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CHAPTER- V

Enumeration and Isolation of Beneficial Soil Microflora

5.1 Background

Isolation of appropriate microbes is very essential before putting them through

screening procedure. The initial step for isolation is to obtain the organism in pure culture

without which, it cannot be considered for characterization. Enrichment of isolation culture is

essential to isolate desired class of microorganisms where selective media is utilized for the

pure culture of required specific strain.

With the growing interest in exploration and exploitation of native microbes having

antagonistic properties as biocontrol agents, research have been undertaken and efforts were

also been made by the scientists in screening various native antagonistic microbial strains

(Barthakur and Dutta, 1992; Ponmurugan and Baby, 2005; Mishra et al., 2005; Phukan et al.,

2007; Ponmurugan et al., 2007) against many tea pathogens.

In continuation of search for few newer strains of phosphate solubilising bacteria

(PSB) (Awasthi et al., 2011) and actinomycetes strains from tea soil, the present work led to

the isolation of beneficial microbes having possible antagonistic potentialities.

5.2 Materials and Methods

5.2.1 Preparation of selective media

Pikovskaya medium as modified by Rao and Sinha (1963) for isolation of PSB and

Kenknight‘s Munaiers medium (Kenknight, 1939) for the isolation of actinomycetes were

prepared as per the recommendation. For screening of the optimum growth medium, the

media were prepared as a liquid form. 1000 ml of media was prepared and distributed in the 5

nos. of 250 ml conical flask that contain 200 ml in each flasks, 5 replicates were made for

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each of the medium. After distribution, the flasks were plugged and sterilized in the autoclave

at 15 PSI for 15 minutes. The flasks were kept for cooling before utilization in further

experimentation.

Pikovkayas Medium (Modified by Rao and Sinha, 1963 for Phosphate solubilizing

organisms)

Quantity/L

Glucose 10.0 gm

Tricalcium Phosphate 5.0 gm

Ammonium Sulphate (NH4SO4) 0.5 gm

Potassium Chloride (KCl) 0.2 gm

Magnesium Sulphate (MgSo4. 7H20) 0.1 gm

Manganese Sulphate (Mn So4) Trace

Ferrous sulphate (Fe So4) Trace

Yeast Extract 0.5 gm

Agar 15 gm

Distil water to adjust the volume

pH 7.0

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Kenknight and Munaiers medium (Kenknight, 1939) for actinomycetes.

Quantity/L

Dextrose 1.0 gm

Potassium Hydrogen Phosphate (KH2PO4) 0.1 gm

Sodium Nitrate (NaNo3) 0.1 gm

Potassium Chloride (KCl) 0.1 gm

Magnesium Sulphate (MgSo4. 7H20) 0.1 gm

Agar 15 gm

Distil water to adjust the volume

pH 7.0-7.2

5.2.2 Isolation of possible antagonistic phosphate solubilising microbes

One ml of the soil suspension of 10-5

dilution prepared in sterilized water was

inoculated on solid medium and then incubated for 4 days at 30°C. Growth of the colonies of

Phosphate solubilizing microorganisms were routinely screened by a plate assay method

using Pikovskaya (PVK) agar (Pikovskaya, 1948). Relative efficiency test of the isolated

strains was carried out by selecting the microorganisms capable of producing a halo/clear

zone on plate due to the secretion of organic acids into the surrounding medium as described

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by Katznelson, et al. (1962). The isolates were inoculated on (PVK) agar plate by spotting

and the plates were incubated at 300C for 24 to 48 hrs. The isolates showing clear zone of

solubilisation around the colony were considered as P solubilizers. The diameter of the zone

of solubilization was measured in centimetres, and the results were expressed as

solubilization efficiency (E) after Nguyen et al., (1992).

Solubilization diameter

E = ------------------------------ x 100.

Growth diameter

The isolates showing maximum phosphate solubilizing efficiency were selected for

further studies. Single cell culture was obtained by repeated streaking in petri plates

containing PDA medium. The colonies not forming clear zone were also selected, assuming

them as (PSB), since they repeatedly grew well in Pikovskaya‘s medium.

5.2.2.1 Morphological study of the PSB

Morphological characterization of the phosphate solubilizers were done as per the

procedures given by Anon (1957) and Bartholomew and Mittewer (1950), for which all PSB

isolates were examined for the colony morphology, cell shape and gram reaction.

5.2.2.1.1 Gram staining

Selected pure phosphate solubilizer isolates were picked from the samples and were

Gram stained. Samples were heat fixed, stained with crystal violet, iodine solution, 95%

ethanol and saffranin solution respectively for 60 sec each with a 5 sec wash interval with

water following Madigan et al.,(1998). Samples were air dried and visualised under a

compound microscope. Recipes of stains and a detailed protocol are outlined in Appendix.

5.2.3 Isolation of possible antagonistic actinomycetes

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One g of thoroughly mixed soil sample was suspended in 100 ml sterile distilled

water and incubated in an orbital shaking incubator at 28°C with shaking at 140 rpm for 30

min. Mixtures were allowed to settle and serial dilutions upto 10-4

were prepared. 1 ml from

10-4

dilution was poured in petriplates (in triplicates) on Kenknight‘s medium and then

incubated at 28°C for 10 days following El-Nakeeb and Lechevalier (1963), Kuster and

Williams, (1964). Individual colonies with characteristics of actinomycetes morphology were

isolated and obtained in pure culture by repeated streaking on PDA plates. The pure isolates

were transferred to PDA slants and preserved at 4°C for further experimentation.

Morphological, cultural and physiological properties of the isolated actinomycetes strains

were studied according to the methods of Shirling and Gotlieb (1966). Actinomycete isolates

were characterized morphologically to the genus level using cover slip method, followed by

comparing the morphology of spore bearing hyphae with entire spore chain as described in

Bergey‘s manual by Locci (1989).

5.2.3.1 Morphological and cultural characterization of actinomycetes

Morphological and cultural characters of the selected actinomycetes strains were

studied by inoculating the selected strain into sterile Kenknights media. The media were

sterilized and poured into sterile petri dish. After solidification of the media, the culture of the

selected strain was streaked on the media surface aseptically and incubated at 27 °C for 7

days. Morphological properties such as colony characteristics, type of aerial hyphae, growth

of vegetative hyphae, pigment production and gram reaction were observed following Haque

et al.,(1992); Yang and Chen ,(1995).

5.2.3.1.1 Cover slip method

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Micromorphological study of the actinomycetes isolates was done by using cover slip

method in which individual cultures were transferred to the base of the cover slips buried in

PDA medium as described by Williams and Cross (1971). The cover slips were inserted at

450 angle on solid media inoculated with actinomycetes strains. The mycelia growth with

branched filaments and sporulating body on the medium extended up to the cover slips. The

cover slips were removed carefully after 8 days of incubation, stained properly and their

detail morphology was observed under microscope using 100 × magnifications.

5.2.3.1.2 Gram staining method

Smears of the selected strains were prepared on a clean glass slide and were allowed

to air-dry and then fixed heat. The heat-fixed smear was flooded with crystal violet and after

one minute, it was washed with water and flooded with mordant Gram‘s iodine. The smears

were decolorized with 95 % ethyl alcohol, washed with water and then counter-stained with

safranin for 45 secs. After washing with water, the smears were dried with tissue paper and

examined under oil immersion (100 xs).

5.2.4 Maintenance of working cultures

The selected PSB and actinomycetes strains were maintained as slant cultures for

routine use. For this, PDA (Peeled potatoes-200gm, Dextrose- 20gm, Agar-15gm and

Distilled water -1000ml) was poured in test tubes, autoclaved at 15 lb pressure for 20 min,

and were allowed to solidify. Pure cultures of each microbial strain were transferred to

solidified agar slants by means of a sterile wire loop. These were then incubated for 24 hrs at

370C and then stored at 4

0C, slant cultures were refreshed every 2 weeks.

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5.3 Results

5.3.1 Isolation of possible antagonistic PSB

Phosphate solubilizing bacterial strains was isolated from the tea estate soils of the

study area using the Pikovskaya agar medium and the isolates showing a clear halo around

the bacterial growth were identified as phosphate (P) solubilizers. A total of 12 PSB isolates

with larger halo sizes (solubilisation efficiency>25%) were selected among the identified

PSB 6 numbers of strains were isolated from rehabilitated soil, 2 from non-rehabilitated soil,

2 from virgin and another 2 from disease infested soil. Among them, 6 of the strains were

isolated from SBZ, 3 strains from UBZ and other 3 strains from NBZ during both summer

and winter (Table 5.1). Among the 12 isolated PSB strains, p-solubilising zone was found to

be maximum in the strains MM/PH/BST and MM/PH/KMP with 0.82 cm and 0.78 cm

respectively, and the lowest were observed in MM/PH/MB-2 and MM/PH/LKS with 0.20cm

each (Table 5.1). Thus solubilization efficiency was recorded highest in MM/PH/BST with

136.5% followed by MM/PH/KMP with 134.48%.

5.3.1.1 Morphological study of the phosphate solubilizing bacteria

The morphological characteristics of the isolated strains of PSB are presented in Table

5.2. The colours of the colonies were shades of white, cream and yellow. None of the strains

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produced any pigments. Most of the strains formed round, smooth and opaque type of

colonies.

By gram‘s staining and microscopic observation, it was confirmed that out of

12 PSB isolates, 8 of them are gram-negative and 4 are gram positive in nature. They were of

different shapes, such as rod (10 numbers) and cocci (2 numbers) (Table5.2).The photographs

of some of the isolated PSB strains are given in Fig 5.1

5.3.2. Isolation of possible antagonistic actinomycetes

The details of the isolated actinomycetes strains from the tea soils of the study

area are presented in the Table 5.3. In accordance with differences in the colony morphology,

total 17 actinomycetes strains were isolated from soil samples. The strains were purified by

visual, microscopic and cultivation.methods and were maintained on the same media, which

was used for isolation, at 4oC. The range of actinomycetes population was from 1.3×10

4cfu/g

to 9.0×104cfu/g most of the population were isolated from the rehabilitated and non-

rehabilitated soils. Among the 17 isolated strains, 9 were isolated from rehabilitated soil, 6

from non-rehabilitated soil, 1 from virgin soil and 1 from disease free area soil. Maximum

numbers of strains were isolated from UAZ (10 numbers).

5.3.2.1 Morphological and cultural characterization of actinomycetes

Morphology of the actinomycete colonies were determined in the selective

medium, where they had been isolated. All isolates grew on Kenknights agar media showing

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typical morphology of actinomycetes. The colonies were slow growing, aerobic, glabrous,

folded and with aerial and substrate mycelia of different colours. All actinomycetes isolates

were found to be Gram- positive and showed branched mycelia in their cell morphology

similar to fungal characters as described by Holt (1989). The cultural characteristics (Pigment

production) and morphological characteristics of different actinomycetes isolates are

presented in (Table 5.4). Among the 17 isolates, the myceliums of 6 strains were substrate

type and others were with aerial type mycelia. The colour of the substrate mycelium and

aerial spore mass was varied and 9 strains produced pigmentation of reddish, brownish and

yellowish colour. Photographs of some of the isolated actinomycetes are given in Fig 5.2

5.4 Discussion

Tea soil contains a variety of microorganisms comprising bacteria, fungi,

actinomycetes, azotobacter, azospirillum and phosphate solubilizers and most of which are

beneficial to plant growth (Baby et al., 2002).

Tea soils are generally rich in phosphate. Presence of PSB has been reported in the tea

soil (Patgiri and Bezbaruah, 1990) and the strains of Bacillus and Pseudomonas were also

found to be inhibitory to phytopathogenic fungi (Kumar and Bezbaruah 1996, 1997).

Presences of actinomycetes having antifungal activity were also previously isolated from the

tea soil (Ponmurugan et al., 2007).

On the basis of their characteristics of phosphate solubilising zone (Sundara Rao and

Sinha, 1963), 12 PSB have been isolated from the tea soil of 19 different tea estates of

Brahmaputra valley in the present study. It was observed that the phosphate solubilizing

efficiency was within the range 32.25-136.5 %.The results showed a wide range of variations

in phosphate solubilizing efficiency which are in proximity to some previous studies in tea

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soils (Kapoor et al., 1989., Singh and Kapoor, 1994).The present study also revealed that

population density of PSB in the soil samples, varied from 0.6-5.4 x 105 cfu/g, which are also

concomitant to previous reports by Venkateswarlu et al. (1984).

Tea soil of the present study area may be ideal for actinomycetes growth as it

generally grow in acidic soil (Stackebrandt et al., 1991). The present study revealed presence

of 17 actinomycetes strains with typical morphological and cultural characteristics in tea soils

of selected tea gardens of the study area. The range of actinomycetes population density was

from 1.3-9.0×104

cfu/g, which comes close to the findings of Ponmurugan et al. (2007). The

actinomycetes were selected on the basis of their inhibition to other microbes in the total

plate count PDA plates.

Much of the work on tea soils have been done on fungi, nitrogen fixers, and

mycorrhiza. However, scientific information on the use of PSB and actinomycetes in tea is

very few. No reports have been recorded till now on the isolation of PSB and actinomycetes

strains especially from rehabilitated or non-rehabilitated soil and most of the PSB and

actinomycetes were previously isolated from rehabilitated area. In the present study much of

the PSB strains were isolated from UAZ while more numbers of actinomycetes were isolated

from SBZ.

PSB and actinomycetes are potential sources of antagonistic agent that can profitably

be utilized in development of bio-inoculants. Use of bio-inoculants against soil-borne

pathogens of tea plants becomes a promising area of research leading to eradication of soil-

borne tea diseases thereby enhancing the growth and yield of the crop. The stain isolated

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from the tea soil of the present study provides scope for further investigation in the area of

bio-inoculants in tea plantation.

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Table 5.1: Phosphate solubilizing bacterial strains isolated from tea soil of

different zones subjected under the investigations

R=Rehabilitated areas, NR=Non-Rehabilitated areas, V=Virgin areas, CA=Charcoal stump

rot affected areas, BA=Brown root rot affected areas.

Sl

No.

Strain code Name of the

T.E

Type of

land

pattern

Season Population

density

(×105

cfu/gm)

Phosphate

Solubilizing

zone(cm)

Solubilization

Efficiency

(%)

1 MM/PH/DLJB-1 Dalowjan NR Winter 2.4 0.51 72.85

2 MM/PH/DLJB-2 Dalowjan R Summer 1.2 0.54 90

3 MM/PH/GPB Gabroopurbat R Winter 2.2 0.39 70.9

4 MM/PH/BST Ghillidary R Summer 0.6 0.82 136.5

5 MM/PH/KC Kakojan CA Winter 1.67 0.27 43.50

6 MM/PH/TY Tyroon BA Winter 0.67 0.49 89.09

7 MM/PH/KMP Kolony R Summer 1.42 0.78 134.48

8 MM/PH/MB-1 Monobug R Summer 4.40 0.25 49.01

9 MM/PH/ MB-2 Monobug V Summer 3.4 0.20 32.25

10 MM/PH/BZL Bazaloni R Summer 2.6 0.46 70.76

11 MM/PH/LKS Lankasi NR Summer 5.4 0.20 47.60

12 MM/PH/SV Sookerating V Summer 4.4 0.37 86.04

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Table 5.2: Morphological characteristics of phosphate solubilizing bacteria isolates.

SL no. Isolates Colony Morphology Cell shape Gram reaction

1 MM/PH/DLJB-1 Creamy, round Rod -ve

2 MM/PH/DLJB-2 White round ,transparent, smooth

Rod -ve

3 MM/PH/GPB Creamy, raised

centre,round,opaque

Rod -ve

4 MM/PH/BST Cream spreading, opaque Rod +ve

5 MM/PH/KC Dull white ,irregular, spreading

cocci -ve

6 MM/PH/TY White, round ,opaque,

smooth

Rod -ve

7 MM/PH/KMP light yellow, round, opaque

Rod +ve

8 MM/PH/MB-1 White,circular,flat,

transparent

Rod +ve

9 MM/PH/ MB-2 White, round, smooth. cocci -ve

10 MM/PH/BZL Creamish, circular,

opaque.

Rod -ve

11 MM/PH/LKS White, brownish margin,

flat.

Rod -ve

12 MM/PH/SV Yellow, smooth, round. Rod +ve

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Table 5.3: Actinomycetes strains isolated from tea soil of different zones subjected

under the investigations

Sl

No.

Strain code Name of the T.E Type of

land pattern

Season Population

density (×105

cfu/gm)

1 MM/PH/AC-01 Sookerating R Summer 6.4

2 MM/PH/AC-02 Budlabheta R Summer 4.6

3 MM/PH/AC-03 Gabroopurbat NR Summer 1.6

4 MM/PH/AC-04 Dherai NR Summer 1.3

5 MM/PH/AC-05 Budlabheta R Summer 4.6

6 MM/PH/AC-06 Lankasi NR Summer 4.4

7 MM/PH/AC-07 Sookerating R Summer 6.4

8 MM/PH/AC-08 Dalowjan R Summer 2.6

9 MM/PH/AC-09 Dhulapadung NR Winter 6.3

10 MM/PH/AC-10 Bazaloni V Summer 4.3

11 MM/PH/AC-11 Monobug R Winter 2.6

12 MM/PH/AC-12 Budlabheta R Winter 2.4

13 MM/PH/AC-13 Lankasi R Summer 3.6

14 MM/PH/AC-14 Gabroopurbat R Summer 9.0

15 MM/PH/AC-15 Sookerating NR Summer 5.6

16 MM/PH/AC-16 Lankasi NR Summer 4.4

17 MM/PH/AC-17 Lattekujan NIR Winter 7.0

R=Rehabilitated areas, NR=Non-Rehabilitated areas, V=Virgin areas,

NIR= Non-infested area of charcoal affected region

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Table 5.4: Morphological characteristics of Actinomycetes isolates.

Sl

No.

Isolates Colour Mycelium

type

Pigment

production

Grams

reaction

1 MM/PH/AC-01 Light greyish Substrate Red +

2 MM/PH/AC-02 Dark grey aerial Reddish brown +

3 MM/PH/AC-03 Brown aerial Light brown +

4 MM/PH/AC-04 White aerial Yellow +

5 MM/PH/AC-05 Yellow substrate Nil +

6 MM/PH/AC-06 Grey substrate Red +

7 MM/PH/AC-07 Pink substrate Brown +

8 MM/PH/AC-08 White aerial Red +

9 MM/PH/AC-09 Whitish grey aerial Nil +

10 MM/PH/AC-10 Pinkish white aerial Nil +

11 MM/PH/AC-11 Yellow aerial Yellow +

12 MM/PH/AC-12 Grey substrate Nil +

13 MM/PH/AC-13 Green substrate Pale yellow +

14 MM/PH/AC-14 Grey aerial Nil +

15 MM/PH/AC-15 Greyish violet aerial Nil +

16 MM/PH/AC-16 Greyish white aerial Nil +

17 MM/PH/AC-17 White aerial Nil +

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Fig 5.1: isolates of Phosphate solubilising bacteria

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Fig 5.2: Isolates of Actinomycetes