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

An overview

von Dr. Prem Jose Vazhacharickal (Autor) Sajeshkumar N.K. (Autor) Jiby John Mathew (Autor) Mili Rose Michel (Autor)

Wissenschaftliche Studie 2017 59 Seiten

Agrarwissenschaften

Leseprobe

Table of contents

Table of figures

Table of tables

List of abbreviations

Abstract

1. Introduction
1.1 Objectives
1.2 Scope of the study
1.3 Taxonomical classification

2. Review of literature

3. Hypothesis

4. Materials and Methods
4.1 Study area
4.2 Sample collection and processing
4.3 Isolation of phosphate solubilisation bacteria
4.4 Screening of isolates for PSE (plate assay method)
4.5 Identification of phosphate solubilisation bacteria
4.6 Antimicrobial assay
4.7 Soil analysis
4.8 Statistical analysis

5. Results and discussion
5.1 Isolation and population dynamics of PSB
5.2 Morphological and biochemical characteristics

6. Conclusions

Acknowledgements

References

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 co-operation 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

Table of figures

Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009).

Figure 2. Map of Kerala showing the various sample collection point of soil samples from differ Piper nigrum varieties. Authors own work.

Figure 3. Pepper plant (Piper nigrum) immature peppercorns. Photo courtesy: Wikipedia.

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.

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.

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.

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.

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.

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.

Figure 10. Piper nigrum L. lateral branch habit; 1-Errect, 2-Horrizontal, 3-Hanging (IPGRI, 1995).

Figure 11. Piper nigrum L. leaf lamina shape; 1-Ovate, 2-Ovate-elliptic, 3-Ovate-lanceolate, 4-Elliptic- lanceolate, 5-Cordate (IPGRI, 1995).

Figure 12. Piper nigrum L. leaf base shape; 1-Round, 2-Cordate, 3-Acute, 4-Oblique (IPGRI, 1995).

Figure 13. Piper nigrum L. leaf margin; 1-Even (entire), 2-Wavy (repand) (IPGRI, 1995).

Figure 14. Piper nigrum L. types of veining; 1-Acrodromous, 2-Campylodromous, 3-Eucamptodromous (IPGRI, 1995).

Figure 15. Piper nigrum L. spike orientation; 1-Errect, 2-Prostrate (IPGRI, 1995).

Figure 16. Piper nigrum L. spike shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical.

Figure 17. Piper nigrum L. fruit shape; 1-Filiform, 2-Cylindrical, 3-Globular, 4-Conical (IPGRI, 1995).

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.

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.

Figure 20. Description of the antibiotic disc diffusion of the isolated microorganisms a) B6, b) B7, c) B8, d) control. Authors own images.

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.

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.

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.

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.

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.

Table of tables

Table 1. Different vernacular names of Piper nigrum L. around the globe and India.

Table 2. Description of the Pepper nigrum L. varieties (P18, P14) and places in Kerala.

Table 3. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

Table 4. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

Table 5. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

Table 6. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

Table 7. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

Table 8. Plant characteristics and properties of Piper nigrum L. varieties (P18, P14) in Kerala.

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.

Table 10. Biochemical charaterization of the isolated phosphate solubilising bacteria B1, B2, B3, B4, B5, B6, B7, B8) in Kerala.

Table 11. Soil general properties of the soil samples during the cold dry season in 2016.

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.

List of abbreviations

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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

* premjosev@gmail.com

1Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kerala, India-686576

Abstract

Black pepper 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.

1. Introduction

Black pepper (Piper nigrum) the flowering wine belongs to the family piperaceae, cultivated for its fruit, which is usually dried and used as spice and seasoning. Morphological characters like plant habit, pubescence, texture and leaf shape of juvenile and mature forms, orientation of the spike and length of peduncle, nature of bract and fruit colour are used as key distinguish different species (Hooker,1886; Kanjilal et al.,1940; Gamble, 1925). Piperaceae is considered to be one of the most primitive families of Angiosperms (Engler, 1893; Rendle, 1925) derived from the herbaceous proto-angiosperms with simple, minute flowers (Heywood and Fleming, 1986; Taylor and Hickery, 1990; Taylor and Hickery, 1992).

Black pepper is belongs to piperaceae family and is known as “king of species” (Rani et al., 2013; Chaithong et al., 2006). This piperaceae family contain approximately 2,000 species (chaithong et al., 2006). Black pepper has high economic value in India and most widely used spices in food industry (Seshachala and Tallapragada, 2012; Seshachala and Tallapragada, 2013). Pepper is mostly used in ayurvedic, herbal, folklore medicine and they are most cultivated in southern part of India, Indonesia and some regions in Tamil Nadu and Kerala (Seshachala and Tallapragada, 2012; Seshachala and Tallapragada, 2013). The pepper leaves are thick, green colour with ovate shape. Flowers are white in colour which produce fruits borne on short, the length of the spikes is from 4 to 12 cm long. Fruits are green when unripe and become red at maturity. The favourable temperature range for crop is 23 - 32°C and the ideal temperature is around 28°C. Optimum soil temperature for root growth is 26°C - 28°C.

The piperaceae family consist of 12 genera and more than 1400 species (Trivedi et al., 2011) and is one of the largest families and widely distributed across the tropical and sub-tropical regions and over the high ranges of Andes and Sub Himalayas. In genus Piper more than 1000 are included, from which 110 are of the Indian origin (Parthasarathy, 2006; Chakraborty, 2011). In India black pepper is cultivated to a large extent in Kerala, Karnataka and Tamil Nadu and to a limited extent in Maharashtra, North eastern states and Andaman and Nicobar Islands (ICAR, 2015). The white pepper of commerce is the product of the same pepper plant, produced by removing the pericarp (Ravindran, 2000). The spices and herb naturally produce secondary metabolites which possess natural antioxidants that help in delaying of aging process and improve immune system (Nahak and Sahu, 2011).

Phosphorus (P) is one of the most important macronutrients and they are essential for the biological growth, development of plants and it is the most essential nutrient for plants (Seshachala and Tallapragada, 2012). 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. They convert insoluble phosphate into soluble to form available to plants (Seshachala and Tallapragada, 2012; Ramachandran et al., 2003). The organic acid and phosphatase enzymes produced by plants and microorganisms are used to convert insoluble phosphate compounds into solubilised form (Sharma, 2005).

The phosphate solubilising bacteria (PSB), which play an important role in enhancing phosphorous availability (Susilowati and Syekhfani, 2014; Baliah and Belgum, 2015; Aipova et al., 2010). Phosphorus provided in the form of fertilizers and it increases phosphate availability (Ejikeme and uzoma, 2013; Patel and Parmar, 2013).

Soil, they are rich in micro and macronutrients and sixteen elements or nutrients are essential for plant growth and reproduction. They are carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium( Mg), iron (Fe), boron (B), manganese (Mn), copper (Cu), zinc (Zn), molybdenum (Mo), and chlorine (Cl) (Roy et al., 2006). Nutrients required for plants to complete their life cycle are considered essential nutrients. Several soil bacteria, particularly belonging to the genera Pseudomonas and Bacillus possess ability to bring insoluble soil phosphate into soluble forms by secreting acids like formic, lactic and acetic (Afzal et al., 2005).

Black pepper require humidity and high rainfall and it is largely cultivated in Kerala and favourable temperature range is 23-32°C (Zacharaiah et al., 2015). In India pepper is mainly cultivated in Karnataka, Kerala and Thamil Nadu (Seshachala et al., 2012). Pepper is mostly dioecious in wild form, but in the cultivated types the plants are mostly gynomonoecious or trimonoecious (Thangaselvabal et al., 2012). A majority of the cultivated monoecious through variation in expression rainging from complete male to complete female is found. Over 75 cultivars of black pepper are being cultivated in India. Karimunda is the most popular cultivar in Kerala. The other important ultivars are Kottanadan, Narayakodi, Aimpiriyan, Neelamundi and Kuthiravally (Devasahayam et al., 2013).

1.1 Objectives

The objective of this research work was to isolate and characterise the phosphate solubilising bacteria from different area of rhizosphere of soil and soil analysis.

1.2 Scope of the study

The study would enlighten the rhizosphere phosphate solubilising microorganisms associated with black pepper varieties which could be further explored as biofertizers.

1.3 Taxonomical classification

Kingdom: Plantae-- planta, plantes, plants, vegetal

Subkingdom: Viridiplantae

Division: Tracheophyta – vascular plants, tracheophytes

Class: Magnoliopsida

Order: Piperales

Family: Piperaceae – peppers

Genus: Piper L.

Species: Piper nigrum L.

Table 1. Different vernacular names of Piper nigrum L. around the globe and India.

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2. Review of literature

In black pepper high variability was also noticed for yield contributing characters like runner shoot production, holding capacity, adventitious root production, lateral branch habit, spike length, type of hermaphroditism, number of spikes per lateral branch, fruit set, dry weight, essential oil, oleoresin and piperine content. Intra-cultivar variability was reported earlier and was used to characterize the cultivars of black pepper (Ratnambal et al., 1985; Pillai et al., 1987; Ravindran et al., 1992a; Ravindran et al., 1997b; Sasikumar et al., 1999a., Mathew and Mathew, 2002; Mathew et al., 2005).

Soil acts as a medium, a habitat for soil organisms, a recycling system for nutrients and organic wastes, a modifier of atmospheric composition, and a medium for plant growth (Dominati et al., 2010). The soil texture is determined by the mineral components of soils that are sand, slit and clay. The mineral constituents of a loam soil might be 40% sand, 40% silt and 20% clay by weight and soil texture affects soil behaviour (Brown, 2003). There are mainly sixteen nutrients that are essential for plant growth and reproduction. Plants need these nutrient for complete their life cycle and are consider as essential nutrients and these enhance the growth of plants but are not necessary to complete the plant's life cycle are considered non-essential. With the exception of carbon, hydrogen and oxygen, which are supplied by carbon dioxide and water, the nutrients derive originally from the mineral component of the soil (Roy et al., 2006).

Phosphorus is an important plant macronutrient, making up about 0.2% of a plant's dry weight and it is a component of key molecules such as nucleic acids, phospholipids, and adenosine triphosphate (ATP). Plants cannot grow without nutrients, which can only grow with available of nutrients. Phosphorus is the second most important macronutrient for plant growth after N (Theodorou and Plaxton, 1993). Photophosphorylation, genetic transfer, transportation of nutrients, and phospholipid cell membranes, these are the process that use phosphorus as macronutrient. When P is compared with the other major nutrients, it is least mobile and available to plants in most soil conditions (Baliah and belgum, 2015; Aipova et al., 2010) Although P is abundant in soils in both organic and inorganic forms, it is frequently a major factor plant growth factor. The bioavailability of soil inorganic phosphorus in the rhizosphere varies considerably with plant species, nutritional status of soil (Khan et al., 2006).

Phosphate solubilising bacteria (PSB) are beneficial bacteria capable of solubilising inorganic phosphorus from insoluble compounds. P-solubilisation ability of rhizosphere microorganisms is considered to be one of the most important traits associated with plant phosphate nutrition and a large portion of soluble inorganic phosphate applied to the soil as chemical fertilizer (Chen et al., 2006). Microorganisms play an important role in enhancing P availability to plant roots and is most sensitive to lead (Pb) and elevated Pb levels in the soil may inhibit soil microbial activity (Susilowati and Syekhfani, 2014). Phosphate solubilising microorganisms (PSM) have attracted the researchers to exploit their potential to utilize phosphate reserves in semi-arid regions and to enhance the crop yields. Phosphate solubilising microorganisms have established their role for optimum growth of plants under nutrient imbalance conditions and phosphate solubilisers are economical, and eco-friendly (Jain and Khichi, 2014)

The development of effective microbial inoculants remains a major scientific challenge but in the past 10 years, several strains of plant growth–promoting bacteria (Acinetobacter, Alcaligenes, Arthrobacter, Azospirillium, Azotobacter, Bacillus, Beijer-inckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium Rhizobium, and Serratia) have been identified. Such strains stimulate growth and yield of apple (Malus domestica L.), high bush blueberry (Vaccinium corymbosum L.), mulberry (Morus alba L.), apricot (Prunus armenia L.), sweet cherry (Prunus avium L.), and raspberry (Rubus ideaus L.) (gunes et al., 2009). Since the beginning of last century, many PSB have been isolated including, for example, those in Bacillus, Pseudomonas, Erwinia, Agrobacterium, Serratia, Flavobacterium, Enterobacter, Micrococcus, Azotobacter, Bradyrhizobium, Salmonella, Alcaligenes, Chromobacterium, Arthrobacter, Streptomyces, Thiobacillus, and Escherichia. The microorganisms functioning similarly also include some fungi in genus Penicillium, Aspergillus, Rhizopus, Fusarium, and Sclerotium (Jain and Khichi, 2014; Gunes et al., 2009).

Several authors attribute the solubilisation of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars. Phosphate solubilising microorganisms are routinely screened by a plate assay method using Pikovaskya (PVK) agar. The PVK medium contains yeast extract and it is desirable to formulate a desirable medium to elucidate the role of microorganisms in phosphorus mineralization (Nautiyal, 1999).The insoluble forms of P such as tricalcium phosphate (Ca3PO4)2, aluminium phosphate (Al3PO4), iron phosphate (Fe3PO4), etc. may be converted to soluble P by P-solubilising organisms inhabiting different soil ecosystems. Several workers have documented their findings in order to better understand as to how the microbial populations cause the solubilisation of insoluble P (Khan et al., 2007; Khan et al., 2014). Of the various strategies adopted by microbes, the involvement of low molecular mass organic acids (OA) secreted by microorganisms has been a well-recognized and widely accepted theory as a principal means of P-solubilisation, and various studies have identified and quantified organic acids and defined their role in the solubilisation process (Khan et al., 2014).

Phosphorus (P) is proven to be one of the key limiting factors of bacterial activity and for primary production in aquatic systems (Quian et al., 2010).Phosphorus also plays an important role in photosynthesis, respiration, energy storage and transfer, cell division etc and also include in mechanism like acid formation, chelating metal ions and exchange reactions (Karpagam and Nagalakshmi, 2014; Aipova et al., 2010). In order to increase crop yields, mineral phosphate fertilizers are regularly incorporated into the soil (Aipova et al., 2010). A single plant growth promoting rhizobacterium (PGPR) may possess one or more than one of these plant beneficial traits. Phosphate solubilising microbes are considered as important members of PGPR and their application in the form of bio fertilizer has been shown to improve growth of cereals and other crops (Gandhi et al., 2014).

Several varieties of phosphate-solubilising microorganisms (PSMs) have been isolated from the rhizospheric soils of crops. Of these, 20%-40% are cultivable soil microorganisms. A majority of the isolated organisms are bacterial organisms, although several fungi are also known to solubilise phosphates and their role in increasing the soil nutrient value is of utmost importance. These bacteria and fungi have the potential to be used as bio fertilizers but Bacteria are more effective in phosphorus solubilisation than fungi (Mohammadi, 2012; Tallapragada and Seshachala, 2012). Most phosphate-solubilising bacteria are highly specific to a host plant and host-specificity in bacterial colonization is an important factor for success in bio fertilization and it is thus interesting to explore phosphate solubilising bacteria naturally abundant in the forests to solve phosphorus deficiency in agriculture (Ruangsanka, 2014).

3. Hypothesis

The current research work is based on the following hypothesis

1) Pepper plant varieties have a promising effect on soil rhizosphere microorganisms
2) Phosphate solubilising bacteria differ according the pepper varieties
3) Morphological and biochemical characteristics of each PSB are varying among the isolates
4) Soil chemical structure has an influence on PSB in soil

4. Materials and Methods

4.1 Study area

Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts. The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al ., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November (Figure 1).

4.2 Sample collection and processing

Various pepper varieties in Kerala were selected based on a baseline survey, information’s collected from various beneficiaries and databases. Two different pepper varieties were selected across Kerala for soil analysis and bacterial isolation. The soil samples (Four samples) were collected from the fields of Piper nigrum rhizosphere from various district of Kerala, India (Figure 2) during the month of December 2016. Samples were collected from the selected sites at a depth of 15 cm from 6 different points within the area. The samples were transported to the laboratory until processing. The samples were dried in hot air oven at 40°C for 48 hrs. The samples were pooled based on locations and were finely powered using a agate pestle and mortar; later stored in airtight polyethylene tubes till analysis. Locations of the sample collection points were recorded using a Trimble Geoexplorer II (Trimble Navigation Ltd, Sunnyvale, California) and data were transferred using GPS Pathfinder Office software (Trimble Navigation Ltd, Sunnyvale, California).

4.3 Isolation of phosphate solubilisation bacteria

The homogenized soil sample were serially diluted up to 10-4 using sterile distilled water and was spread on Pikovskaya’s agar medium (PVK). The plates were incubated at 30°C for 4 days. After incubation the phosphate solubilising microorganism were selected based on the zone of clearance around the colonies and purified by repeated culturing and maintained on Pikovskaya’s agar slants at 4°C.

4.4 Screening of isolates for PSE (plate assay method)

The pure culture was point inoculated into Pikoskaya’s agar plates and incubated at 30°C for 10 days. The solubility of phosphate was observed as a zone of clearance with a diameter that was measured in millimetres. The microbial phosphate solubilisation was analyzed by determine the P solubilisation (PSE)

The efficient P solubilising microbes then further identified.

4.5 Identification of phosphate solubilisation bacteria

The homogenized soil sample were serially diluted up to 10-4 using sterile distilled water and was spread on Pikovskaya’s agar medium (PVK). The plates were incubated at 30°C for 4 days. After incubation the phosphate solubilising microorganism were selected based on the zone of clearance around the colonies and purified by repeated culturing and maintained on Pikovskaya’s agar slants at 4°C.

4.5.1 Gram’s staining

The isolate was characterized for its Gram staining characteristics as per the following standard procedure: Take the smear on the glass slide with the help of inoculating loop let it be air dry. The slides were fixed, crystal violet solution added for 30 seconds followed by washing. Followed by Gram’s iodine for 60 seconds. Wash it with 95% Ethyl alcohol, Add saffranin for 30 seconds after this wash it with the distilled water. Air dry it with the help of blotting paper and observed under microscope. The pink colonies will show the gram negative bacteria and the purple colonies will show the gram positive bacteria.

4.5.2 Biochemical tests

Biochemical tests were performed for the identification of the isolates.

4.5.2.1 Indole production test

The test was used to differentiate the bacteria into two based on the ability to produce indole by hydrolysis of tryptophan. Here the tryptophan broth was prepared and inoculated with the culture organism. Then it was incubated at 37°C for 2 days. After the incubation added 0.5 ml kovac’s reagent into the tubes and observed it for 2 minutes. The colour change was appeared as pink or red color at the junction of the medium and reagent was positive.

4.5.2.2 Methyl Red Test and Voges-proskauer Test

The both tests were performed simultaneously on the same medium (MR and VP). It was called as MR-VP test. Here the MR-VP broth was prepared to each isolate and control for both testes. Then the tubes were inoculated with the desired isolates. After inoculation tubes were incubated at 35°C for 48 hours. Then 5 drops of methyl red indicator was added into one tube of each isolate and a control. Then observed colour change for the results. To the rest of tubes of each isolates and control, added 10 drops of VP1 reagent followed by 2-3 drops of VP2 reagent. Gently shake the tubes after removing the cotton plugs and kept for 15-30 minutes to complete the reaction. Then the colour change was observed as red within few minutes. The reaction is positive.

4.5.2.3 Catalase activity for H2O2 Production

This test was performed to identify the catalase activity of the isolates. Here picked a loopful of culture from petriplate and transferred into a few drops of water. Then added a few drops of 3% hydrogen peroxide (H2O2) over the culture. Then wait for 20 seconds for the observation. It could be find bubble formation due to the rapid evolution of oxygen within seconds represents positive reaction. On the other hand there is no bubble formation.

4.5.2.4 Citrate utilization Test

This test is based on the ability of an organism to utilize citrate as its only source of carbon and ammonia as nitrogen source. In this test first Simmon’s citrate agar slants were prepared and inoculated by stabbing the isolates into base of the slant: thereafter, streaked the surface. Then the slants were incubated at 37°C for 48 hrs. After incubation slants were observed, in positive reaction there is growth and colour change from green to blue along the slant and no growth and colour change in negative reaction.

4.5.2.5 Carbohydrate Fermentation Test (Sucrose, Lactose, Glucose)

This test is recommended for identification of an unknown species. The acid, alkali or gas production results a visible change in the inoculated broth. This experiment was performed by taking three sets of tubes containing 3 different kinds of broth namely sucrose, lactose, and glucose broth each contains sufficient amount of other components and bromothymol blue as pH indicator. Then the Durham tube was inserted into each tube after sterilization and cooling. Each set of test tubes were inoculated with appropriate isolates (B1, B2, B3, B4, B5, B6, B7 and B8) and incubated at 37°C for 24 hrs. Then examined colour change and gas production of the medium for results.

4.6 Antimicrobial assay

Antimicrobial assay was performed by agar disc diffusion method using standard antibiotic discs (Himedia Laboratory, Mumbai, India). This test is used by spread plate technique. Place Muller-hinton agar (MHA) plate in the laminar air flow chamber. Inoculate 0.1ml organism from the nutrient broth using sterile pipette. Using L- rod spread the inoculate into the entire surface, horizontally, vertically, and around the outer edge of the plate to ensure a heavy growth over the entire agar surface. Allow all cultures to dry for 5 minutes. Using forceps apply the antibiotic disc by placing them on the marked agar surface and gently press the disc for proper location. Incubate all plate culture in an invert incubation for 48 hours at 37°C and examine them by obtaining clear zones.

4.7 Soil analysis

4.7.1 Estimation of Organic Carbon (Walkley- Black method)

The soil, ground up so as to pass through a 0.5 mm mesh sieve, was placed in a 500 ml Erlenmeyer flask. The amount of soil used in the determination was calculated based on initial information on the carbon (C) concentration in the soil and ranged from 0.1 to 0.5 g. Ten ml of potassium dichromate (K2Cr2O7) and 20 ml of concentrated sulfuric acid (H2SO4) were added to the soil while stirring it to ensure good mixing of the soil with the reagents. After a 30 min rest, 200 ml of distilled water, 10 ml of concentrated phosphoric acid (H3PO4) and 1 ml of 0.16 % diphenylamine were added. The excess dichromate that was not reduced in the reaction was determined by volumetric titration using ammonium ferrous sulfate.

4.7.2 Estimation of Available Phosphorous (Ascorbic acid blue colour method)

Five gram of soil was taken in a 100 ml shaking bottle. 50 ml of Bray No. 1 Solution was added. It was shaken for five minutes and filtered through a Whatman No.42 filter paper. Transferred 50 ml filtrate into a 50 ml volumetric flask. The pH was adjusted to three with 4N sodium carbonate (Na2CO3) or 4 ammonium chloride (NH4Cl), 2, 4- dinitrophenol being used as an indicator. It became yellow as pH three is approached from the acid side. Added a few drops of indicator to solution and if yellow colour was formed, 4N hydrocholoric acid (HCl) was added drop wise until it becomes colourless. If indicator gives a colourless solution indicating a solution pH below three then 4N Na2CO3 can be added drop wise just until a yellow colour is developed, and finally 4N HCl was added and yellow becomes faint. Added 5 ml molybdate reagent, followed by 4ml freshly prepared ascorbic acid. Make up the volume. Mixed well and kept it for colour development. Intensity of colour was determined in a photo electric colorimeter or spectrophotometer at 730-840 nm.

4.7.3 Estimation of Available Potassium

Five gram of soil was taken in a shaking bottle and 50 ml neutral 1N ammonium acetate (NH4CH3CO2) solution was added. It was kept shaken for five minutes in a reciprocating shaker and after five minutes it was filtered through a Whatman No.42 filter paper. Pipette out ten ml of aliquot and make up the volume to 50 ml with distilled water. Reading was taken in a flame photometer using potassium filter. The equipment was standardized initially with distilled water to indicate a reading of zero in the meter dial of flame photometer. Then a 10 ppm potassium solution was used which is to be read as hundred in the meter.

4.7.4 Estimation of Available Sulphur

Shake 10g of air-dried processed soil with 50 ml of 0.15% CaCl2 solution in a 250ml conical flask for 30 minutes. Filter the extract through WatmanNo.42 filter paper and estimate the sulphate content turbid metric procedure.

4.7.5 Estimation of Iron, Zinc, Copper and Manganese

Two gram of soil was shaken with twenty ml of 0.1 M HCl for five minutes. It was filtered through Whatman No.42 filter paper. The filtrate was collected and the contents of Fe, Mn, Zn, and Cu were estimated using AAS.

4.7.6 Estimation of available Boron

Scoop 10 cm3 or weigh 10 g of air-dried, sieved soil into a Low-B boiling flask capable of use with a water-reflux condensing apparatus. Add 20 ml of B extracting solution to the flask, attach to the condenser. Heat the flasks until initiation of boiling, and then reflux the suspension for 5 - 15 minutes. Allow to cool slightly (2-3 minutes) before removal from condenser. For funnel-flask extractions, remove the funnel and immediately place the flask with soil and extracting solution on a balance and add hot deionized water to attain original recorded weight. Swirl to mix. Immediately transfer the solution to a plastic centrifuge tube and centrifuge for 15 minutes at 2,700 g. Decant and aliquot from the supernatant of the centrifuge tube for analysis. It may be necessary to filter the supernatant solution to remove particulate matter. Alternatively, filter the suspension, while still warm, through Whatman No. 42 filter paper, catching the filtrate in plastic sample bottles. Inspect supernatant solution or filtrate for clarity and refilter if necessary.

Pipette a 1 ml aliquot of soil extract into a plastic tube or small plastic beaker. Add 2 ml of the Buffer-masking solution and mix thoroughly by swirling. Add 2 ml of Azomethine-H solution and mix contents thoroughly. Allow mixture to stand 30 minutes, then measure absorbance at a wavelength of 420 nm. Prepare a standard curve by adding 1 ml of each of the working standards to a plastic tube or beaker; add Buffer-masking solution and Azomethine-H in an identical manner as with soil extracts. Determine absorbance of standards and compare results of samples with those from a standard curve.

4.7.7 Estimation of Electrical Conductance

The clear supernatant of 1:2.5 soil water suspensions prepared for pH measurement can be used for estimation of electrical conductivity. Conductivity meter was calibrated using potassium chloride (KCl) solution and the cell content was determined. The conductivity of the supernatant liquid was determined.

4.7.8 Estimation of pH

The pH meter was calibrated using two buffer solutions, one was buffered with neutral pH and the other was buffered based on the range of calibrate pH in the soil. The buffer solution was taken in the beaker. The electrode was inserted alternately in the beakers containing two buffer solutions and adjusted the pH. The instrument indicating pH as per the buffers is used to test the samples. Ten gram of soil sample was taken in a 100 ml beaker and 20 ml of calcium chloride (CaCl2) solution was added. The soil was allowed to absorb calcium chloride solution without stirring. Then it was thoroughly stirred for ten seconds using a glass rod. The suspension was stirred for thirty minutes and the pH was recorded on the calibrated pH meter.

4.8 Statistical analysis

The survey results were analyzed and descriptive statistics were done using SPSS 12.0 (SPSS Inc., an IBM Company, Chicago, USA) and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, USA).

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Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009).

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Figure 2. Map of Kerala showing the various sample collection point of soil samples from differ Piper nigrum varieties. Authors own work.

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Figure 3. Pepper plant (Piper nigrum) immature peppercorns. Photo courtesy: Wikipedia. https://en.wikipedia.org/wiki/Black_pepper.

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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. https://en.wikipedia.org/wiki/Black_pepper.

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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. https://en.wikipedia.org/wiki/Black_pepper.

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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.

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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.

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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.

[...]

Details

Seiten
59
Jahr
2017
ISBN (eBook)
9783668476257
ISBN (Buch)
9783668476264
Dateigröße
8.3 MB
Sprache
Englisch
Katalognummer
v370222
Institution / Hochschule
Mar Augusthinose College
Note
1.5
Schlagworte
Rhizosphere; Phosphate solubilising bacteria; Black pepper; Pikovaskya agar

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