concentration was estimated (Albdaiwi et al., 2019), 1mL of the
22
supernatant, a drop of orthophosphoric acid and 2mL of Salkowski’s reagent (50mL, 35%
23
perchloric acid and 1mL, 0.5M FeCl3) were added and mixed. Colour was measured by
24
spectrophotometrically at 530nm.
25 26
2.3.2 Siderophore Production
27
Bacterial isolates were streaked in CAS agar medium and incubated at 28± 10oC for 48h.
28
The siderophore production was observed as orange halos around the colonies (Tirry et al.,
29
2018).
30
.
31 4
1
2.3.3 Phosphate Solubilization
2
Phosphate solubilization test was done using modified Pikovskaya medium which
3
consists of 10g glucose, 5g tribasic phosphate (Ca5HO13P3), 0.5g (NH4)2SO4,0.2gKCl, 0.1g
4
MgSO4.7H2O, 0.0001g MnSO4 and FeSO4, 0.5g yeast extract, and 15g agar, in 1000mL distilled
5
water. Bacterial isolates were spot inoculated and after 4 days of incubation, bacterial zone
6
formation was observed. The diameter of halo clear zones were measured and solubilization
7
efficiency (SE) and solubilization index (SI) were evaluated using following formulae (García et
8
al., 2017).
9 10
SE= Solubilization diameter /growth diameter SI= Colony diameter + halozone diameter/colony diameter
11 12
2.3.4 Hydrogen Cyanide Production
13
The bacterial isolates were checked for hydrogen cyanide production. In nutrient broth
14
containing 4.4g glycine/1, the isolates were streaked in agar plate. On the top of the plate,
15
Whatmann No.1 filter paper immersed in 2% sodium carbonate in 0.5% picric acid solution was
16
kept and sealed with parafilm and incubated at 28±2ºC for 4 days and colour changed was
17
observed (Pandey and Gupta, 2019).
18 19
2.3.5 Production of Ammonia
20
Bacterial isolates in 10mL peptone were inoculated and incubated for 72 h at 28±2ºC. To
21
these tunes, Nessler’s reagent (0.5mL) was added and colour change was observed (Pandey and
22
Gupta, 2019).
23 24
2.3.6 Biological Nitrogen Fixation
25
The bacterial strain was subjected to screening of nitrogen fixation activity using glucose
26
nitrogen free minimal medium (G-NFMM). Single colony was inoculated into G-NFMM
27
containing BTB (Bromothymol blue solution) and after one week incubation, colour change was
28
recorded (García et al., 2017).
29 30
2.4 Identification of Pesticide Tolerant PGPR
31
2.4.1 Plate Assay 5
1
Rhizobacterial strains were isolated from host plant rhizosphere with dilution plate
2
technique (Dworkin and Foster, 1958), whereas pesticide was applied intentionally. The
3
modified minimal salt agar medium containing different concentrations of 1 to 20% (100 to
4
2000mg L-1) of pesticide as sole source of carbon was used for isolation of pesticide tolerant
5
PGPR strains. The bacterial isolates were spot inoculated in agar plates with pesticide. The
6
growth or inhibition of bacterial strains at highest concentration of pesticide was considered as
7
the maximum tolerance level (MTL).
8 9 10
2.5 Optimization of Different Parameters for Pesticide Tolerance 2.5.1 Growth at Different Time Course
11
The effect of pesticides on the growth of bacterial isolates was examined. Exponentially
12
grown cultures were inoculated liquid culture medium (presence and absence) and incubated at
13
28ºC for 24, 48, 72, 96, 120 and 144 h. and the control was also maintained. Growth at different
14
times was determined by measuring the absorbance at 540 nm (Kale and Allen, 1989).
15 16
2.5.2 Growth at Different pH
17
For pH optimization, the rhizobacterial strains were grown in nutrient broth in different
18
levels of pH (5, 6, 7, 8 and 9). The medium with different pH was prepared and inoculated with
19
respective rhizobacterial strains. These culture were kept at 28 ± 2°C for 72h and optical density
20
was measured at 600 nm (Singh et al., 2014).
21 22
2.5.3 Salt Tolerance Test
23
Bacterial isolates salt tolerance levels were examined by osmo adaptation assay. The
24
bacterial isolates were grown in nutrient broth with different salt concentrations (1, 2, 3, 4, 5 and
25
6 dS m-1) and incubated for 72h at 28±2°C and optical density was measured at 540 nm (Sharma
26
et al., 2016).
27 28
2.5.4 Determination of Antibiotic Resistance
29
The antibiotic resistance of pesticide tolerant bacterial isolates of Bacillus sp.1 and
30
Lysinibacillus sp. against to three different antibiotics [Chloramphenicol (30µg), Vancomycin
31
(30µg) and Norfloxacin (30µg)] was carried out by disc diffusion method (Bauer et al., 1966). 6
1
These inoculums were maintained at 4°C in Luria-Bertani medium. The bacterial cultures were
2
reactivated in Muller Hinton Broth at 37°C for 24h. Bacterial culture was swabbed on the
3
surface of MHA with known potency antibiotic disc which was incubated at 37°C for 48h, the
4
zone of incubation (diameter in mM) around the antibiotic disc were measured.
5 6
2.6 In vivo Study of Trigonellafoenum - graecum
7
2.6.1 Nursery Experiment
8
All the seeds were sterilized with 70% ethanol and washed with 0.1% sodium hypo
9
chloride and rinsed with sterilized distilled water. The rhizobacterial cells were removed by
10
centrifuged at 10000 rpm for 10min at 4ºC and washed in distilled water. The pellet was
11
resuspended in sterile water and then diluted for a bacterial suspension concentration of 108
12
CFU/mL (OD 595= 0.3) (Kanthaiah and Velu, 2019).
13
The fenugreek seeds (1g seeds) were sterilized with 70% ethanol. Seeds were coated with
14
different treatments (Table 1) as follows: Test 1 - seeds (1g) + carboxyl methyl cellulose (100
15
mg), Test 2 – seeds (1g) + pesticide, Test 3 - pesticide tolerant PGPR (FOW1) + seeds(1g) +
16
carboxyl methyl cellulose (100 mg), Test 4 - pesticide tolerant PGPR (FOW7) + seeds (1g) +
17
carboxyl methyl cellulose (100 mg), Test 5 - pesticide tolerant PGPR (FOW7) + seeds (1g) +
18
pesticide + carboxyl methyl cellulose (100 mg), and Test 6 - pesticide + seeds (1g) + carboxyl
19
methyl cellulose (100 mg). All these seed were air dried for 12 hours. Dried seeds were
20
transplanted in poly bags (top diameter 300 mM, 160 mM bottom and height 16 cm) containing
21
soil and kept for experiments under nursery conditions for 30 days. After the growth period, the
22
crop’s morphometric analyses were done for the following parameter for each treatments plants-
23
shoot height, root length, shoot and root fresh weight, number of leaves and number of
24
adventitious roots. The experiments were done in duplicates and statiscally analysed.
25 26
3. Results and Discussion
27
3.1 Isolation and Identification of Rhizobacteria
28
Bacterial strains were isolated from the fenugreek field soil samples. Based on the colony
29
morphology, size, colour and biochemical characterization, seven bacterial isolates were
30
identified, such as Bacillus sp.1 (FOW1), Azospirillum sp. (FOW2), Acetobacter sp. (FOW3),
7
1
Klebsiella sp. (FOW4), Burkholderia sp. (FOW5), Bacillus sp.2 (FOW6) and Lysinibacillus sp.
2
(FOW7).
3 4
3.2 Screening for Plant Growth Promoting Rhizobacteria
5
3.2.1 Indole Acetic Acid Production All the seven isolates were produced different levels of IAA at different days (Fig. 1). On
6 nd
7
the 2 day, highest level of IAA was produced by Lysinibacillus sp. (FOW7), whereas on the 4th
8
day maximum IAA was produced by Azospirillum sp. (FOW2), followed in 6th day maximum
9
level of IAA was produced by Azospirillum sp. (FOW2) and Acetobacter sp. (FOW3) and on the
10
8th day by Bacillus sp.1 (FOW1), Azospirillum sp. (FOW2), Acetobacter sp. (FOW3) and
11
Lysinibacillus sp. (FOW7). On the 10th day Azospirillum sp. (FOW2), Acetobacter sp. (FOW3)
12
and Lysinibacillus sp. (FOW7) were produced maximum level of IAA. Maximum level of IAA
13
results in better shoot growth of the fenugreek plant. A similar result by several rhizobacteria
14
was reported (Lwin et al., 2012). Tryptophan as precursor has been reported for the increased
15
level of IAA production d in PGPR (Tien and Blackburn, 1996; Xie and Wang, 1996; Park et al.,
16
2005; Naz et al., 2009; Yadav et al., 2010). Production of high level of IAA by florescent
17
Pseudomonas has been reported. A similar finding of high level of IAA production has reported
18
by other researchers (Ahmad et al., 2005).
19 20
3.2.2 Siderophore Production
21
Among the seven PGPR isolates, 4 isolates namely, Bacillus sp. 1 (FOW1), Acetobacter
22
sp. (FOW3) Bacillus sp. 2 (FOW6) and Lysinibacillus sp. (FOW7) were produced siderophore.
23
The production of siderophore is one of the biocontrol mechanisms under iron limiting condition
24
by PGPR groups. Therefore, the iron limiting condition in the environment would suppress the
25
growth of pathogenic organisms (Tirry et al., 2018).
26 27
3.2.3 Phosphate Solubilization
28
Phosphate solubilization indication was shows maximum by Bacillus sp.1 (FOW1),
29
Acetobacter sp. (FOW3), Klebsiella sp. (FOW4), Burkholderia sp. (FOW5) and Bacillus sp.2
30
(FOW6) (Table 2). Experimental results was shows that Bacillus sp. has synthesized growth
31
hormones that might be the most probable mean to promote plant growth phosphate solubilizers. 8
1
Phosphate solubilisation is caused by phytohormones and thus increased the growth of plant
2
(García et al., 2017).
3
.
4
Phosphate is a second most major plant nutrient, it could compensate the need of
5
expensive P fertilizers, bioinoculants are an alternative which was shows P solubilization
6
activity, but the soil phosphorus in the form of insoluble phosphates are not taken up by the
7
plants. The capability of bacteria to solubilize the mineral phosphate is of great interest to
8
agricultural researcher as it makes the availability of phosphorous and iron for plant growth.
9
Plant growth promoting rhizobacteria are solubilize the precipitated phosphate and improves
10
phosphate availability to plant. Free living phosphate solubilizing bacteria are releases phosphate
11
from spare soluble inorganic and organic phosphate compounds in soil and avails phosphate for
12
the plants (Yadav et al., 2010; Gopalakrishnan et al., 2013).
13 14
3.2.4 Hydrogen Cyanide Production
15
Among the seven isolates, only Bacillus sp.1 (FOW1) bacterial isolate had showed
16
positive for hydrogen cyanide (HCN) production and it acts as an inducer of plant resistance.
17
Hydrogen cyanide production was one of the plant growth promoting and plant disease
18
controlling traits, the HCN is a volatile metabolite produced by soil bacterial biocontrol agents
19
(BCAs) against soil borne pathogen (Jayaprakashvel et al., 2010). Biological control of
20
pathogens is achieved by rhizobacteria which produces hydrogen cyanide (Voisard et al., 1989).
21 22
3.2.5 Ammonia Production
23
All the seven isolates were efficient in ammonia production and showed the positive
24
results (Table 3). Plant growth is influenced by ammonia production in PGPR (Yadav et al.,
25
2010). The diazotrophic microorganism converts nitrogen to ammonia so that it can be
26
assimilated by plants (Ravi et al., 2002).
27 28
3.2.6 Biological Nitrogen Fixation
29
The isolated rhizobacteria were screened for their ability to fix nitrogen in the solid
30
medium in which Bacillus sp.1 (FOW1), Acetobacter sp. (FOW3) and Lysinibacillus sp. (FOW7)
31
showed maximum nitrogen activity than other isolates. Few microorganisms can fix biological 9
1
nitrogen fixation (BNF) which is the important source of nitrogen. Nitrogen is the key element of
2
plant nutrient required for plant growth. The N element is plenty in the earth’s atmosphere;
3
however most of the tropical soils are deficient in available N (Senthil et al., 2011). Nitrogen
4
fixation can be done by several associative and free living rhizosphere microorganisms and it is
5
recognized for their important role in plant growth (Boddy et al., 1996).
6 7
3.3 Characterization of Pesticide Tolerant PGPR
8
3.3.1 Plate Assay
9
The experimental results were showed that pesticide had a negative impact on growth of
10
most isolates at higher level. Rhizobacterial isolates were showed variation in growth with
11
increasing levels of pesticide. Most of the rhizobacterial strains were showed maximum growth
12
at low pesticide concentration and their growth decreased with increasing concentration of
13
pesticide. Among the seven isolates, the maximum growth was observed in Bacillus sp.1
14
(FOW1) and Lysinibacillus sp. (FOW7), when compared with Azospirillum sp. (FOW2),
15
Acetobacter sp. (FOW3), Burkholderia sp. (FOW5) and Bacillus sp.2 (FOW6) (Table 4). Based
16
on these results, Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) were optimised for
17
maximum growth with different parameters such as time, pH and salt tolerance.
18 19
3.3.2 Time Course for Growth of the Bacterial Isolates
20
Growth pattern of bacterial both (Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7)
21
were isolates in pesticide (1%) containing medium at different time intervals were studied.
22
Growth of the isolates at the lowest concentrations was comparable to that of control. Maximum
23
growth for Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7 were observed after 24, 48, 72,
24
96 and 120h, respectively. Both isolates were tested for time courses growth. Growth of the
25
isolates at the maximum concentration was observed in 24 and 48 h (Fig.2) and at the minimum
26
concentration was comparable to that of the control (Kale and Allen, 1989).
27 28
3.3.3 Growth at Different pH Levels
29
The present study Bacillus sp. 1 (FOW1) had showed maximum grow that alkaline pH 9.
30
Both strains were showed in poor growth at acidic pH. The maximum grow that pH 9 was
31
observed in the case of Bacillus sp.1 (FOW1) (Fig. 3). And both bacterial growth was negatively 10
1
affected at acidic pH and showed variable response with increasing pH level up to alkaline range.
2
This variation in growth of rhizobacterial strains at different pH levels, may be attributed to
3
metabolic modification with high acid production, raising the transporters and enzymes, and
4
changes in the outer layers of cell for proton retention (Padan et al., 2005).
5 6
3.3.4 Salt Tolerance
7
The results were showed that two strains (Bacillus sp.1 (FOW1) and Lysinibacillus sp.
8
FOW7) have showed maximum growth at lowest salinity level (Fig.4). At maximum cell growth
9
was observed in the case of FOW7, and followed by FOW1 showed poor cell density. Though,
10
the salinity had an adverse effect on growth of bacteria, likewise the growth of rhizobacterial
11
isolates decreased with each levels were increased when in salinity. However, some isolates
12
showed more growth even at higher salt concentrations. This difference in their growth at higher
13
salinity levels might be due to the ability of bacterial strains to tolerate salt stress
14
(Bhakthavatchalu et al., 2013).
15 16
3.3.5 Antibiotic Resistance
17
In the experiment, two isolates for MIC with three different antibiotics (chloramphenical,
18
vancomycin and noarfxlacin) at its 1mg concentrations. The MIC for Bacillus sp.1. (FOW1)
19
chloramphenicol - 20mM, vancomycin - 17mM, noarfloxacin- 3mM, the Bacillus sp.1. (FOW1)
20
was highly resistant chloramphenicol and vancomycin. The isolate Lysinibacillus sp.(FOW7) has
21
shown MIC in chloramphenical 18mM, vancomycin 6mM, noarfloxacin 22mM and the Bacillus
22
sp.1 (FOW1) was highly resistant to chloramphenical and noarfloxacin (Table 5).
23
Microorganisms could adapt to change in environmental condition by horizontal gene a transfer
24
mechanism, which was provides them with new traits, so that they can survey and colonize their
25
new environments (De Gelder et al., 2008).
26 27
3.4 Nursery Experiment
28
Trigonellafoenum - graecum seeds were inoculated with selected pesticide and selective
29
isolates of Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) were sowed into the
30
experimental soil. After 15 and 30th days, plants were harvested and indexes were observed
31
(Tables 6 and 7), and plant all the morphometric growths were increased significantly in all the 11
1
treatment when compared to control. The synergistic effect of Bacillus sp. and Lysinibacillus sp.
2
were apparent in co-inoculated trial, for growth enhancement of fenugreek leaves. Tolerant
3
PGPR strains were used for growth promotion under pot experiment and this is a good tool for
4
effective plant growth. Plant growth promoting rhizobacteria are highly advantageous for plant
5
growth and can serve as potential degraders for pesticides. The beneficial effects of the tolerant
6
PGPR and crops have been reported (Rathaur et al., 2012). Studies were showed that tolerant
7
PGPR improves soil aeration, water holding capacity and stimulates microorganisms in soil,
8
readily available leading plant nutrients for good yield and quality of plants (Jha and Saraf,
9
2011).
10 11
4. Conclusion
12
In the present study was purposeful characterized the pesticide tolerant bacteria which
13
were highly contaminated sources of samples with frequently pesticidal used vegetable
14
production practices. Isolated bacterial strains were showed resistant to the pesticides exhibited
15
in synthetic media namely Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) and the results
16
of the present study suggest that the isolates are able to grow in presence of pesticides due to its
17
biodegradation property. In conclude that these PGPR characteristic isolates may be evaluated
18
for their ability of bioremediation under pesticide stress with other multifarious traits and the
19
future research on pesticide-rhizobacteria interaction at molecular level is needed to identify
20
which enzymes or genes are affected in rhizobacteria under pesticide stress.
21 22
Acknowledgement
23
The authors are great full to DST-PURSE, Bharathidasan University for financial support.
24 25
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21 22 23 24 25 26 27 28 29 30 31 18
1
Figure Legends
2
Figure 1 Indole Acetic Acid Production
3
FOW1- Bacillus sp.1, FOW2- Azospirillum sp., FOW3- Acetobacter sp., FOW4- Klebsiella sp.,
4
FOW5- Burkholderia sp., FOW6- Bacillus sp.2 and FOW7- Lysinibacillus sp..
5 6
Figure 2 Growth at Different Time Course
7
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.; Concentration of pesticide-1%
8 9 10
Figure 3 Growth at different pH levels
11
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.
12 13
Figure 4 Salt Tolerance Test
14
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 19
1
Figure 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
20
1
Figure 2
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21
1
Figure. 3
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 22
1
Figure 4
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 23
1
Table Legends
2
Table 1 - Nursery experimental setup
3
Table 2 - Phosphate solubilization
4
Table 3 - Ammonia production
5
Table 4 - Plate Assay using different concentration of insecticide
6
Table 5 - Determination of antibiotic resistant test
7
Table 6 - Morphometric analysis of host plant after 15 days
8
Table 7 - Morphometric analysis of host plant after 30 days
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 24
Table 1
1 2
S. No.
Particular of treatments
T1
Non- treated seeds
T2
Seed + Pesticide
T3
Seed+ Tolerant isolate (FOW1)
T4
Seed + Tolerant isolate (FOW7)
T5
Seed+ Pesticide + Tolerant isolate (FOW1)
T6
Seed+ Pesticide + Tolerant isolate (FOW7)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25
Table 2
1
Isolates
Zone of Inhibition (mM)
Bacillussp 1 (FOW1) Azospirillumsp (FOW2)
11 -
Acetobactersp (FOW3) Klebsiellasp (FOW4) Burkholderiasp(FOW5) Bacillussp 2 (FOW6) Lysinibacillussp (FOW7)
10 10 10 10 -
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26
Table 3
1
2
Isolates
Strength of ammonia production
Bacillussp 1 (FOW1)
+
Azospirillumsp (FOW2)
++
Acetobactersp (FOW3)
+
Klebsiellasp (FOW4)
+
Burkholderiasp(FOW5)
+++
Bacillussp 2 (FOW6)
+
Lysinibacillussp (FOW7)
+
+ slightly positive, ++ medium positive, +++ strongly positive
3 4
27
Table 4
1 2 3
Concentration of pesticide Isolates
1%
3%
5%
7%
10%
12%
14%
16%
18%
20%
Growth (mM) Bacillussp 1 (FOW1) Azospirillums p (FOW2) Acetobacters p (FOW3) Klebsiellasp (FOW4) Burkholderia sp(FOW5) Bacillussp 2 (FOW6) Lysinibacillus sp (FOW7)
16
13
12
12
10
6
5
7
6
6
18
16
13
10
9
6
5
-
-
-
6
12
7
8
7
-
-
-
-
-
9
9
7
9
7
6
-
-
-
-
9
9
8
7
6
-
-
-
-
-
7
8
8
7
8
-
-
-
-
-
15
15
13
12
10
8
6
7
7
7
28
Table 5
1 2
List of Antibiotic disc
Bacillus sp 1 (FOW1)
Lysinibacillus sp (FOW7)
Diameter (mM) 20
18
Vancomycin
17
6
Noarfloxacin
3
22
Chloramphenical
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
29
Table 6
1 2
Root Shoot fresh fresh weig weight ht (gm) (gm)
Plant Trea height tmen (cm) ts
Shoot height (cm)
Root leng th (cm)
T1
6.2
1.2
1
0.12
0.06
0.2
0.02
2
0
T2
8.7
7.5
1.2
0.126
0.08
0.26
0.02
2
0
T3
9.4
1.3
8.1
0.197
0.020
0.35
0.03
2
0
T4
8.7
1
7.7
0.107
0.01
0.23
0.010
3
0
T5
7.4
6.3
1.1
0.103
0.06
0.23
0.02
2
0
T6
6.9
5.8
1.1
0.102
0.06
0.21
0.02
2
0
3 4 5 6 7 8 9 10 11 12 30
Shoot Dry weight (gm)
Root Dry weight (gm)
Number Number of of leaves adventitio us roots
Table 7
1 2
Shoot Dry weight (gm)
Treat ments
Plant height (cm)
Shoot height (cm)
Root lengt h (cm)
Shoot fresh weigh t (gm)
Root fresh weight (gm)
T1
9.5
5.5
4
0.085
0.012
0.04
T2
13
5.5
7.5
0.195
0.010
T3
15.1
9.0
6.1
0.016
T4
18
10.6
8.2
T5
13.5
7
T6
13.2
6.8
Root Dry Numbe Number of weight r of adventitio (gm)
leaves
us roots
0.01
5
1
0.10
0.012
4
3
0.241
0.01
0.2
6
3
0.318
0.022
0.25
0.02
7
5
6.5
0.213
0.016
0.15
0.01
5
4
7
0.239
0.012
0.239
0.01
5
3
3 4 5 6 7 8 9 10 11 12 13
31
AUTHORS STATEMENT
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Biocatalysis and Agricultural Biotechnology.
Dr. Velu Rajesh Kannan Corresponding Author
S1878-8181(19)31263-0
DOI:
https://doi.org/10.1016/j.bcab.2020.101493
Reference:
BCAB 101493
To appear in:
Biocatalysis and Agricultural Biotechnology
Received Date: 12 October 2019 Revised Date:
16 December 2019
Accepted Date: 2 January 2020
Please cite this article as: Nathiya, S., Janani, R., Kannan, V.R., Potential of plant growth promoting Rhizobacteria to overcome the exposure of pesticide in Trigonellafoenum - graecum (fenugreek leaves), Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2020.101493. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Potential of Plant Growth Promoting Rhizobacteria to Overcome the Exposure of Pesticide
2
in Trigonellafoenum - graecum (Fenugreek leaves)
3 4
S. Nathiya, R. Janani, V. Rajesh Kannan*
5 6
Rhizosphere Biology Laboratory, Department of Microbiology, Bharathidasan University,
7
Tiruchirappalli - 620 024, Tamil Nadu, India.
8 9
*Corresponding Author:
10
Dr. V. Rajesh Kannan
11
Email id: [email protected]
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1
1
Abstract
2
Agrochemicals are generally used as additional supplements to the soil for improving soil
3
fertility throughout the world. In this present study was carried out to characterize the pesticide
4
tolerance plant growth promoting rhizobacteria (PGPR) were isolated from fenugreek leaves.
5
Seven bacterial isolates were obtained namely FOW1 to FOW7 and characterized on their
6
colony morphology, size and colour. Among the seven, FOW1 and FOW7 isolates were found to
7
grown well in nutrient medium containing 10 to 20% concentration of pesticide. All the seven
8
isolates were produced indole-3- acetic acid in the range of 56 to 97µg/mL and only two isolates
9
(FOW1 and FOW7) displayed the phosphate solubilising activity in Pikovskaya agar, whereas
10
six isolates were showed positive for production of siderophore. Out of seven isolates, two
11
exhibited in vitro plant growth promotion activities and indicated that these isolates can be
12
exploited as biofertilizers and microbial inoculants for crops as they enhanced plant growth via
13
diverse mechanisms and offered an attractive strategy to replace synthetic fertilizers and
14
pesticides. The morphological features such as germinating ability, shoot length, root length
15
were enhanced in the presence of PGPR and was maximum at 1%.
16
Keywords: Pesticide; PGPR; Fenugreek seed; Rhizosphere; Tolerance.
17 18
1. Introduction
19
Pesticide is a single or combination of toxic chemical or chemical agent that are
20
deliberately let into the environment in order for the control/ destruction of harmful pests.
21
Pesticides are not only toxic to living pests and the ecosystem too and ultimately resulting in
22
their bioaccumulation. Early reports have established the presence of several pesticides such as
23
DDT, lindane and dieldrin in even food products beyond their permissible limits ( Linhart et al.,
24
2019). Few microorganisms on long term exposure show resistance to pesticides and they can be
25
effectively used in the remediation of pesticide contaminated soils (Khan et al., 2009). Microbes
26
perform efficiently in the presence of specific pesticides by using them as nutrients and energy
27
source (Pramanik et al., 2018). Microorganisms degrade these pesticides and use them as a
28
carbon source. Their ability to degrade pesticide is an important phenomenon through which
29
these chemicals are eliminated from the environment and control the environmental pollution
30
(Backer et al., 2018). These bacterial strains can be used in modern agriculture as inoculants
31
under stress conditions such as heavy metal stress (Bhatt and Vyas, 2014), herbicide stress, 2
1
(Ahemad and Khan, 2011a), insecticides stress (Ahemad and Khan, 2011b), fungicides stress,
2
(Ahemad and Khan, 2012) and salinity stress (Tank and Saraf, 2010). Therefore, those microbes
3
are essential component in recycling process and important to maintain soil fertility (Glick,
4
2012).
5
Bioremediation is transformation or degradation of pollutants by microbes into non-
6
hazardous or less toxic substances. It mainly depends on microbial enzymatic attack on the
7
pollutants and converts them to less toxic products. On this strategy which not only proves
8
beneficial in reducing organophosphates accumulation but it’s also enhances plant growth up to
9
in quality aspects. Thus, the course of bioremediation is addressed by plant growth promoting
10
rhizobacteria (PGPR). The PGPR colonises on the roots of the plants and help in promoting plant
11
growth (DalCorso et al., 2019). The PGPR may exert their effects directly or indirectly by
12
several metabolites, singly or in combination with several other factors (Shaheen and Sundari,
13
2013). Generally, PGPR functions by synthesizing particular compounds, inhabiting certain
14
nutrient uptake and guarding the plants from diseases (Castellanos Rozo et al., 2013). Thus it
15
becomes more beneficial to apply such a strategy which remediate the pollutants as well enhance
16
plant growth. The PGPR has been explored on biotechnological applications in various uses such
17
as biofertilizers, biocontrol agents, phytostimulators and bioremediators based on their mode of
18
action in agriculture practices (Jariyal et al., 2018). The PGPR is known for the production of
19
growth hormones, vitamins and other amino acids that influence the growth of the plant by
20
increasing its nutrient availability (Kumar et al., 2011).
21
Addition that the PGPRs like Pseudomonas, Azospirillum, Agrobacterium, Bacillus,
22
Enterobacter and Flavobacterium strains that degrade organic and inorganic contaminants in soil
23
(Myresiotis et al., 2011; Joseph et al., 2012). While the inorganic amendments to agricultural soil
24
improves the efficiency of fertility in certain limits (Htwe et al., 2019) and the excess or
25
unutilized
26
available on biodegradation of soil applied pesticides by PGPR strains and their effects on
27
bacterial growth (Pandey and Gupta, 2019) and the indigenous soil microbial community (Liang
28
et al., 2019). Therefore, the present study was conducted to isolate and characterize the pesticide
29
tolerant rhizobacteria for multiple plant growth promoting traits.
those inorganic nutrients leads to fatal the soil organisms, the limited data are
30 31 3
1
2. Materials and Methods
2
2.1 Sample Source
3
Soil samples were collected from the Trigonellafoenum graecum rhizosphere which was
4
regular cultivation field in Othakadai Village of Tiruchirappalli district in Tamil Nadu, India.
5
Organochlorine pesticide was purchased from certified agrochemical shop in Tiruchirappalli.
6 7
2.2 Isolation and Identification of PGPR
8
Bacterial isolates were obtained from fenugreek plants rhizosphere soils through dilution
9
plate technique (Albdaiwi et al., 2019) in detail, one gram of rhizosphere soil samples were taken
10
and mixed in 10mL of sterile water and then serially diluted. The dilutions of 10-3, 10-4 and 10-5
11
were used for bacterial isolation. Approximately 0.1mL sample was spread over the nutrient agar
12
medium the plates were incubated at 30oC for up to 48h. The colonies were identified based on
13
their morphology and biochemical tests (Aditi et al., 2017; Tirry et al., 2018). The isolates were
14
stored at -4°C for further analyses.
15 16
2.3 Screening of Plant Growth Promoting Rhizobacteria
17
2.3.1 Indole Acetic Acid Production
18
The bacterial strains were cultured in nutrient broth supplement with 500µg tryptophan
19
mL-1 and incubated at 27ºC about two weeks. Approximately 1mL of cultures were withdrawn at
20
different time intervals (2nd - 10th day with 2 days interval) and centrifuged at 6000rpm for
21
30min. Indole acetic acid concentration was estimated (Albdaiwi et al., 2019), 1mL of the
22
supernatant, a drop of orthophosphoric acid and 2mL of Salkowski’s reagent (50mL, 35%
23
perchloric acid and 1mL, 0.5M FeCl3) were added and mixed. Colour was measured by
24
spectrophotometrically at 530nm.
25 26
2.3.2 Siderophore Production
27
Bacterial isolates were streaked in CAS agar medium and incubated at 28± 10oC for 48h.
28
The siderophore production was observed as orange halos around the colonies (Tirry et al.,
29
2018).
30
.
31 4
1
2.3.3 Phosphate Solubilization
2
Phosphate solubilization test was done using modified Pikovskaya medium which
3
consists of 10g glucose, 5g tribasic phosphate (Ca5HO13P3), 0.5g (NH4)2SO4,0.2gKCl, 0.1g
4
MgSO4.7H2O, 0.0001g MnSO4 and FeSO4, 0.5g yeast extract, and 15g agar, in 1000mL distilled
5
water. Bacterial isolates were spot inoculated and after 4 days of incubation, bacterial zone
6
formation was observed. The diameter of halo clear zones were measured and solubilization
7
efficiency (SE) and solubilization index (SI) were evaluated using following formulae (García et
8
al., 2017).
9 10
SE= Solubilization diameter /growth diameter SI= Colony diameter + halozone diameter/colony diameter
11 12
2.3.4 Hydrogen Cyanide Production
13
The bacterial isolates were checked for hydrogen cyanide production. In nutrient broth
14
containing 4.4g glycine/1, the isolates were streaked in agar plate. On the top of the plate,
15
Whatmann No.1 filter paper immersed in 2% sodium carbonate in 0.5% picric acid solution was
16
kept and sealed with parafilm and incubated at 28±2ºC for 4 days and colour changed was
17
observed (Pandey and Gupta, 2019).
18 19
2.3.5 Production of Ammonia
20
Bacterial isolates in 10mL peptone were inoculated and incubated for 72 h at 28±2ºC. To
21
these tunes, Nessler’s reagent (0.5mL) was added and colour change was observed (Pandey and
22
Gupta, 2019).
23 24
2.3.6 Biological Nitrogen Fixation
25
The bacterial strain was subjected to screening of nitrogen fixation activity using glucose
26
nitrogen free minimal medium (G-NFMM). Single colony was inoculated into G-NFMM
27
containing BTB (Bromothymol blue solution) and after one week incubation, colour change was
28
recorded (García et al., 2017).
29 30
2.4 Identification of Pesticide Tolerant PGPR
31
2.4.1 Plate Assay 5
1
Rhizobacterial strains were isolated from host plant rhizosphere with dilution plate
2
technique (Dworkin and Foster, 1958), whereas pesticide was applied intentionally. The
3
modified minimal salt agar medium containing different concentrations of 1 to 20% (100 to
4
2000mg L-1) of pesticide as sole source of carbon was used for isolation of pesticide tolerant
5
PGPR strains. The bacterial isolates were spot inoculated in agar plates with pesticide. The
6
growth or inhibition of bacterial strains at highest concentration of pesticide was considered as
7
the maximum tolerance level (MTL).
8 9 10
2.5 Optimization of Different Parameters for Pesticide Tolerance 2.5.1 Growth at Different Time Course
11
The effect of pesticides on the growth of bacterial isolates was examined. Exponentially
12
grown cultures were inoculated liquid culture medium (presence and absence) and incubated at
13
28ºC for 24, 48, 72, 96, 120 and 144 h. and the control was also maintained. Growth at different
14
times was determined by measuring the absorbance at 540 nm (Kale and Allen, 1989).
15 16
2.5.2 Growth at Different pH
17
For pH optimization, the rhizobacterial strains were grown in nutrient broth in different
18
levels of pH (5, 6, 7, 8 and 9). The medium with different pH was prepared and inoculated with
19
respective rhizobacterial strains. These culture were kept at 28 ± 2°C for 72h and optical density
20
was measured at 600 nm (Singh et al., 2014).
21 22
2.5.3 Salt Tolerance Test
23
Bacterial isolates salt tolerance levels were examined by osmo adaptation assay. The
24
bacterial isolates were grown in nutrient broth with different salt concentrations (1, 2, 3, 4, 5 and
25
6 dS m-1) and incubated for 72h at 28±2°C and optical density was measured at 540 nm (Sharma
26
et al., 2016).
27 28
2.5.4 Determination of Antibiotic Resistance
29
The antibiotic resistance of pesticide tolerant bacterial isolates of Bacillus sp.1 and
30
Lysinibacillus sp. against to three different antibiotics [Chloramphenicol (30µg), Vancomycin
31
(30µg) and Norfloxacin (30µg)] was carried out by disc diffusion method (Bauer et al., 1966). 6
1
These inoculums were maintained at 4°C in Luria-Bertani medium. The bacterial cultures were
2
reactivated in Muller Hinton Broth at 37°C for 24h. Bacterial culture was swabbed on the
3
surface of MHA with known potency antibiotic disc which was incubated at 37°C for 48h, the
4
zone of incubation (diameter in mM) around the antibiotic disc were measured.
5 6
2.6 In vivo Study of Trigonellafoenum - graecum
7
2.6.1 Nursery Experiment
8
All the seeds were sterilized with 70% ethanol and washed with 0.1% sodium hypo
9
chloride and rinsed with sterilized distilled water. The rhizobacterial cells were removed by
10
centrifuged at 10000 rpm for 10min at 4ºC and washed in distilled water. The pellet was
11
resuspended in sterile water and then diluted for a bacterial suspension concentration of 108
12
CFU/mL (OD 595= 0.3) (Kanthaiah and Velu, 2019).
13
The fenugreek seeds (1g seeds) were sterilized with 70% ethanol. Seeds were coated with
14
different treatments (Table 1) as follows: Test 1 - seeds (1g) + carboxyl methyl cellulose (100
15
mg), Test 2 – seeds (1g) + pesticide, Test 3 - pesticide tolerant PGPR (FOW1) + seeds(1g) +
16
carboxyl methyl cellulose (100 mg), Test 4 - pesticide tolerant PGPR (FOW7) + seeds (1g) +
17
carboxyl methyl cellulose (100 mg), Test 5 - pesticide tolerant PGPR (FOW7) + seeds (1g) +
18
pesticide + carboxyl methyl cellulose (100 mg), and Test 6 - pesticide + seeds (1g) + carboxyl
19
methyl cellulose (100 mg). All these seed were air dried for 12 hours. Dried seeds were
20
transplanted in poly bags (top diameter 300 mM, 160 mM bottom and height 16 cm) containing
21
soil and kept for experiments under nursery conditions for 30 days. After the growth period, the
22
crop’s morphometric analyses were done for the following parameter for each treatments plants-
23
shoot height, root length, shoot and root fresh weight, number of leaves and number of
24
adventitious roots. The experiments were done in duplicates and statiscally analysed.
25 26
3. Results and Discussion
27
3.1 Isolation and Identification of Rhizobacteria
28
Bacterial strains were isolated from the fenugreek field soil samples. Based on the colony
29
morphology, size, colour and biochemical characterization, seven bacterial isolates were
30
identified, such as Bacillus sp.1 (FOW1), Azospirillum sp. (FOW2), Acetobacter sp. (FOW3),
7
1
Klebsiella sp. (FOW4), Burkholderia sp. (FOW5), Bacillus sp.2 (FOW6) and Lysinibacillus sp.
2
(FOW7).
3 4
3.2 Screening for Plant Growth Promoting Rhizobacteria
5
3.2.1 Indole Acetic Acid Production All the seven isolates were produced different levels of IAA at different days (Fig. 1). On
6 nd
7
the 2 day, highest level of IAA was produced by Lysinibacillus sp. (FOW7), whereas on the 4th
8
day maximum IAA was produced by Azospirillum sp. (FOW2), followed in 6th day maximum
9
level of IAA was produced by Azospirillum sp. (FOW2) and Acetobacter sp. (FOW3) and on the
10
8th day by Bacillus sp.1 (FOW1), Azospirillum sp. (FOW2), Acetobacter sp. (FOW3) and
11
Lysinibacillus sp. (FOW7). On the 10th day Azospirillum sp. (FOW2), Acetobacter sp. (FOW3)
12
and Lysinibacillus sp. (FOW7) were produced maximum level of IAA. Maximum level of IAA
13
results in better shoot growth of the fenugreek plant. A similar result by several rhizobacteria
14
was reported (Lwin et al., 2012). Tryptophan as precursor has been reported for the increased
15
level of IAA production d in PGPR (Tien and Blackburn, 1996; Xie and Wang, 1996; Park et al.,
16
2005; Naz et al., 2009; Yadav et al., 2010). Production of high level of IAA by florescent
17
Pseudomonas has been reported. A similar finding of high level of IAA production has reported
18
by other researchers (Ahmad et al., 2005).
19 20
3.2.2 Siderophore Production
21
Among the seven PGPR isolates, 4 isolates namely, Bacillus sp. 1 (FOW1), Acetobacter
22
sp. (FOW3) Bacillus sp. 2 (FOW6) and Lysinibacillus sp. (FOW7) were produced siderophore.
23
The production of siderophore is one of the biocontrol mechanisms under iron limiting condition
24
by PGPR groups. Therefore, the iron limiting condition in the environment would suppress the
25
growth of pathogenic organisms (Tirry et al., 2018).
26 27
3.2.3 Phosphate Solubilization
28
Phosphate solubilization indication was shows maximum by Bacillus sp.1 (FOW1),
29
Acetobacter sp. (FOW3), Klebsiella sp. (FOW4), Burkholderia sp. (FOW5) and Bacillus sp.2
30
(FOW6) (Table 2). Experimental results was shows that Bacillus sp. has synthesized growth
31
hormones that might be the most probable mean to promote plant growth phosphate solubilizers. 8
1
Phosphate solubilisation is caused by phytohormones and thus increased the growth of plant
2
(García et al., 2017).
3
.
4
Phosphate is a second most major plant nutrient, it could compensate the need of
5
expensive P fertilizers, bioinoculants are an alternative which was shows P solubilization
6
activity, but the soil phosphorus in the form of insoluble phosphates are not taken up by the
7
plants. The capability of bacteria to solubilize the mineral phosphate is of great interest to
8
agricultural researcher as it makes the availability of phosphorous and iron for plant growth.
9
Plant growth promoting rhizobacteria are solubilize the precipitated phosphate and improves
10
phosphate availability to plant. Free living phosphate solubilizing bacteria are releases phosphate
11
from spare soluble inorganic and organic phosphate compounds in soil and avails phosphate for
12
the plants (Yadav et al., 2010; Gopalakrishnan et al., 2013).
13 14
3.2.4 Hydrogen Cyanide Production
15
Among the seven isolates, only Bacillus sp.1 (FOW1) bacterial isolate had showed
16
positive for hydrogen cyanide (HCN) production and it acts as an inducer of plant resistance.
17
Hydrogen cyanide production was one of the plant growth promoting and plant disease
18
controlling traits, the HCN is a volatile metabolite produced by soil bacterial biocontrol agents
19
(BCAs) against soil borne pathogen (Jayaprakashvel et al., 2010). Biological control of
20
pathogens is achieved by rhizobacteria which produces hydrogen cyanide (Voisard et al., 1989).
21 22
3.2.5 Ammonia Production
23
All the seven isolates were efficient in ammonia production and showed the positive
24
results (Table 3). Plant growth is influenced by ammonia production in PGPR (Yadav et al.,
25
2010). The diazotrophic microorganism converts nitrogen to ammonia so that it can be
26
assimilated by plants (Ravi et al., 2002).
27 28
3.2.6 Biological Nitrogen Fixation
29
The isolated rhizobacteria were screened for their ability to fix nitrogen in the solid
30
medium in which Bacillus sp.1 (FOW1), Acetobacter sp. (FOW3) and Lysinibacillus sp. (FOW7)
31
showed maximum nitrogen activity than other isolates. Few microorganisms can fix biological 9
1
nitrogen fixation (BNF) which is the important source of nitrogen. Nitrogen is the key element of
2
plant nutrient required for plant growth. The N element is plenty in the earth’s atmosphere;
3
however most of the tropical soils are deficient in available N (Senthil et al., 2011). Nitrogen
4
fixation can be done by several associative and free living rhizosphere microorganisms and it is
5
recognized for their important role in plant growth (Boddy et al., 1996).
6 7
3.3 Characterization of Pesticide Tolerant PGPR
8
3.3.1 Plate Assay
9
The experimental results were showed that pesticide had a negative impact on growth of
10
most isolates at higher level. Rhizobacterial isolates were showed variation in growth with
11
increasing levels of pesticide. Most of the rhizobacterial strains were showed maximum growth
12
at low pesticide concentration and their growth decreased with increasing concentration of
13
pesticide. Among the seven isolates, the maximum growth was observed in Bacillus sp.1
14
(FOW1) and Lysinibacillus sp. (FOW7), when compared with Azospirillum sp. (FOW2),
15
Acetobacter sp. (FOW3), Burkholderia sp. (FOW5) and Bacillus sp.2 (FOW6) (Table 4). Based
16
on these results, Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) were optimised for
17
maximum growth with different parameters such as time, pH and salt tolerance.
18 19
3.3.2 Time Course for Growth of the Bacterial Isolates
20
Growth pattern of bacterial both (Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7)
21
were isolates in pesticide (1%) containing medium at different time intervals were studied.
22
Growth of the isolates at the lowest concentrations was comparable to that of control. Maximum
23
growth for Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7 were observed after 24, 48, 72,
24
96 and 120h, respectively. Both isolates were tested for time courses growth. Growth of the
25
isolates at the maximum concentration was observed in 24 and 48 h (Fig.2) and at the minimum
26
concentration was comparable to that of the control (Kale and Allen, 1989).
27 28
3.3.3 Growth at Different pH Levels
29
The present study Bacillus sp. 1 (FOW1) had showed maximum grow that alkaline pH 9.
30
Both strains were showed in poor growth at acidic pH. The maximum grow that pH 9 was
31
observed in the case of Bacillus sp.1 (FOW1) (Fig. 3). And both bacterial growth was negatively 10
1
affected at acidic pH and showed variable response with increasing pH level up to alkaline range.
2
This variation in growth of rhizobacterial strains at different pH levels, may be attributed to
3
metabolic modification with high acid production, raising the transporters and enzymes, and
4
changes in the outer layers of cell for proton retention (Padan et al., 2005).
5 6
3.3.4 Salt Tolerance
7
The results were showed that two strains (Bacillus sp.1 (FOW1) and Lysinibacillus sp.
8
FOW7) have showed maximum growth at lowest salinity level (Fig.4). At maximum cell growth
9
was observed in the case of FOW7, and followed by FOW1 showed poor cell density. Though,
10
the salinity had an adverse effect on growth of bacteria, likewise the growth of rhizobacterial
11
isolates decreased with each levels were increased when in salinity. However, some isolates
12
showed more growth even at higher salt concentrations. This difference in their growth at higher
13
salinity levels might be due to the ability of bacterial strains to tolerate salt stress
14
(Bhakthavatchalu et al., 2013).
15 16
3.3.5 Antibiotic Resistance
17
In the experiment, two isolates for MIC with three different antibiotics (chloramphenical,
18
vancomycin and noarfxlacin) at its 1mg concentrations. The MIC for Bacillus sp.1. (FOW1)
19
chloramphenicol - 20mM, vancomycin - 17mM, noarfloxacin- 3mM, the Bacillus sp.1. (FOW1)
20
was highly resistant chloramphenicol and vancomycin. The isolate Lysinibacillus sp.(FOW7) has
21
shown MIC in chloramphenical 18mM, vancomycin 6mM, noarfloxacin 22mM and the Bacillus
22
sp.1 (FOW1) was highly resistant to chloramphenical and noarfloxacin (Table 5).
23
Microorganisms could adapt to change in environmental condition by horizontal gene a transfer
24
mechanism, which was provides them with new traits, so that they can survey and colonize their
25
new environments (De Gelder et al., 2008).
26 27
3.4 Nursery Experiment
28
Trigonellafoenum - graecum seeds were inoculated with selected pesticide and selective
29
isolates of Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) were sowed into the
30
experimental soil. After 15 and 30th days, plants were harvested and indexes were observed
31
(Tables 6 and 7), and plant all the morphometric growths were increased significantly in all the 11
1
treatment when compared to control. The synergistic effect of Bacillus sp. and Lysinibacillus sp.
2
were apparent in co-inoculated trial, for growth enhancement of fenugreek leaves. Tolerant
3
PGPR strains were used for growth promotion under pot experiment and this is a good tool for
4
effective plant growth. Plant growth promoting rhizobacteria are highly advantageous for plant
5
growth and can serve as potential degraders for pesticides. The beneficial effects of the tolerant
6
PGPR and crops have been reported (Rathaur et al., 2012). Studies were showed that tolerant
7
PGPR improves soil aeration, water holding capacity and stimulates microorganisms in soil,
8
readily available leading plant nutrients for good yield and quality of plants (Jha and Saraf,
9
2011).
10 11
4. Conclusion
12
In the present study was purposeful characterized the pesticide tolerant bacteria which
13
were highly contaminated sources of samples with frequently pesticidal used vegetable
14
production practices. Isolated bacterial strains were showed resistant to the pesticides exhibited
15
in synthetic media namely Bacillus sp.1 (FOW1) and Lysinibacillus sp. (FOW7) and the results
16
of the present study suggest that the isolates are able to grow in presence of pesticides due to its
17
biodegradation property. In conclude that these PGPR characteristic isolates may be evaluated
18
for their ability of bioremediation under pesticide stress with other multifarious traits and the
19
future research on pesticide-rhizobacteria interaction at molecular level is needed to identify
20
which enzymes or genes are affected in rhizobacteria under pesticide stress.
21 22
Acknowledgement
23
The authors are great full to DST-PURSE, Bharathidasan University for financial support.
24 25
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1
Figure Legends
2
Figure 1 Indole Acetic Acid Production
3
FOW1- Bacillus sp.1, FOW2- Azospirillum sp., FOW3- Acetobacter sp., FOW4- Klebsiella sp.,
4
FOW5- Burkholderia sp., FOW6- Bacillus sp.2 and FOW7- Lysinibacillus sp..
5 6
Figure 2 Growth at Different Time Course
7
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.; Concentration of pesticide-1%
8 9 10
Figure 3 Growth at different pH levels
11
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.
12 13
Figure 4 Salt Tolerance Test
14
FOW1- Bacillus sp.1; FOW7- Lysinibacillus sp.
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 19
1
Figure 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
20
1
Figure 2
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21
1
Figure. 3
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 22
1
Figure 4
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 23
1
Table Legends
2
Table 1 - Nursery experimental setup
3
Table 2 - Phosphate solubilization
4
Table 3 - Ammonia production
5
Table 4 - Plate Assay using different concentration of insecticide
6
Table 5 - Determination of antibiotic resistant test
7
Table 6 - Morphometric analysis of host plant after 15 days
8
Table 7 - Morphometric analysis of host plant after 30 days
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 24
Table 1
1 2
S. No.
Particular of treatments
T1
Non- treated seeds
T2
Seed + Pesticide
T3
Seed+ Tolerant isolate (FOW1)
T4
Seed + Tolerant isolate (FOW7)
T5
Seed+ Pesticide + Tolerant isolate (FOW1)
T6
Seed+ Pesticide + Tolerant isolate (FOW7)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25
Table 2
1
Isolates
Zone of Inhibition (mM)
Bacillussp 1 (FOW1) Azospirillumsp (FOW2)
11 -
Acetobactersp (FOW3) Klebsiellasp (FOW4) Burkholderiasp(FOW5) Bacillussp 2 (FOW6) Lysinibacillussp (FOW7)
10 10 10 10 -
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26
Table 3
1
2
Isolates
Strength of ammonia production
Bacillussp 1 (FOW1)
+
Azospirillumsp (FOW2)
++
Acetobactersp (FOW3)
+
Klebsiellasp (FOW4)
+
Burkholderiasp(FOW5)
+++
Bacillussp 2 (FOW6)
+
Lysinibacillussp (FOW7)
+
+ slightly positive, ++ medium positive, +++ strongly positive
3 4
27
Table 4
1 2 3
Concentration of pesticide Isolates
1%
3%
5%
7%
10%
12%
14%
16%
18%
20%
Growth (mM) Bacillussp 1 (FOW1) Azospirillums p (FOW2) Acetobacters p (FOW3) Klebsiellasp (FOW4) Burkholderia sp(FOW5) Bacillussp 2 (FOW6) Lysinibacillus sp (FOW7)
16
13
12
12
10
6
5
7
6
6
18
16
13
10
9
6
5
-
-
-
6
12
7
8
7
-
-
-
-
-
9
9
7
9
7
6
-
-
-
-
9
9
8
7
6
-
-
-
-
-
7
8
8
7
8
-
-
-
-
-
15
15
13
12
10
8
6
7
7
7
28
Table 5
1 2
List of Antibiotic disc
Bacillus sp 1 (FOW1)
Lysinibacillus sp (FOW7)
Diameter (mM) 20
18
Vancomycin
17
6
Noarfloxacin
3
22
Chloramphenical
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
29
Table 6
1 2
Root Shoot fresh fresh weig weight ht (gm) (gm)
Plant Trea height tmen (cm) ts
Shoot height (cm)
Root leng th (cm)
T1
6.2
1.2
1
0.12
0.06
0.2
0.02
2
0
T2
8.7
7.5
1.2
0.126
0.08
0.26
0.02
2
0
T3
9.4
1.3
8.1
0.197
0.020
0.35
0.03
2
0
T4
8.7
1
7.7
0.107
0.01
0.23
0.010
3
0
T5
7.4
6.3
1.1
0.103
0.06
0.23
0.02
2
0
T6
6.9
5.8
1.1
0.102
0.06
0.21
0.02
2
0
3 4 5 6 7 8 9 10 11 12 30
Shoot Dry weight (gm)
Root Dry weight (gm)
Number Number of of leaves adventitio us roots
Table 7
1 2
Shoot Dry weight (gm)
Treat ments
Plant height (cm)
Shoot height (cm)
Root lengt h (cm)
Shoot fresh weigh t (gm)
Root fresh weight (gm)
T1
9.5
5.5
4
0.085
0.012
0.04
T2
13
5.5
7.5
0.195
0.010
T3
15.1
9.0
6.1
0.016
T4
18
10.6
8.2
T5
13.5
7
T6
13.2
6.8
Root Dry Numbe Number of weight r of adventitio (gm)
leaves
us roots
0.01
5
1
0.10
0.012
4
3
0.241
0.01
0.2
6
3
0.318
0.022
0.25
0.02
7
5
6.5
0.213
0.016
0.15
0.01
5
4
7
0.239
0.012
0.239
0.01
5
3
3 4 5 6 7 8 9 10 11 12 13
31
AUTHORS STATEMENT
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Biocatalysis and Agricultural Biotechnology.
Dr. Velu Rajesh Kannan Corresponding Author