Isolation of phosphate solubilsing bacteria from ...

Isolation of phosphate solubilsing bacteria from rhizosphere of different black pepper (Piper nigrum) varieties in Kerala: an overview

Prem Jose Vazhacharickal, Sajeshkumar N.K, Jiby John Mathew and Mili Rose Michel

Acknowledgments

ACKNOWLEDGEMENTS

Firstly we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully. We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. V.J.Joseph, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the cooperation of many farmers. We are gratefully indebted to our teachers, parents, siblings and friends who were there always for helping us in this project.

Prem Jose Vazhacharickal*, Sajeshkumar N.K, Jiby John Mathew and Mili Rose Michel *Address for correspondence Assistant Professor Department of Biotechnology Mar Augusthinose College Ramapuram-686576 Kerala, India [email protected]

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Table of contents

Table of contents Table of contents..............................................................................................................ii Table of figures ............................................................................................................... iii Table of tables.................................................................................................................vi List of abbreviations ....................................................................................................... vii Isolation of phosphate solubilising bacteria from rhizosphere of different black pepper (Piper nigrum) varieties in Kerala: an overview ............................................................... 1 Abstract ........................................................................................................................... 1 1. Introduction ................................................................................................................. 2 1.1 Objectives .............................................................................................................. 4 1.2 Scope of the study ................................................................................................. 4 1.3 Taxonomical classification ..................................................................................... 4 2. Review of literature...................................................................................................... 5 3. Hypothesis .................................................................................................................. 8 4. Materials and Methods ................................................................................................ 9 4.1 Study area.............................................................................................................. 9 4.2 Sample collection and processing .......................................................................... 9 4.3 Isolation of phosphate solubilisation bacteria ......................................................... 9 4.4 Screening of isolates for PSE (plate assay method) .............................................. 9 4.5 Identification of phosphate solubilisation bacteria ................................................ 10 4.6 Antimicrobial assay .............................................................................................. 12 4.7 Soil analysis ......................................................................................................... 12 4.8 Statistical analysis ................................................................................................ 15 5. Results and discussion.............................................................................................. 42 5.1 Isolation and population dynamics of PSB ........................................................... 42 5.2 Morphological and biochemical characteristics .................................................... 42 6. Conclusions............................................................................................................... 43 Acknowledgements ....................................................................................................... 43 References .................................................................................................................... 44 ii

Table of tables

Table of figures Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009). ............................................. 15 Figure 2. Map of Kerala showing the various sample collection point of soil samples from differ Piper nigrum varieties. Authors own work. .............................................. 16 Figure 3. Pepper plant (Piper nigrum) immature peppercorns. Photo courtesy: Wikipedia.................................................................................................................. 17 Figure 4. Black pepper (Piper nigrum) description a) tree bearing half mature fruits, b) plant climbing on support, c) spike and leaf, d) black, green, pink and white peppercorns, e) different types of peppercorns. Photo courtesy: Wikipedia............. 18 Figure 5. Black pepper (Piper nigrum) description a) black pepper grains, b) white pepper grains, c) peppercorn close-up, d) handheld pepper mills, e) roughly cracked black peppercorns. Photo courtesy: Wikipedia. ........................................................ 19 Figure 6. Black pepper (Piper nigrum) description a) fully ripened fruits, b) mature fruits, c) green mature fruits separated from spike, d) red fully mature fruits separated from spike, e) mature and fully ripened fruits with spike, f) Dried peppercorns. Authors own images. .......................................................................... 20 Figure 7. Description of Karimunda (P14) a) immature spike and fruits, leaves; b) and c) immature spike, d) and e) fully mature spike and fruit, f) mature spike. Authors own images. ................................................................................................ 21 Figure 8. Description Panniyoor1 (P18) a) and c) pepper plant with spike and leaves, b) pepper plant showing the support tree, d) leaf and semi matured fruits with spike, f) tip of spike showing semi matured fruit. Authors own images. .............................. 22 Figure 9. Description Panniyoor1 (P18) continued, a) and b) spike and leaves, c), d), e), f), g) and h) spike showing fully matured fruit. Authors own images. .................. 23 Figure 10. Piper nigrum L. lateral branch habit; 1-Errect, 2-Horrizontal, 3-Hanging (IPGRI, 1995). .......................................................................................................... 24

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Table of tables

Figure 11. Piper nigrum L. leaf lamina shape; 1-Ovate, 2-Ovate-elliptic, 3-Ovatelanceolate, 4-Elliptic- lanceolate, 5-Cordate (IPGRI, 1995). ..................................... 24 Figure 12. Piper nigrum L. leaf base shape; 1-Round, 2-Cordate, 3-Acute, 4-Oblique (IPGRI, 1995). .......................................................................................................... 24 Figure 13. Piper nigrum L. leaf margin; 1-Even (entire), 2-Wavy (repand) (IPGRI, 1995). ....................................................................................................................... 25 Figure 14. Piper nigrum L. types of veining; 1-Acrodromous, 2-Campylodromous, 3Eucamptodromous (IPGRI, 1995). ........................................................................... 25 Figure 15. Piper nigrum L. spike orientation; 1-Errect, 2-Prostrate (IPGRI, 1995).... 26 Figure 16. Piper nigrum L. spike shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4Conical. .................................................................................................................... 26 Figure 17. Piper nigrum L. fruit shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995). .......................................................................................................... 26 Figure 18. Soil sample description a) soil sample 1 ; MKID, b) soil sample 2; MKKD, c) soil sample 3; MPAD, d) soil sample 4; MPID. Authors own images. ................... 27 Figure 19. Description of the antibiotic disc diffusion of the isolated microorganisms a) B1, b) B2, c) B3, d) B4, e) B5, f) control. Authors own images. ........................... 34 Figure 20. Description of the antibiotic disc diffusion of the isolated microorganisms a) B6, b) B7, c) B8, d) control. Authors own images................................................. 35 Figure 21. Description of the carbohydrate tests (dextrose, lactose, sucrose; a), b) and c)) and catalase activity of the isolated microorganisms d), B1, e) B2, f) B3. Authors own images. ................................................................................................ 37 Figure 22. Description of the catalase activity of the isolated microorganisms a) B4, b) B5, c) B6, d) B7, e) B8, f) control. Authors own images. ...................................... 38 Figure 23. Description of the IMViC test (citrate, VP, MR, indole) of the isolated microorganisms a) B1, b) B2, c) B3, d) B4, e) B5, f) B6, g) B7, h) B8. Authors own images...................................................................................................................... 39

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Figure 24. Description isolated microorganisms from different soil samples under varying dilutions a), b), c) and d) soil sample 1; MKID; 10-1 dilution to 10-4, e), f), g) and h) soil sample 2; MKKD; 10-1 dilution to 10-4. Authors own images. .................. 40 Figure 25. Description isolated microorganisms from different soil samples under varying dilutions a), b), c) and d) soil sample 3; MPAD; 10-1 dilution to 10-4, e), f), g) and h) soil sample 4; MPID; 10-1 dilution to 10-4. Authors own images. ................... 41

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Table of tables

Table of tables Table 1. Different vernacular names of Piper nigrum L. around the globe and India.. 5 Table 2. Description of the Pepper nigrum L. varieties (P18, P14) and places in Kerala. ...................................................................................................................... 28 Table 3. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 29 Table 4. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 29 Table 5. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 29 Table 6. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 30 Table 7. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 30 Table 8. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala. .................................................................................................................. 30 Table 9. Colony morphology, gram staining and motility properties of the isolated phosphate solubilising bacteria B1, B2, B3, B4, B5, B6, B7, B8) in Kerala. ............. 31 Table 10. Biochemical charaterization of the isolated phosphate solubilising bacteria B1, B2, B3, B4, B5, B6, B7, B8) in Kerala. ............................................................... 32 Table 11. Soil general properties of the soil samples during the cold dry season in 2016. ........................................................................................................................ 33 Table 12. Antibiotic disc diffusion of the isolated bacteria (B1, B2, B3, B4, B5, B6, B7, B8) using antibiotic discs and zone of inhibition in cm. ...................................... 36

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List of abbreviations

List of abbreviations %

: Percentage

°C

: Centigrade

AAS

: Atomic absorption spectrophotometer

ATP

: Adenosine triphosphate

AXM

: Amoxycillin

AZM

: Azithromycin

B

: Boron

B1

: Bacterial isolate 1

B2


B3


B4


B5


B6


B7


B8


C

: Carbon

CaCl2

: Calcium chloride

cm

: Centimetre

CTX

: Cefotaxine

Cu

: Copper

Fe

: Iron

g

: Gram

H2O2

: Hydrogen peroxide

H3PO4

: Phosphoric acid

HCl

: Hydrochloric acid

K

: Potassium

K2Cr2O7

: Potassium dichromate

KCl

: Potassium chloride

m

: Metre

M

: Molarity

Mg

: Milligram

MHA

: Mueller hinton agar

ml

: Millilitre

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List of abbreviations

mm

: millimeter

Mn

: Manganese

Mo

: Molybdenum

MR

: Methyl red

N

: Nitrogen

N

: Normality

Na2CO3

: Sodium carbonate

NH4CH3CO2

: Ammonium acetate

NH4Cl

: Ammonium chloride

nm

: Nanometre

OA

: Organic acid

P

: Phosphorous

P

: Popular

P14

: Karimunda

P18

: Paniyoor 1

Pb

: Lead

PGPR

: Plant growth promoting rhizobacterium

pH

: Hydrogen ion concentration

Po

Popular

ppm

: Parts per million

PSB

: Phosphate solubilising bacteria

PSM

: Phosphate solubilising microorganisms

PVK agar

: Pikovaskya agar

S

: Sulphur

SE

: Standard error

VP

: Voges Prausker

Zn

: Zinc

µl

: Micro litre

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Isolation of phosphate solubilising bacteria from rhizosphere of different black pepper (Piper nigrum) varieties in Kerala: an overview

Isolation of phosphate solubilising bacteria from rhizosphere of different black pepper (Piper nigrum) varieties in Kerala: an overview Prem Jose Vazhacharickal1*, Sajeshkumar N.K1, Jiby John Mathew1 and Mili Rose Michel1 * [email protected] 1

Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

Abstract Black pepper is belongs to piperaceae family and is known as “king of species”. This piperaceae family contain approximately 2,000 species. Phosphorus is one of the most important micronutrients and they are essential for the biological growth, development of plants and it is the most essential nutrient for plants. Phosphate deficiency is wide spread and phosphate fertilizers universally required in the form of inorganic P fertilizers, only a small portion is utilized by plants and the remaining are in insoluble form and they are solubilised by the microbes present in the soil. Soil, they are rich in micro and macronutrients and sixteen elements or nutrients are essential for plant growth and reproduction. Several soil bacteria, particularly belonging to the genera Pseudomonas and Bacillus posses ability to bring insoluble soil phosphate into soluble forms by secreting acids like formic, lactic and acetic. The rhizosphere soil sample were serially diluted up to 10-4 using sterile distilled water and plated on Pikovskaya’s agar medium by pour plate method. The P solubilising isolates was evaluated on agar plates of Pikovaskya growth medium by solubilising the tricalcium phosphate of the medium. After incubation the phosphate solubilising microorganisms were selected based on different colonies. Out of so many bacterial isolates, 8 isolates were selected for the further study to perform qualitative and quantitative analysis. Phosphate solubilising microorganisms are possible to use as bio fertilizer for all crops. Among the isolates some of are almost identical in biochemical test but they have different morphology characters. Keywords: Rhizosphere; Phosphate solubilising bacteria; Black pepper; Pikovaskya agar.

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