results and discussion - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/29464/9/09... ·...

Results and Discussion

A. Fungal Diversity During Different Seasons

1. Summer season

A total of 54 species belonging to 16 genera and two sterile mycelia were isolated.

Among them Deuteromycotina fungi dominated over the member of Ascomycotina and

Zygomycotina. Out of total isolates 4 species (3 genera) belong to Zygomycotina, 2 species

(2 genera) belong to Ascomycotina and 48 species (12 genera) belong to Deuteromycotina.

(Table-1).

In the cave Aspergillus taxa were found dominant. The genus Aspergillus were

represented by 16 species, Penicillium was represented by 10 species, Curvularia by 4

species, Fusarium and Cladosporium by 3 species, Alternaria, Dreshclera, Rhizopus,

Trichoderma by 2 species and Periconia, Monilia, Mucor, Choanephora cucurbatarum,

Emericella nidulans, Phanerochaete chrysosporium, Talaromyces flavus represented by one

species.

From different sources maximum diversity was found from guano deposits (29

species from 65 colonies) followed by leaf litter deposits (25 species from 62 colonies) and

logs and twigs deposits (22 species from 61 colonies).

Table – 1: Fungal isolates from different sources (summer season).

Name Of Fungi Guano

deposits

Leaf litter

deposits

Log and twig

deposits

Zygomycotina Colony

number

Colony

number

Colony number

Choanephora Cucurbitarum 1

Mucor hemalis 2

Rhizopus oryzae 1

Rhizopus stolonifer 1

Ascomycotina

Emericella nidulans 1 1

Talaromyces flavus 2

Deuteromycotina

Alternaria alternata 2 1 4

Alternaria crassa 1

Aspergillus albus 2

Aspergillus astus 4

Aspergillus aureus 2

Aspergillus awamoori 1

Aspergillus flavus 1

Aspergillus fumigatus 2 2

Aspergillus japonicus 3

Aspergillus luchensis 2 1

Aspergillus nidulans 5

Aspergillus niger 2 4 2

Aspergillus parasiticus 4 2

Aspergillus sulphureus 1 1

Aspergillus sydowii 2

Aspergillus terreus 1 1

Aspergillus versicolor 3 2

Aspergillus sp I 2

Cladosporium cladosporides 3 14

Cladosporium oxysporium 2 9

Cladosporium-sphaerospermum 10

Curvularia clavata 4 5

Curvularia geniculata 2 2

Curvularia pallecense 2

Curvularia lunata 1 1

Drechslera bicolor 4

Drechslera tetramer 1

Fusarium oxysporum 1

Fusarium moniliform 1

Fusarium caucasicum 2

Monilia sp 1

Penicillium brevicompactum 2 4 1

Penicillium citrinum 5 2 3

Penicillium crysogenum 2

Penicillium digitatum 2

Penicillium meleagrinum var.

viridiflavum

1 3

Penicillium multicolor 1 1

Penicillium notatum 2 3

Penicillium oryzae 2 2

Penicillium rubrum 3

Penicillium rugulosum 1 2

Periconia sp 1

Phanerochaete chrysosporium 2

Trichoderma viridae 1

Trichoderma atroviridae 1

Mycelia sterilia

Mycelia sterilia (White) 4

Mycelia sterilia (Peach) 3

Fig- 1: Species count in summer season.

Fig – 2: Dominant fungal groups in summer season.

Fig – 3: Colony count in summer season.

2. Post rainy season

0

5

10

15

20

25

30

Guano Deposit

Leaf Litter

deposit

log and twig

deposits Sp

ecie

s co

un

t in

su

mm

er

seaso

n

Sample name

species count

7%

3%

86%

4%

Dominant fungal groups in summer season

Zygomycotina Ascomycotina

Deuteromycotina Mycelia sterilia

38%

33%

29%

Colony count of fungi in summer season

Guano Deposit Leaf Litter deposit log and twig deposits

A total of 71 species belonging to 24 genera and two sterile mycelia were isolated.




(Table-3).

During post rainy season Aspergillus taxa were found dominant. The genus

Aspergillus were represented by 21 species, Penicillium was represented by 12 species,

Curvularia, alternaria and Cladosporium by 4 species, Fusarium and by 3 species,

Dreshclera, Rhizopus, Trichoderma, Mucor and Acremonium by 2 species and Periconia,

Monilia,, Choanephora cucurbatarum, Emericella nidulans, Phanerochaete chrysosporium,

Talaromyces flavus, Circinella simplex, Cunninghamella elegans, Absidia Corymbifera,

Thielavia basicola, Nigrospora, Chaetomium globussum, Aureobasidium pullalans

represented by one species.

From among different resources maximum diversity was found from guano deposits (51

species from 90 colonies) followed by leaf litter deposits (47 species from 88 colonies) and

logs and twigs deposits (30 species from 43 colonies) (Table- 2).

Table- 2: Fungal isolates from different sources (post rainy season).

Name Of Fungi Guano

deposits

Leaf litter

deposits

Log and twig

deposits

Colony

number

Colony

number

Colony

number

Absidia corymbifera 1 2

Choanephora cucurbitarum 1

Circinella simplex 1

Cunninghamella elegans 2 1

Mucor hemalis 1 1

Mucor racemosum 2 1

Rhizopus oryzae 1

Rhizopus stolonifer 1

Ascomycotina

Chaetomium globussum 1 2



Thielavia basicola 2

Deuteromycotina

Acremonium persicinum

2

Acremonium strictum 2


Alternaria crassa 2 1

Alternaria radicina 1 1

Alternaria spI 1 1

Aspergillus albus 2 1 1

Aspergillus astus 2

Aspergillus aureus 1

Aspergillus awamoori 2 1 1


Aspergillus fumigatus 2 1 2

Aspergillus japonicus 1 2

Aspergillus luchensis 3 1 1



Aspergillus niveus 2

Aspergillus ochraceous 2 1

Aspergillus parasiticus 2 2 1

Aspergillus phoensis 1 1

Aspergillus sclerotium 1 1

Aspergillus stellatus 1

Aspergillus sulphureus 2 1

Aspergillus sydowii 1 3

Aspergillus terreus 2 1

Aspergillus versicolor 3

Aspergillus sp I 2

Aureobasidum pullalans 2

Cladosporium

cladosporides

6 10 2

Cladosporium oxysporium 8 5 2

Cladosporium-

sphaerospermum

5 1

Cladosporium herbarum 1 1

Curvularia clavata 1

Curvularia geniculata 2

Curvularia pallecense 2 2

Curvularia lunata 1 1 2

Drechslera bicolor 1

Fig- 4: Species count in post rainy season.

0

10

20

30

40

50

60

Guano Deposit Leaf Litter deposit

log and twig deposits

Sp

ecie

s co

un

t in

Po

st r

ain

y

seaso

n

Sample name

Drechslera tetramer 1

Fusarium oxysporum 1

Fusarium moniliform 1 1

Fusarium caucasicum 1

Monilia sp 1 1 1

Nigrospora oryzae 2

Penicillium brevicompactum 2 2 2


Penicillium crysogenum 2


Penicillium meleagrinum

var. viridiflavum

3 1 2

Penicillium multicolor 2 1

Penicillium notatum 1

Penicillium oryzae 2

Penicillium oxalicum 2

Penicillium purpogenum 1

Penicillium rubrum 2 2

Penicillium rugulosum 2

Periconia sp 1

Phanerochaete

chrysosporium

2 3

Trichoderma viridae 1 2

Trichoderma atroviridae 1 1

Mycelia sterilia

Mycelia sterilia (White) 2 1

Mycelia sterilia (Orange) 1

Fig- 5: Dominant fungal groups in post rainy season.

Fig – 6: Colony count of fungi in post rainy season.

3. Winter season

A total of 59 species belonging to 21 genera and four sterile mycelia were isolated.




(Table- 2).

During post rainy season Aspergillus taxa were found dominant. The genus Aspergillus

were represented by 17 species, Penicillium was represented by 12 species, Curvularia by 4

species, Alternaria and Fusarium by 3 species, Cladosporium, Acremonium and

11% 5%

81%

3%

Dominant fungal groups in post rainy

season Zygomycotina Ascomycotina Deuteromycotina Mycelia sterilia

43%

37%

20%

Colony count of fungi in post rainy

season Guano Deposit Leaf Litter deposit log and twig deposits

Trichoderma by 2 species, Dreshclera, Rhizopus, Trichoderma, Mucor and Acremonium by

2 species and Monilia, Emericella nidulans, Phanerochaete chrysosporium, Talaromyces

flavus, Circinella simplex, Cunninghamella elegans, Absidia Corymbifera, Thielavia

basicola, Nigrospora, Chaetomium globussum and Aureobasidium pullalans represented by

one species.

From among different resources maximum diversity was found from guano deposits (46

species from 81colonies) followed by leaf litter deposits (40 species from 91 colonies) and

logs and twigs deposits (21 species from 31 colonies) (Table – 3).

Table-3: Fungal isolates from different sources (winter season).

Name Of Fungi Guano

deposits

Leaf Litter

deposits

Log and twig

deposits

Zygomycotina Colony

number

Colony

number

Colony

number

Absidia corymbifera 1 `

Circinella simplex 2 1

Cunninghamella elegans 1

Mucor hemalis 1 2

Ascomycotina

Chaetomium globussum 1



Thielavia basicola 1

Deuteromycotina

Acremonium persicinum 1

Acremonium strictum 1 1


Alternaria crassa 2 1 1

Alternaria radicina 2

Aspergillus albus 1 1

Aspergillus astus 2

Aspergillus aureus 2 1

Aspergillus awamoori 2 2 1


Aspergillus fumigatus 2 3 2

Aspergillus japonicus 1 1

Aspergillus luchensis 2 2 1



Aspergillus ochraceous 1 1

Aspergillus phoensis 2 2

Aspergillus sclerotium 1

Aspergillus stellatus 2

Aspergillus sulphureus 1 1 1

Aspergillus terreus 1

Aspergillus versicolor 1

Aureobasidum pullalans 1

Cladosporium oxysporium 5 5

Cladosporium herbarum 10 5

Curvularia clavata 3 2

Curvularia geniculata 3

Curvularia pallecense 1 2 1

Curvularia lunata 2 2

Drechslera bicolor 1 2

Drechslera tetramer 1 4

Fusarium oxysporum 1 2

Fusarium moniliform 2 1

Fusarium caucasicum 1 2

Monilia sp 3

Nigrospora oryzae 1

Penicillium brevicompactum 1


Penicillium crysogenum 3 2



viridiflavum

3 4 3

Penicillium multicolor 2 2 1

Penicillium notatum 2

Penicillium oryzae 1 3

Penicillium oxalicum 1 1

Penicillium purpogenum 1

Penicillium rubrum 2 1

Penicillium rugulosum 1 1

Phanerochaete chrysosporium 2

Trichoderma viridae 2 1 2

Trichoderma atroviridae 3 2

Mycelia sterilia

Mycelia sterilia (White) 2

Mycelia sterilia (Orange) 2

Mycelia sterilia (Peach)

1

Mycelia sterilia (Dull White) 2

Fig – 7: Species count in winter season.

Fig – 8: Dominant fungal groups in winter season .

Fig- 9: Colony count in winter season.

Percentage Frequency

0

10

20

30

40

50

Guano Deposit Leaf Litter

deposit log and twig deposits S

pec

ies

cou

nt

in w

inte

r se

aso

n

Sample name

7% 6%

81%

6%

Dominant fungal groups in Winter

Season

Zygomycotina Ascomycotina

Deuteromycotina Mycelia sterilia

40%

45%

15%

Colony count of fungi in winter

season

Guano Deposit Leaf Litter deposit log and twig deposits

During study maximum percentage frequency was shown by Alternaria alternata,

Aspergillus niger and penicillium citrinum (100%) followed by Penicillium meleagrinum

var. viridiflavum and Aspergillus fumigatus (90%), Penicillium brevicompactum,

Curvularia lunata, Cladosporium oxysporium, Aspergillus awamoori, Aspergillus

sulphureus (78%), Alternaria crassa, Emercilla nidulans (66%), Emericella nidulans and

Penicillium rugulosum (66%) Curvularia clavata, Cladosporium cladosporides, Aspergillus

parasiticus and penicillium rugulosum (57%). Rest fungal species had shown below 50% of

frequency.

Fig – 10: Percentage frequency of fungal species in all three seasons.

Percentage Density

During study maximum percentage density had shown by Cladosporium oxysporium

(5.88%) ,Cladosporium cladosporides (5.71%), penicillium citrinum (3.75%), Aspergillus

niger (3.43%), Penicillium meleagrinum var. viridiflavum (3.26%), Alternaria alternata and

Aspergillus fumigatus (2.61%), Curvularia clavata (2.45%), Penicillium brevicompactum

(2.28%), Aspergillus awamoori and Aspergillus parasiticus (1.79%), Curvularia lunata

0%

20%

40%

60%

80%

100%

120%

(1.6%), Aspergillus sulphureus, Alternaria crassa and Emericella nidulans (1.30%)

and Penicillium rugulosum (1.14%).

Fig- 11: Percentage density of fungal species in all three seasons.

B. Bacterial Diversity During Different Seasons

1. Summer Season

A total of 25 isolates were isolated during summer season. From among different

sources maximum diversity was found from Guano deposits (20 species) followed by leaf

litter deposits (19 species) and log and twig deposits (14 species), (Table- 4).

Cream colony I, Brevundimonas diminuta, Bacillus thuringiensis, Bacillus cereus,

Cream rough colony II, Cream rough colony III, Orange colony, Transparent colony 4 were

dominant and isolated from all three samples.

2. Post Rainy Season

0.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

A total of 32 isolates were isolated during post rainy season. From among different

sources maximum diversity was found from Guano deposits (25 species) followed by leaf

litter deposits (23species) and log and twig deposits (19 species), (Table- 4).


Transparent colony II, Whitish cream colony, Creamish yellow colony II, Lemon yellow

colony I and Transparent colony 4 were dominant and isolated from all three samples.

3. Winter Season

A total of 28 isolates were isolated during post rainy season. From among different

source maximum diversity was found from Guano deposits (22 species) followed by leaf

litter deposits (21species) and log and twig deposits (13 species), (Table- 4).


Transparent colony 4, Light yellow colony II, White colony III, Yellowish rough colony

were dominant and isolated from all three samples.

Table- 4: Bacterial isolates from different sources in different seasons

Isolated

Bacteria

Summer season Post rainy season Winter season

Number of colony

Number of colony

Number of

colony

Guano

deposits

Leaf

litter

deposits

Log and

twig

deposits

Guano

deposits

Leaf

litter

deposits

Log and

twig

deposits

Guano

deposits

Leaf

litter

deposits

Log and

twig

deposits

Bacillius

cereus

++ + ++ ++ ++ + ++ +

Bacillus

thuringiensis

++ +++ ++ ++++

Brevundimona

s diminuta

++++ ++ + + +++ + ++ ++ ++

Brown smooth + ++ + +

colony

Cream colony

4

+ +

Cream colony

I

++ ++ ++ +++ + + + ++ +

Cream colony

II +++ + ++ + +

Cream colony

III

+ +++ ++

Cream rough

colony I

++ + +++ +

Cream rough

colony II

+ + + ++ + ++

Cream rough

colony III

++ + ++ +++ ++ ++ +

Creamish

yellow colony

+ ++ + ++++ +++ +

Creamish

yellow colony

II

+ + ++ ++

Lemon yellow

colony I

++ ++++ + ++ + +

Lemon yellow

colony II

++ + ++ + + +

Light pink

colony

+ +

Light yellow

colony I

+ +

Light yellow

type II

+++ + + ++ + +

Milky white

colony

++ ++ ++

Orange colony + + + ++ ++

Pink colony + + +

Transparent

colony 4

+ ++ + + + + + + +

Transparent

colony I

++ ++ + +

Transparent

colony II

++ +++ ++ ++ + ++ +

Transparent

colony III

+ ++ ++ +

White colony I ++ + + +

White colony

II

++ +++ ++ ++

White colony

III

++ + ++ ++ +

White rough

colony

+ + ++ +

Whitish cream

colony

++ ++ + + ++ ++ +

Yellowish

rough colony

++ ++ ++ + + ++ +

Yellowish

smooth colony

+ + + ++ + + +

+ = poor, ++ = moderate, +++ = good, ++++ = excellent.

Table – 5: Colony morphology and cell morphology of isolated bacteria Isolated bacteria Colony morphology Cell morphology

Colour Size Shape Texture Cell

shape

Cell

grouping

Gram

reaction

Bacillus cereus Dull white Small Irregular Rough Rod In chain Positive

Bacillus

thuringiensis

White Medium Rounded Smooth Rod Single Positive

Brevundimonas

diminuta

Light brown Small Rounded Smooth Rod In chain Negative

Brown rough

colony

Brown Large Irregular Rough Rod In chain Positive

Brown smooth

colony

Brown Small Rounded Smooth Spiral Irregular

colony

Negative

Cream colony I Cream Small Rounded Smooth Rod In chain Negative

Cream colony III Cream Small Irregular Rough Round Paired Positive

Cream colour II Cream Medium Irregular Rough Rod Irregular

colony

Negative

Cream rough

colony I

Cream Large Irregular Rough Round Single Negative

Cream rough II Cream Large Irregular Rough Round Single Negative

Cream rough III Cream Small Irregular Rough Cocci In chain Positive

Creamish yellow

colony

Creamish

yellow

Small Rounded Smooth Round In chain Negative

Creamish yellow

colony II

Creamish

yellow

Small Rounded Smooth Elliptical Irregular

colony

Negative

Lemon yellow

colony I

Lemon

yellow

Small Rounded Smooth Rod Irregular

colony

Negative

Lemon yellow

colony II

Lemon

yellow

Medium Rounded Rough Rod Paired Negative

Light pink colony Light pink Medium Irregular Rough Round Paired Positive

Light yellow

colony I

Light yellow Small Irregular Smooth Round Single Positive

Light yellow

colony II

Light yellow Medium Rounded Smooth Rod In chain Negative

Milky white

colony

White Large Rounded Smooth Rod Irregular

colony

Negative

Orange colony Orange Medium Rounded Smooth Round Tetrad Positive

Pink colony Pink Small Rounded Smooth Rod In chain Negative

Transparent 4 Transparent Medium Irregular Smooth Cocci Irregular

colony

Positive

Transparent colony

I

Transparent Small Rounded Smooth Round Irregular

colony

Negative

Transparent colony

II

Transparent large Rounded Smooth Rod Single Negative

Transparent colony

III

Transparent Small Rounded Smooth Rod In chain Negative

White colony I White Medium Rounded Smooth Rod Single Negative

White colony II White Large Rounded Rough Rod Single Positive

White colony III White Small rounded Smooth Round Irregular

colony

Negative

White rough

colony

White Medium Irregular Rough Round Single Positive

Whitish cream Whitish Large Irregular Rough Rod In chain Positive

colony yellow

Yellowish rough

colony

Yellowish Medium Rounded Rough Round Single Positive

Yellowish smooth

colony

Yellowish Small Rounded Smooth Round In chain Negative

Fig-12: Species count in summer season from different sources.

Fig-13: Species count in post rainy season from different sources.

0

5

10

15

20

25

Guano deposits

leaf litter deposits

Log and twig deposits Sp

eci

es

Co

un

t In

Su

mm

er

Seas

on

Sample Name

0

5

10

15

20

25

30

Guano deposits leaf litter deposits

Log and twig deposits

Sp

ecie

s co

un

t in

po

st r

ain

y

seaso

n

Sample Name

Fig-14: Species count in winter season from different sources.

Fig-15: Percentage occurrence of gram negative and positive bacteria.

Percentage Frequency

During study maximum percentage frequency was shown by Bacillus cereus,

Bacillus thuringiensis, Brevundimonas diminuta, Cream colony I and Transparent colony 4

(100%) followed by Yellowish rough colony, Yellowish smooth colony, Whitish cream

colony, Transparent colony II and Cream rough colony III (78%), Cemon yellow colony I,

Lemon Yellow colony II, Light yellow colony II, Cream rough colony II and Creamish

yellow colony (66%). Result Bacterial species had shown below than 50% of frequency.

0

5

10

15

20

25

Guano deposits leaf litter deposits Log and twig deposits

Sp

ecie

s co

un

t in

win

ter

seaso

n

Sample name

59%

41%

Percentage occurence of gram

negative and positive bacteria

Gram negative bacteria Gram positive bacteria

Fig- 16: Percentage Frequency of bacterial species in all three seasons.

The results revealed that guano of bat are mostly preferred by some members of

Deuteromycotina and Zygomycotina viz species of Aspergillus, Penicillium, Alternaria,

Curvularia and Mucor. It may be due to higher percentage of partially digested cellulose,

lignin and pectin. The alimentary canal of bat provide shelter to various types of symbiotic

and commensale bacteria which help in digesting the complex organic materials. On the

other hand the members of Deuteromycotina viz, Cladosporium, Fusarium, Phanerochaete,

Trichoderma also preferred to grow over leaf litter and log and twig deposits that contain a

greater percentage of lignin and cellulose indicating that these mold species are ligninolytic

as well as cellulolytic.

In the same way in bacteria also it was found that bat guano are mostly preferred

by bacteria. During studies we have also noticed that the degradation of log and twig

deposits were preferred by only few groups of fungi and bacteria, thus indicating that lignin,

(as log and twig contains more percentage of lignin) is hard to degrade because of its

complex aromatic structure. Similar work has been reported by Koilraj and his co-workers

(1999) where they studied fungal diversity inside caves of southern India. They had isolated

thirty-five species of sporulating mesophilous fungi and seven types of non-sporulating

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100%

fungi from the soil samples collected at the entrance, twilight and dark zones of six caves.

Novakova (2009) also studied diversity of fungi from Domica cave system where they have

isolated total of 195 fungal taxa from various cave substrates. Similarly diversity of

thermophilous soil microfungi in forest and cave ecosystems of Taiwan has also been

studied by Hsu and coworkers (2001). Rajput et al. (2012) isolated bacterial species from

different sediments of Kotomsar cave of Bastar, Chhattisgarh. Adetutu et al. (2011) studied

fungal diversity in areas accessible and not accessible to tourists at Naracoorte caves.

Fusarium sp., Trichoderma sp. and Neonectria sp. were only cultured form tourist

inaccessible area. Shapiro and Pringle (2010) isolated different fungal species (Bionectria,

Cladophora, Fusarium, Hypocrea, Mortierella, Paraconiothyrium, Penicillium and

Podospora) from caves in Kentucky and Tennessee.

A. Evaluation of Pectin Degradation Potency of Fungal Isolates:

To acertain pectin degradation potency of fungal isolates the organisms were first

grown separately on Hankin agar plates containing citrus pectin as sole carbon source and

incubated for 5 days. Three replicates of each organism were taken and average of growth /

digestion zone diameter were considered. To get mycelial dry weight the organisms were

grown separately in Hankin broth media containing citrus pectin as sole carbon source and

incubated for 5 days. The results are shown in Table – 6.

Table – 6: Zone of digestion and mycelial dry weight in pectin media.

Isolated fungi Zone of

digestion

(diameter)

Mycelial dry

weight per 50ml

Mycelial dry

weight per 1000ml

Alternaria alternata 40.26 mm 0.34gm 6.8 gm

Alternaria radicina 28 mm 0.12gm 2.4 gm

Aspergillus awamoori 26.74 mm 0.09gm 1.8 gm

Aspergillus japonicas 28.6 mm 0.11gm 2.2 gm

Aspergillus fumigatus 31.76 mm 0.18gm 3.6 gm

Aspergillus luchensis 30.16 mm 0.14gm 2.8 gm

Aspergillus niger 28.7 mm 0.12gm 2.4 gm

Aspergillus ochraceous 36.76mm .23gm 4.6 gm

Aspergillus parasiticus 27.5 mm 0.10gm 2.0 gm

Aspergillus stellatus 28.51mm 0.11gm 2.2 gm

Aspergillus versicolor 32.5mm 0.16gm 3.2 gm

Curvularia clavata 25.4 mm 0.07gm 1.4 gm

Curvularia lunata 26.12 mm 0.08gm 1.6 gm

Curvularia pallesence 25.4 mm 0.06gm 1.2 gm

Penicillium citrinum 36.26 mm 0.26gm 5.2 gm

Penicillium digitatum 27.43mm 0.12gm 2.4 gm


viridiflavum

37.5 mm 0.29gm 5.8 gm

Penicillium multicolor 29.19mm 0.14gm 2.8gm

Penicillium notatum 27.2 mm 0.10gm 2.0 gm

Penicillium oryzae 21.1 mm 0.05gm 1.0 gm

Penicillium rugulosum 20.46 mm 0.04gm 0.08gm

Phoma shorgina 21.51 mm 0.05gm 1.0gm

Phoma exigua 22.44 mm 0.06gm 1.2 gm

Phanerochaete crysosporium 28.4 mm 0.09gm 1.8 gm

Trichoderma viridae 31.22 mm 0.10gm 2.0 gm

Results show that out of total species studied 25 species were found to produce

pectin degrading enzyme in substantial tune. Out of 25 species, best grown species were

Alternaria alternata (plate- 2.1 and 8.1), Penicillium mealagrinum var. viridiflavum (plate-

2.3 and 8.3), Penicillium. citrinum (plate- 2.2 and 8.2), Aspergillus ochraceous, Aspergillus

fumigatus Aspergillus niger and Phanerochaete chyrsosporium. They exhibited greater

zone of digestion and highest mycelial weight, when compared with other pectinase

producing fungi.

Dube and gour (1975) studied extracellular pectic enzymes produced by

Macrophomina phaseolina on Richard's solution containing sucrose, Richard's solution

containing pectin and potato pectin medium. They found that organisms showed maximum

pectolytic enzyme secretion in the media containing pectin. Thus it indicates that pectolytic

enzymes are inductive in nature. Fonseca and Said (1999) studied effect of pectinases,

produced by Penicillium frequentans on pectin. The fungus was found to secrete one endo

and one exopolygalacturonase during the first 10 hr of incubation either in media

supplemented with pectin or in the absence of carbohydrate source. Phutela et al. (2005)

screened 120 different fungal isolates for pectinase and polygalacturonase production and

found that Aspergillus fumigatus was best for pectinase and polygalacturonase production

where as Akbar et al. (2012) isolated and screened different fungal strains from agro

industrial waste products and performed plate assay method by using pectin as sole carbon

source as well as quantitative estimation. According to their result Aspergillus carbonarius

showed maximum pectolytic activity. Patil and Choudhary (2010) worked on pectinase

producing microorganisms and observed that Pencillium sp are potent polygalacturonase

producer. In the same way Banu et al. (2010) also isolated ten fungi from municipal waste

soil samples and screened for pectinase production. They observed maximum pectinase

production by Penicillium chrysogenum. Abassi and Fazeilpoor (2010) found Aspergillus

niger as better pectinase producer then Trichoderma reesi. Akhter et al. (2011) also carried

solid state fermentation with 7 fungal strains and among them Aspergillus niger was found

as effective pectinase producer. Adeleke et al. (2012) evaluated the potency of fungi to

produce pectinase and cellulase by using orange peels as substrate. They used CMC and

citrus pectin as sole carbon source and their study showed that Penicillium atroventum,

Aspergillus Flavus and Aspergillus oryzae were highest pectinase and cellulase producer.

Okafor et al. (2010) studied pectolytic activity on Aspergillus clavatus, Aspergillus niger,

Fusarium sp., Penicillium chrysogenum and Trichoderma sp. using citrus pectin as sole

carbon source. They observed maximum pectinase production by Aspergillus niger and

Penicillium chrysogenum. Desouky (2007) isolated five fungi and screened for their ability

to produce pectinolytic and cellulolytic activity using quantitative and qualitative analysis.

They identified Penicillium hordei as best cellulase producer and Aspergillus ustus as best

pectinase producer.

B. Evaluation of Cellulose Degradation Potency of Fungal Isolates

The organisms were grown on basal agar plates containing CMC (sole carbon source)

to study zone of digestion and then grown in basal media containing CMC as sole carbon

source to obtain mycelial dry weight. Incubation period of 7 days were given for all the test

organisms. Averages of three replicates were considered for each organism. The results are

shown in Table – 7.

Table – 7: Zone of digestion / mycelial dry weight in cellulose media.

Isolated Fungi Zone of

digestion

(diameter)

Mycelial dry

weight per 50 ml

Mycelial dry

weight per

1000ml

Alternaria alternata 36.56 mm 0.08gm 1.6 gm

Alternaria crassa 27.12 mm 0.03gm 0.6 gm

Aspergillus fumigatus 32.42mm 0.06gm 1.2 gm

Aspergillus luchensis 25.4 mm 0.02gm 0.4 gm

Aspergillus niger 34.67mm 0.06gm 1.2 gm

Aspergillus ochraceous 25.4mm 0.03gm 0.6gm

Aspergillus parasiticus 32.4mm 0.05gm 1.0 gm

Curvularia lunata 26.67mm 0.03gm 0.6 gm

Penicillium brevicompactum 26.62mm 0.03gm 0.6 gm

Penicillium citrinun 42.16mm 0.14gm 2.8 gm

Penicillium crysogenum 27.49mm 0.03gm 0.6 gm


viridiflavum

43.26mm 0.15gm 3.0gm

Penicillium notatum 34.24mm 0.05gm 1.00 gm

Penicillium rubrum 29.62mm 0.04gm 0.8 gm

Phoma exigua 25.4mm 0.028gm 0.56 gm

Trichoderma atro

viridae

44.24mm 0.17gm 3.4 gm

Trichoderma viridae 45.22mm 0.19gm 3.8gm

Results show that out of total species studied 17 species were found to produce

cellulose degrading enzyme in substantial tune. Present studies revealed that Trichoderma

viridae, Trichoderma atroviridae, Penicillium meleagrinum var. viridiflavum (plate- 3.3 and

9.3), Penicillium citrinum (plate- 3.2 and 9.2), and Alternaria alternata (plate-3.1and 9.1)

possess greater cellulolytic potency as they exhibited greater zone of digestion and highest

mycelial dry weight as compared to rest of the isolated organisms. Hankin and Anagnostakis

(1976), using same method, also reported cellulolytic ability of some fungi viz.

Trichoderma viridae, Penicillium sp, Peziza ostracoderma and fusarium sp. and some

bacteria like Streptomyces, Cellulomonas etc. Kasana et al. (2008) also screened cellulase-

producing microorganisms that was routinely done on carboxymethylcellulose (CMC)

plates and were flooded either with 1% hexadecyltrimethyl ammonium bromide or with

0.1% Congo red followed by 1 M NaCl. Ghanbary et al. (2010) working on laboratory

stimulation of cellulose degradation by soil Aspergilli reported that A.niger and A.niveus

had highest ability for cellulose degradation. In present studies also 5 species of Aspergillus

were found to have cellulose degradation ability to a moderate extent. Picart et al (2008)

isolated and characterized a new fungal strain from a rotten rag, and identified it as

Stachybotrys atra. The production of cellulases by this strain was analyzed and

characterized. Ahmed et al. (2009) partially purified three cellulases namely exoglucanase

(EXG), endoglucanase (EG) and β-glucosidase (BGL) from Trichoderma harzianum.

Cellulase production by different species of Trichoderma, Fusarium, Aspergillus,

Penicillium and Alternaria alternata (mutant) were also reported by Macris (1983), Sazci et

al. (1986) and Jahangeer et al. (2005). Present studies also revealed that two species viz

Penicillium meleagrinum var. viridiflavum and p. citrinum, which were not reported earlier

as cellulase producer were found to be potent cellulose degraders. Sridevi et al. (2009)

tested Aspergillus niger for production of cellulase from sawdust, wheat straw, sugarcane

bagasse and ricebran, and concluded that wheat bran had yielded more cellulase whereas

Juwaied et al. (2011) used Aspergillus niger and Trichoderma viridae and reported

sugarcane as a potential substrate for production of cellulase. Ikeda et al. (1967, 1973) had

also reported the isolation of a homogeneous cellulolytic enzyme from A. niger.

C. Evaluation of Lignin Degradation Potency of Fungal Isolates

The organisms were grown on basal agar plates containing lignin as sole carbon source

to study colonial growth and then grown in basal media containing lignin sulphonic acid

sodium salt as sole carbon source to obtain mycelial dry weight. Incubation period of 9 days

were given for all the test organisms. Averages of three replicates were considered for each

organism. The results are shown in Table–8.

Table – 8: mycelial growth / mycelial dry weight in lignin media.

Name of fungi Growth rate Mycelial dry

weight per 50 ml

Mycelia dry weight

per 1000ml

Alternaria alternata ++ 0.10gm 2.0gm

Aspergillus fumigatus + 0.04gm 0.8 gm

Aspergillus luchensis + 0.028gm 0.56 gm

Aspergillus niger + 0.036gm 0.72 gm

Curvularia lunata + 0.03gm 0.6 gm

Fusarium caucassam + 0.02gm 0.4 gm

Penicillium citrinum ++ 0.08gm 1.6 gm

Penicillium meleagrinum

var. viridiflavum

++ 0.11gm 2.2 gm

Phanerochaete

chrysosporium

+++ 0.12gm 2.4 gm


In ligninolytic test total 9 species grown well in media containing lignin as sole

carbon source. Phanerochaete crysosporium showed best ligninolytic activity, followed by

Penicillium mealagrinum var. viridiflavum (plate – 4.3 and 10.3) ,Alternaria alternata

(plate- 4.1 and 10.1) and Penicillium citrinum (plate- 4.2 and 10.2).

Wu et al. (2005) explored lignin-degrading capacity of attached-growth white-rot

fungi. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity

for lignin degradation. Kalmis et al. (2008) reported ligninolytic enzyme activities of

different fungal species (six commercial and 13 wild) in solid and liquid culture media.

Among the wild strains, only Pleurotus ostreatus-1 (MCC45), P. ostreatus-2 (MCC40),

Pleurotus eryngii-1 (MCC25) and P. eryngii-2 (MCC26), and commercial strains P.

ostreatus, P. sajor-caju, P. eryngii presented lignin peroxidase (LiP) activity. Pant and

Adholeya (2007) isolated two fungal strains producing ligninolytic enzymes and having the

potential to decolorize distillery effluent from the soil of a distillery effluent contaminated

site. The correlation between decolorization and ligninolytic abilities of white-rot fungi has

been commented upon by several authors (Banat et al. (1996), Revankar and Lele (2006)

and Zhang et al. (2006) ). MnP activity was reported in Alternaria alternata with a possible

role in humic acid degradation by Rezacova et al. (2006), Since, it has been reported that

fungal ligninolytic enzymes play a major role in humic acid and melanoidin decolorization,

by Blondeau (1989), different Penicillium sp have also been reported, to produce

ligninolytic enzymes, by Sack and Gunther (1993) though its degrading mechanism is

considered to be different from that of the white-rot fungi (Hy et al. 2005). Cadmaleiv et al.

(2005) isolated two extracellular oxidases (laccases) from the extracellular fluid of the

fungus Panus (Lentinus) tigrinus cultivated in low-nitrogen medium supplemented with

birch sawdust. Dhouib et al. (2005) reported the ability of autochthonous fungi from Tunisia

to produce ligninolytic enzymes and these fungal strains were firstly screened on solid

media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of

lignin-modifying enzymes as specific color reaction whereas Ching et al. (2006) also

studied laccase activities of Penicillium simplicissimum H5 during solid-state fermentation

with rice straw. Mabrouk et al. (2010) studied lignin degradation on marine ascomycetes

and concluded that Trematosphaeria mangrovei showed highest ligninolytic activity.

D. Evaluation of Pectin Degradation by Bacterial Isolates

The organisms were grown on basal agar plates containing citrus pectin as sole

carbon source to study colonial growth and then grown in basal media containing citrus

pectin as sole carbon source to obtain colonial dry weight. Incubation period of 3 days were

given for all the test organisms. Average of three replicates was considered for each

organism. The results are shown in Table- 9.

Table- 9: Colonial growth and mycelial weight in pectin media.

Isolated Bacteria Growth rate Colonial dry weight per 50 ml

Colonial dry weight per 1000 ml

Bacillus cereus +++ 0.11gm 2.2 gm Bacillus thuringiensis +++ 0.12 gm 2.4 gm Brevundimonas diminuta ++++ 0.14 gm 2.8 gm

Cream colony I ++ 0.08 gm 0.16 gm Cream colony II + 0.03 gm 0.06 gm Cream rough colony II ++ 0.07 gm 0.14 gm Cream rough colony II + 0.04 gm 0.08 gm Cream rough colony III ++ 0.06 gm 0.12 gm Creamish yellow colony I ++ 0.08 gm 0.16 gm Creamish yellow colony II ++ 0.068 gm 0.136 gm Lemon yellow colony I + 0.03 gm 0.06gm Lemon yellow colony II ++ 0.07gm 0.14 gm Orange colony + 0.05 gm 0.10gm Transparent colony 4 + 0.029 gm 0.058 gm Transparent colony II ++ 0.06 gm 0.12 gm White colony II + 0.04 gm 0.08 gm Whitish cream colony + 0.03 gm 0.06 gm Yellowish rough colony ++ 0.07 gm 0.14 gm


Out of 32 species 18 species grown well in media containing pectin as sole

carbon source, but Brevundimonas diminuta (plate- 5.1 and 11.1), Bacillus thuringiensis

(plate- 5.2 and 11.2), Bacillus cereus (plate- 5.3 and 11.3), Cream colony I, Lemon yellow

colony and Yellowish rough colony were proved to be best pectinase producer as their

growth rate in pectin media was more as compared to other bacteria and even they showed

highest dry weight. Kobayashi et al. (2000) purified the first bacterial exo-poly

galacturonases from Bacillus sp. strain KSM-P443. Mohmoud et al. (2008) studied

pectinolytic enzymes and their production, characterization and applications and isolated

fifty two bacterial isolates, out of which Paenibacillus polymyxa showed the highest

pectinolytic activities and the exo-polygalactouronase was found to play major role.

Bayoumi et al. (2008) reported the nutritional and environmental condition requirements for

production of Polygalacturonase by Bacillus firmus-I-4071 under solid state fermentation

conditions using potato peels. These results are connected with that recorded by Kapoor et

al. (2000) who found that, Bacillus sp. MG-CP-2 produce an alkaline and thermostable

Polygalacturonase in degumming of ramie (Boehmeria nivea) and Sunn hemp (Crotalaria

juncea). Kapoor et al. (2000) and Bayoumi et al. (2008) reported that maximum

polygalacturonase production were achieved when media were supplemented with citrus

pectin as sole carbon source. However Beg et al. (2000a, 2000b) found that, pectinase

production from Streptomyces sp. QG-11-3 was enhanced by wheat bran. Geetha et al.

(2012) worked on bacterial species for pectinase production and identified them as Bacillus

sp. and Pseudomonas sp. According to them Bacillus sp. showed best pectinase activity

when grown in media with one percent pectin concentration. Kumar et al. (2012) studied

pectinase production by bacteria and observed that Bacillus sp MFW7 showed best

pectolytic activity. Janani et al. (2011) also studied pectinase producing bacteria from

agricultural waste dump soils and isolated ten bacterial strains. Three best pectinase

producing strains were identified as Bacillus sp whereas Kumar and Sharma (2012) reported

Cocci sp. as potent producer for pectinase production.

E. Evaluation of Cellulose Degradation by Bacterial Isolates

The organisms were grown on basal agar plates containing CMC (sole carbon

source) to study zone of digestion and then grown in basal media containing CMC as sole

carbon source to obtain mycelial dry weight. Incubation period of 5 days were given for all

the test organisms. Average of three replicates were considered for each organism. The

results are shown in Table –10.

Table- 10: Colonial growth and dry weight in cellulose media

Isolated Bacteria Growth rate Colonial dry

weight per 50 ml

Colonial dry

weight per 1000 ml

Bacillus cereus ++ 0.08 gm 0.16 gm

Bacillus thuringiensis ++ 0.07gm 0.14gm

Brevundimonas diminuta ++ 0.09 gm 0.18 gm

Cream colony I + 0.02 gm 0.04 gm

Cream rough colony II + 0.04 gm 0.08 gm

Cream rough colony III + 0.035 gm 0.07 gm

Creamish yellow colony I + 0.04 gm 0.08 gm

Lemon yellow colony II + 0.028 gm 0.56 gm

Transparent colony II + 0.03 gm 0.06 gm

Yellowish rough colony ++ 0.02gm 0.04gm


From total isolated species of bacteria, 10 species grown well on media containing

CMC as sole carbon source, thus giving an impression that 10 species of bacteria are potent

degraders. According to the result, Brevundimonas diminuta (plate- 6.1 and 12.1), Bacillus

thuringiensis (plate- 6.2 and 12.2), Bacillus cereus (plate- 6.3 and 12.3)and yellowish rough

colony were found to grow best in CMC media and showed highest dry weight. Wj et al.

(2005) also isolated 15 mesophilic cellulose-degrading bacteria from flower stalks-vegetable

waste co-composting system. Bacillus pasteurii and Bacillus cereus, Halobacillus,

Aeromicrobium and Brevibacterium showed high cellulase activity. Wongwilaiwalin et al.

(2010) also analysed thermophilic lignocellulose degrading microbial consortium with the

co-existence of eight major microbes, comprising anaerobic bacterial genera Clostridium

and Thermoanaerobacterium along with aerobic/facultative anaerobic Rhodocyclaceae

bacterium, bacilli, and uncultured bacteria. The microbial consortium showed efficient

degradation activity of cellulose as substrate. Ariffin et al. (2006) screened bacteria for

production of cellulase and used CMC as substrate. They got better production of cellulase

by Bacillus pumilus whereas Kim et al. (2012) observed three strains of bacteria showing

higher potential for cellulose degradation and identified them as Bacillus subtilis strains. Bai

et al. (2012) isolated bacteria from cowdung and analyzed their ability to produce cellulase.

Their study revealed that Bacillus subtilis strain (CELPTKL) was found as potent cellulase

producer.

F. Evaluation of Lignin Degradation by Bacterial Isolates

The organisms were grown on basal agar plates containing lignin sulphonic acid

sodium salt as sole carbon source to study colonial growth and then grown in basal media

containing lignin as sole carbon source to obtain colonial dry weight. Incubation period of 7

days were given for all the test organisms. Average of three replicates were considered for

each organism. The results are shown in Table–11.

Table- 11: Colonial growth and dry weight in lignin media

Isolated bacteria Growth rate Colonial dry weight

per 50 ml

Colonial dry weight per

1000 ml

Bacillus cereus + 0.03 gm 0.6 gm

Bacillus thuringiensis + 0.04 gm 0.08 gm

Brevundimonas

diminuta

+ 0.06 gm 1.2 gm

Cream colour I + 0.02 gm 0.4 gm

Creamish yellow

colony II

+ 0.02 gm 0.4 gm

Transparent colony II + 0.01 gm 0.2 gm


From total isolated species of bacteria, 6 species grown well on media containing

lignin as sole carbon source, thus giving an impression that 6 species of bacteria are potent

degraders. According to the result, Brevundimonas diminuta (plate- 7.1 and 13.1), Bacillus

thuringiensis (plate- 7.2 and 13.2), Bacillus cereus (plate- 7.3 and 13.3) and Creamish

yellow colony II were found to grow best in lignin media and showed highest dry weight.

Gonzalez et al. (1986) used lignin related compounds as a sole carbon source. They

isolated bacterial strains and tested their ability to proliferate in lignin media. Deschamps et

al. (2004) isolated lignin degrading bacteria directly by an enrichment culture technique

using an industrial kraft lignin (Indulin AT) as the sole carbon source. According to their

result, Aeromonas sp. degraded 98% of lignin (1g/l) after 5 days of incubation. Our studies

revealed that Brevundimonas diminuta degraded lignin upto .6g/l after 7 days of incubation.

Thus to some extent it agrees with the results of Aeromonas sp. Hassan and Hanafy 2009

studied biodegradation of lignin by Bacillus species. They also used lignin as sole carbon

source. They identified different genera including Corynebacterium, Agrobacterium,

Pseudomonas, Aeromonas, Klebsiella and Enterobacter. Mandryk et al. (2009) also used

lignin to support the growth and production of antimicrobial substances by 19

microorganisms and organisms from seven genera: Bacillus, Pseudomonas, Streptomyces,

Beauveria, Paecilomyces, Trichoderma and Trichosporon grew on the tested media. P.

aurantiaca S-1 has only shown to grow well in lignin media. Odier et al. (1981) selected

eleven gram-negative aerobic bacteria out of 122 isolates for their ability to assimilate

poplar dioxane lignin isolated from the soil and without a co substrate. Lignin degradation

was confirmed by Pseudomonas sp.

From above results we have observed that some of the species are very good

degraders particularly of cellulose like Trichoderma viridae and T.atroviridae but had not

shown that much potency for pectin and lignin. In the same way Phanerochaete

crysosporium was proved to be a very good lignin degrader, but not as a cellulose and pectin

degrader. But we observed that some species had shown moderate to good degradation in all

three media, and thus proved to be ligninolytic, cellulolytic as well as pectolytic. Penicillium

mealagrinum var. viridiflavum, Alternaria alternata, Penicillium citrinum, Aspergillus

fumigatus, Aspergillus niger had shown positive result equally in all three media. In the

same way Brevundimonas diminuta, Bacillus thuringiensis and Bacillus cereus were found

to be comparatively potent degrader of lignin, cellulose as well as pectin.

Quantitative Test for Pectin, Cellulose and Lignin Utilization by Selected

Isolates.

After evaluating bacteria and fungi for their capacity to degrade lignin, cellulose and

pectin, the target microorganisms were further selected for quantitative estimation. The

selection of the organisms were done on the basis of their comparatively higher degradative

potency on lignin, pectin and cellulose as compared to other studied organisms. Out of total

degraders best three fungal and three bacterial potent degraders were selected. To confirm

utilization of pectin, cellulose and lignin the test organisms were grown in respective basal

broth media. After stipulated incubation period the filtrates were studied for absorbancy

using spectrophotometer and absorbancy were matched with standard solutions of different

concentrations. Results were shown in Table-12 and Table-13 as follows:

Table- 12: Utilization of pectin, cellulose and lignin by fungal organisms.

Isolated fungi Pectin

Incubation period 5

days (gram / litre)

Cellulose

Incubation period 7

days (gram / litre)

Lignin

Incubation period 9

days (gram / litre)

Alternaria alternata 4.4 gm / l ±

0.01gm/l

1.1gm / l ± 0.02 gm/l 1.2 gm / l ± 0.02

gm/l

Penicillium citrinum 4.5 gm / l ±

0.01gm/l

2.1 gm / l ± 0.02

gm/l

1.1 gm / l ± 0.02

gm/l

Penicillium

meleagrinum var.

viridiflavum

4.3 gm / l ±

0.01gm/l

2.2 gm/ l ± 0.02 gm/l 1.6 gm / l ± 0.02

gm/l

Table – 13: Utilization of pectin, cellulose and lignin by bacterial organisms

Isolated bacteria Pectin (5gm/ l)

Incubation period 3

days ( gram / litre )

Cellulose (10 gm / l)

Incubation period 5

days (gram / litre)

Lignin (10 gm / l)

Incubation period 7

days (gram / litre)

Brevundimonas

diminuta

1.3gm / l ± 0.01gm/l 0.8gm / l ± 0.02 gm/l 0.6 gm / l ± 0.02

gm/l

Bacillus

thuringiensis

1.1gm / l± 0.01gm/l 0.6gm / l ± 0.02 gm/l 0.3gm / l ± 0.02

gm/l

Bacillus cereus 1.10 gm / l±

0.01gm/l

0.5gm / l ± 0.02 gm/l 0.2 gm / l ± 0.02

gm/l

Penicillium meleagranum var. viridiflavum, Penicillium citrinum and Alternaria

alternata from fungal isolates and Brevundimonas diminuta, Bacillus thuringiensis and

Bacillus cereus from bacterial isolates were selected as test organisms for further

molecular studies.

MOLECULAR STUDIES

Observations RAPD of Fungal Samples

Fig - 17: PCR amplification of fungi.

Identification Report

Consolidated fungal identification report by RAPD

RAPD of Bacterial Samples

Fig.-19: PCR amplification of bacteria

Identification Report

Consolidated bacterial identification report by RAPD

RFLP of fungal samples

Fig – 21: PCR product of fungal isolates.

Fig – 22: RFLP of PCR product of fungal isolates.

RFLP of bacterial samples

Fig- 23: PCR product of bacterial isolates.

Fig- 24: RFLP of PCR product of bacterial isolates.

RAPD of Fungi Sample F1

Sample F2

Sample F3

Above complete molecular analysis of three fungal samples from the cave Mandeepkhol,

revealed abundance of Penicillium citrinum (NRRL 35449), Alernaria alternata and

Penicillium meleagrinum var.viridiflavum. Dendogram of all three fungi shows that

Alernaria alternata and Penicillium meleagrinum var. viridiflavum are phylogenetically

more closure than Penicillium citrinum.

RAPD of Bacteria

Sample –B1

Sample- B2

Sample –B3

Above molecular analysis revealed that the bacterial strain reported from cave Mandeepkhol

where Brevundimonas diminuta (B34), Bacillus thuringiensis (YY23) and Bacillus cereus

(DS 16) and Bacillus thuringiensis and Bacillus cereus are phylogenetically more closure

than Brevundimonas diminuta.

RFLP of Fungi

We conclude that finding of RFLP for all three samples of fungi are inconclusive and needs

larger size analysis.

RFLP of Bacteria

We conclude that finding of RFLP for all three samples of bacteria are inconclusive and

needs larger size analysis.

Investigation of the microbial ecology of natural and anthropogenically influenced

environments on the basis of culture and colony characteristics are important methods, but

they are extremely biased in their evaluation of microbial genetic diversity due to the

selection of a particular population of microorganisms. The adversity in ascribing an

organism to a biologically meaningful category should be well considered before the use of

any molecular identification tool. One has to be aware of the evolutionary history and

taxonomic position of the specimen under study. It is very important to understand the order

of branching and ages of divergence (phylogeny) of the organisms in examination and to be

familiarized with the nomenclatures used in earliers studies. Terminologies such as “strain”,

“ ariant”, “subspecies” or “breed” could be highly nonobjective in some situations and can

be used as synonymous terms by different investigators to describe the same biological

entity. Many biochemical and molecular methods has been applied to reveal the microbial

community composition over time and space. These new approaches allow linkage between

ecological processes in the environment with specific microbial populations and help us to

acknowledge and resolve many important questions in microbial ecology such as what

factors and resources govern the enormous genetic and metabolic diversity in an

environment. With the advent of recent advances in genomics and sequencing technologies,

microbial community analyses using culture-independent molecular techniques have

initiated a new era of microbial ecology.

In modern taxonomic studies, methods for the identification of species that rely on

DNA or protein sequence analysis presuppose the neutral theory of molecular evolution, in

which different lineages diverge over evolutionary times by the accumulation of molecular

changes (most of them neutral) (Kimura, 1968). These methods are based on the assumption

that individuals from a same species carry specific DNA (or protein) sequences that are

different from those found in individuals from other species. However, the distribution of a

given molecular variant in time and in space will be influenced by the reproductive success

of individuals, migratory events and random genetic drift. Therefore, it should be realized

that a continuous genetic variability does exist among individuals of a species. The level of

intra-species diversity in the locus under study has to be properly assessed before

undertaking any taxonomic identification in order to guarantee that there is no overlap

between intra-species variation and interspecies divergence. Furthermore, different loci have

variable rates of evolution owing to the action of processes such as mutation and

recombination (Sunnucks, 2000). Therefore, to choose the appropriate loci is vital to the

success of the identification.

Molecular techniques are the major tools for the analysis of microorganisms from various

sources. The techniques provide ways to screen for a vast array of agents in a single test

(Field and Wills, 1998). The food industries, water processors, agronomical agencies and

analytical laboratories have taken up these methods for rapid differentiation of species,

strain identification and definition of strain relatedness from infected samples. Molecular

methods vary with respect to discriminatory power, reproducibility, ease of use, and ease of

interpretation (Lasker, 2002).

Molecular analyses of environmental communities have revealed that the cultivable

fraction represents less than one percent of the total number of prokaryotic species present

in any given sample. Various molecular methods based on direct isolation and analysis of

nucleic acids, proteins, and lipids from environmental samples are available that are capable

of revealing structural and functional information about microbial communities. Molecular

techniques such as genetic fingerprinting, metagenomics, metaproteomics,

metatranscriptomics, and proteogenomics are nowadays becoming vital for discovery and

characterization of vast microbial diversity and also important for understanding their

interactions with biotic and abiotic environmental factors. Majority of microbial

communities in nature have not been cultured in the laboratory. Thus the primary source of

information for these uncultured but viable organisms is their biomolecules like nucleic

acids, lipids, and proteins. Techniques like culture-independent nucleic acid approaches

include, “analyses of whole genomes or selected genes such as 16S for prokaryotes and 18S

rRNA (ribosomal RNA) for eukaryotes.” omparati e analyses of rRNA signatory

molecules, have classified the cellular life into two primary domains, viz. eukaryotic

(Eukarya) and prokaryotic (Bacteria and Archaea) (Hugenholtz 2002). The field of

microbial ecology has seen tremendous progress for a while, and a varied number of

molecular techniques have been developed for describing and characterizing the

phylogenetic and functional diversity of microorganisms (Fig.). Depending on their

capability of revealing the microbial diversity structure and function, these techniques have

been broadly classified into, partial community analysis approaches and whole community

analysis approaches.

Fig: Molecular tools for characterization of structural and functional diversity of

microbes in the environment.

Source: Gurdeep Rastogi and Rajesh K. Sani. Molecular Techniques to Assess Microbial Community

Structure, Function, and Dynamics in the Environment. Chapter:2. I. Ahmad et al. (eds.), Microbes

and Microbial Technology: 29 Agricultural and Environmental Applications, Springer Science+Busi

ness Media, LLC 2011.

Traditional methods of bacterial enumeration and typing are often insufficient for the

identification of specific strains in complex, mixed-strain microbial consortia. The aim of

typing is to identify stable sub-groups or clones within the same species using markers that

are reproducible, accurate and conveniently detected.

Several kinds of molecular techniques have been used to study genetic diversity, for

example, labeled probes to detect restriction fragment length polymorphism (RFLP)

(Hegedeus and Khachatourians, 1993; Maurer et al., 1997) and methods based on the

polymerase chain reaction (PCR) such as the use of random amplified polymorphic DNA

(RAPD) (Fegan et al., 1993; Bidochka et al., 1994; Piatti et al., 1998; Freire, 2001; Jensen

et al., 2001; Gaitan et al., 2002), simple sequences repeat (SSR or microsatellites) analysis

(Kretzner et al., 2000; Enkerli et al., 2001, 2005) and internal transcribed spacer (ITS)-

rDNA sequence RFLP analysis (Rakotonirainy et al., 1994; Buscot et al., 1996; Coates et

al., 2002a). Even though they have different and complementary discriminatory power

RAPD, SSR and RFLP analysis have been very effective in detecting genetic diversity in

several species (Couteaudier et al., 1998; Glare and Inwood, 1998; Coates et al., 2002b;

Enkerli et al., 2005). In Chile, however, there has been no previous molecular

characterization of entomopathogenic fungi populations. The PCR technique combined with

RAPD analysis (Welsh and McClelland, 1990; Williams et al., 1990) has been extensively

used to determine the genetic diversity of various entomopathogenic fungi (Cobb and

Clarkson, 1993; Bidochka et al., 1994; Neuvéglise et al., 1994; Neuvéglise and Brygoo,

1994; Bidochka et al., 1995; Hodge et al., 1995; Fungaro et al., 1996; Maurer et al., 1997)

including Metarhizium (Fegan et al., 1993; Leal et al., 1994) and to relate genetic diversity

to pathogenicity index (Bridge et al., 1997) and to identify strains (Milner et al., 2002).

Using DNA-fingerprinting techniques like randomly amplified polymorphic DNA

(RAPD), RFLP, ribotyping and DNA (or RNA)-sequencing, microorganisms can be

identified up to strain level (Anderson and Kohn, 1995; Anderson et al., 1992; Appel and

Gordon, 1996).

The basic principle of molecular biology used in a variety of methods with possible

applications in microbial species identification includes hybridization of complementary DN

A oligonucleoti des (DNA hybridization). Some of the early assays were based on solid-

phase hybridizations performed on nitrocellulose or nylon membranes between whole

genomic or synthetic DNA probes of known origin and DNA extracted from the target

sample (Falkow and Moseley, 1982; Scholl and Jollick, 1987; Torok et al., 1995). The

RFLP analysis is widely used for the detection of interspecies variation at the DNA

sequence level which is consised in the generation of species-specific band profiles through

the digestion of DNA with one or more restriction endonucleases (Botstein et al., 1980).

The distinctive and exclusive RFLP profile of each species is the resultant of the unique

genomic distribution of recognition sites (generated or removed by single-base substitution

reactions) and the distance between them (that varies due to large genomic rearrangements,

such as translocations, transposable elements or tandem duplications). On the other hand,

RAPD profiles are generated by the random PCR amplification of DNA segments using

short primers of arbitrary nucleotide sequence of usually 9 to 10 nucleotides long (Livak et

al., 1992; Williams et al., 1990). Different fingerprinting techniques are based on the use of

primers that are specifically designed to hybridize within repetitive genomic regions. The

differential genomic distribution of repetitive elements between species is responsible for

species-specific profiles. These repetitive regions includes, mammalian-wide interspersed

repeats (MIR) (Buntjer and Lenstra, 1998; Jurka et al., 1995; Melancon et al., 1996),

repetitive extragenic palindromic sequences (REP) (Versalovic et al., 1991) and

enterobacterial repetitive intergenic consensus sequences (ERIC) (Hulton et al., 1991). The

RAPD method does not require prior sequence information for PCR primer design but is

extremely dependent on variations in laboratorial conditions (such as template DNA

concentration, PCR and electrophoretic settings, etc), requiring carefully developed

laboratory protocols to be reproducible. An imperfect hybridization between the primer and

the target site may result in a completely different banding profile.

The choice of genetic marker can have a substantial impact on the analysis and

interpretation of data. For questions relating to the roles played by population size, mating

systems, and gene flow, selectively neutral genetic markers are preferred. For questions

relating to the effects of selection, selected markers should be used. An exception to these

guidelines exists when working with fungi that reproduce predominantly asexually,

producing a population structure that is composed largely of clonal lineages. In such cases, a

neutral marker such as a DNA fingerprint may be used to address questions about selection

because of the complete correspondence between genotype (e.g., DNA fingerprint) and

phenotype (selected marker, e.g., pathotype). However, a complete correspondence between

genotype and phenotype may not always be assumed, because several studies have shown

that different pathotypes can arise within the same clonal lineage (Drenth et al., 1996;

Goodwin et al., 1995) usually as a result of selection. Although the advantages and

disadvantages of different types of genetic markers have been discussed elsewhere (Kohn,

1992; McDonald and McDermott, 1993; Rosewich and McDonald, 1994). In population

genetics, the best genetic markers are those which are selectively neutral, highly

informative, reproducible, and relatively easy to assay. RFLPs, and RAPDs have been used

extensively in eukaryotes. The majority of fungal studies currently are conducted with

DNA-based genetic markers. If the organism in under consideration can be grown in pure

culture on artificial media, the choice of DNA marker is open but if the organism cannot be

grown on artificial media, then polymerase chain reaction (PCR)-based methods are likely

to be more appropriate due to limitations on the amount of tissue that can be isolated for

DNA extraction. Different categories of genetic markers provide widely varying

interpretations of the genetic structure of populations. In case of Mycosphaerella

graminicola, there is a large amount of diversity for nuclear RFLP loci, which suggests high

levels of gene diversity and random mating populations (McDonald et al., 1995). DNA

fingerprints based on hybridization of one probe to repetitive DNA sequences had greater

resolution to differentiate clones then multilocus haplotypes based on hybridization of many

single-copy probes for M. graminicola and Phaeosphaeria nodorum (Boeger et al., 1993;

McDonald et al., 1994). The greater resolution of DNA fingerprints in these cases likely

reflects the larger number of loci assayed by the repetitive probes. Generally it is best to use

the widest practical array of genetic markers to obtain the most comprehensive

understanding of the genetic structure of populations.

Randomly amplified polymorphic DNA (RAPD) technique is amplification-based

nucleic acid fingerprinting techniques (concurrent detection of multiple loci without

assignment of a genotype) that use an in vitro enzymatic reaction to specifically amplify a

multiplicity of target sites in one or more nucleic acid molecules (Micheli et al., 1994). The

amplification reaction is generally carried out by short synthetic oligonucleotides of

arbitrary or semi-arbitrary sequence that produce a collection of amplified products of

largely non-allelic nature. Several authors have reported on the application of RAPD

technique in microorganisms (Babalola, 2002). It uses random primers (Williams et al.,

1990) and can be applied to any species without requiring any information about the

nucleotide sequence. The amplification products from the analysis exhibited polymorphism

and thus can be used as genetic markers. Since presence of a RAPD band, does not provide

any distinction between hetero -and homozygous states, the fragments are scored as

dominant Mendelian elements. Nowrouzian et al., in 2001, designed a RAPD typing method

for the identification of E. coli strains in normal human intestinal microflora. The band

pattern generated in the analysis represents genome characterization of a particular bacterial

strain (Welsh and McClelland, 1990). Also RAPD has the potential for analyzing

phylogenetic relationships among closely related species (Williams et al., 1990) and can

distinguish between strains within a species.

RAPD markers (Williams et al., 1990) have become popular because this PCR

technology is relatively easy to implement. RAPDs can be assayed by using very small

amounts of fungal biomass, which makes them an ideal tool for obligate biotrophs. Since a

large number of amplicons can be screened in a relatively short period of time, RAPDs are

especially useful in differentiating clonal lineages for fungi that reproduce asexually.

Further, RAPD data are convinient to interpret because they are based on amplification or

non-amplification of specific DNA sequences (amplicons), producing a binary data set that

is easy to enter into a spreadsheet for analysis. But RAPDs have many drawbacks despite

their relative ease of use. There are several technical limitations associated with RAPDs that

make them difficult to reproduce between labs and sometimes within labs. Some of these

limitations can be overcome with proper controls, like replicate DNA preparations, Southern

analysis, and conversion of RAPD amplicons into sequence characterized amplified regions

(SCARs) (Paran and Michelmore, 1993). There also are analytical problems associated with

RAPDs (Lynch and Milligan, 1994). RAPDs have only two alleles (amplification or non-

amplification) for each amplicon locus. This condition is ideal for genetic mapping, but it is

a drawback for the measurement of genetic diversity affected by the number of alleles at a

locus. RAPDs are dominant markers, therefore they cannot differentiate between

homozygotes and heterozygotes without a progeny test. This is not a case with haploid

fungi, but it can be a problem with many basidiomycetes and oomycetes that are

heterokaryons, diploid or polyploid. Some of the analytical problems resulting from

dominance and two alleles per locus can be overcome by increasing the number of

individuals assayed (by 2 to 10x compared to isozymes and RFLPs) and using only RAPD

amplicon loci that have intermediate allele frequencies (Lynch and Milligan, 1994). In spite

of their drawbacks, RAPDs are powerful tools that are especially useful for fungi that are

obligate parasites or that have a population structure composed of clonal lineages. Through

advanced PCR-based markers it may become possible to amplify specific DNA sequences

from soil or root samples and make a direct assessment of the genetic structure of

populations of soil borne fungi without first making pure cultures. But if a fungus can be

cultured readily on artificial media, then RFLPs offer many advantages for population

genetics studies.

RFLPs in nuclear and mitochondrial genomes have been used in many studies of

plant-pathogenic fungi. Since RFLPs are based on DNA-DNA hybridization, they are

technically more difficult than RAPDs but they offer the advantage of being more

reproducible as compared to RAPDs and yet, like RAPDs, RFLPs are easy to interpret. In

addition, they are co-dominant markers and exhibit a potentially unlimited number of alleles

per locus. One disadvantage of RFLPs is that they require relatively large amounts (5 to 10

μg) of DNA from each indi idual, so the fungus must be grown in pure culture prior to

DNA extraction. RFLP analysis requires more technical expertise, including cloning,

Southern blotting, and labeling of probes. RFLPs are most useful for fungi that can be

grown easily in pure culture. Since probes can be chosen to show a high degree of

specificity to the target fungus, some contamination by DNA from other fungi, or bacteria

can be tolerated. The properties of co-dominance and multiple alleles make RFLP markers

advantageous compared to RAPDs for most studies in population genetics.

RFLP fingerprinting technique is regarded as the most sensitive method for strain

identification and several bacterial strains have been widely studied using this technique.

Kabadjova et al., in 2002, devised a rapid PCR-RFLP-based identification scheme for four

closely related Carnobacterium species (C. divergens, C. piscicola, C. gallinarum, and C.

mobile) that are of interest to the food industry. By this method, three of the isolates that

were previously incorrectly identified as C. divergens (INRA 508, INRA 586, and INRA

515) were reclassified as C. piscicola. Similarly, four isolates previously misidentified as C.

piscicola (INRA 545, INRA 572, INRA 722, and ENSAIA 13) were reclassified as C.

divergens based on the patterns obtained by the 16S-23S ISR-RFLP methods. Wang et al.,

in 2000 and Penrose et al., in 2000, have already proved the role of PCR and Southern

hybridization in assessment of the effect of introduction of 1-aminocyclopropane-1-

carboxylic acid deaminase genes on disease-suppressive capabilities of Pseudomonas

fluorescens strain CHAO. Manceau and Horvais, 1997, used RFLP analysis of rRNA

operons to assess phylogenetic diversity among strains of Pseudomonas syringae pv.

tomato. They successfully established the close relationships existing between P. syringae

and P. viridiflava species.

The nucleotide sequence of 16S rDNA has been wide accustomed perceive

phylogenetic relationships among prokaryotes (Barry et al., 1990; Weisburg et al., 1991).

Graham et al., 1991 applied phylogenetic relationships to species-specific identification of

bacterium by hybridizing between DNA and 16S rRNA, and orientating the nucleotide

sequences of 16S rDNA. The phylogenetic relationships of assorted true bacteria (Vibrio)

species have additionally been characterised thoroughly by using the sequences of 16S

rDNA (Dorsch et al., 1992; Kita-Tsukamoto et al., 1993). However, these processes are

certainly inappropriate for routine identification of microbial strains because of the length of

time needed. Therefore, restriction fragment length polymorphism (RFLP), by utilizing 16S

rDNA genes amplified by polymerase chain reaction (PCR), has been introduced for speedy

and accurate identification of bacterium at the species level (Ralph et al., 1993; Choudhury

et al., 1994; Moyer et al., 1994; Urakawa et al., 1999). The randomly amplified polymorphic

DNA (RAPD) PCR technique, developed to be used in the detection of V. vulnificus, also

clearly differentiated this true bacteria (Vibrio) species from the other members of the genus

Vibrio (Warner and oliver, 1999).

Molecular biological methods that utilize 16S ribosomal ribonucleic acid (rRNA)

gene sequences are commonly used for the identification and classification of

microorganisms. rRNA sequences are primarily used in ranking biological phylogenetic

nomenclature including that of microorganisms (Hwang et al., 2011). 16S rRNA is a type of

RNA that plays a major role in synthesis of protein macromolecules (Jayaraman et al.,

1981). Since the mechanism of protein synthesis does not differ much from one organism to

another, the RNA that assists the protein production also does not vary allot. The variations

generally occur in predictable locations. It is observed that the nucleotide sequences of some

portions of the 16S ribosomal deoxyribonucleic acid (rDNA) are highly conserved. An

advantage of sequencing ribosomal RNA (rRNA) genes is that the presence of preserved

region (for instance, 18S rRNA in eukaryotes and the 16S rRNA in prokaryotes) adjacent

with extremely variable segments (such because the internal transcribed spacers) permitting

the resolution of relationships among each distantly and closely organic process connected

species, respectively.

Banking on above information and large scale finding of different authors, we

conclude that recognition of all three bacteria (Brevundimonas diminuta, Bacillus

thuringiensis and Bacillus cereus) and all three fungi (Penicillium citrinum, Alterneria

alternata and Penicillium meleagrinum var. viridiflavum) from the Mandeepkhol cave are

justified. The phylogenetic relationships of organism are also scientifically justified. Thus

we conclude that the cave is plenty of microbial diversity irrespective of spleleological

conditions. This is indication of old existence of the cave which needs further geological

investigation.

Conclusion

Speleology is one of the most interesting field in science that is attracting attention

of a large number of researchers all over the world. Each cave, whether it is frequently

visited by visitors or remain untraversed , has got its own unique ecological niche. Every

cave has got its own geographical, geological, atmospheric and biological features on the

basis of which they differ from one another.

In present studies, the selected Mandeepkhol cave, located in deep forest and not

frequently visited by human population, provides shelter to a large number of bats and

receives a huge amount of dried plant parts alongwith water streams from surrounding hilly

area. The guano and plant parts, deposited inside the cave, serve as substrates for the growth

of variety of molds and bacterial decomposers which degrade the complex materials at quite

faster rate in an atmosphere where 100% humidity and a temperature of 26 ºC -28 ºC prevail

almost throughout the year.

Present studies, related to microbial diversity, revealed that Mandeepkhol cave

provides shelter to variety of microorganisms. Our study in this cave will help a

microbiologist to know about the diversity of fungi and bacteria present inside cave. During

study we have got different species of bacteria and fungi which were further evaluated for

their potency to degrade pectin, cellulose and lignin. Results showed that among fungi -

Alternaria alternata , Penicillium citrinum , Penicillium meleagrinum var. viridiflavum,and

bacteria - Brevundimonas diminuta, Bacillus cereus, Bacillus thuringiensis showed quite

higher potency for degradation of pectin, cellulose and lignin. Through molecular studies

(RFLP/ RAPD) microorganisms got genetically marked. Use of these microorganisms will

help in faster degradation of complex compounds and plant biomass in mesophilic

conditions, thus may help in waste management and recycling of carbon compounds. In this

way these organisms may be instrumental in organic farming in mesophilic conditions, as

organic farming requires faster rate of degradation for manure formation.

These studies will pave ways for genetic manipulation in these potent decomposers and will

be helpful in raising such strains which can degrade variety of complex compounds.

results and discussion - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/29464/9/09... ·...

Documents