characterization of functional activity in composted...

Indian Journal of Biotechnology

Vol 8, January 2009, pp 97-109

Characterization of functional activity in composted casing amendments used in

cultivation of Agaricus bisporus (Lange) Imbach

Devendra K Choudhary*, Pavan K Agarwal

1, and Bhavdish N Johri

1

Department of Microbiology, G B Pant University of Agriculture and Technology, Pantnagar 263 145, India 1Department of Biotechnology, Barkatullah University, Bhopal 462 026, India

Received 23 August 2007; revised 20 August 2008; accepted 16 October 2008

In cultivation of button mushroom [Agaricus bisporus (Lange) Imbach], casing layer that is nutritionally deficient to

compost is believed to trigger the fruit body formation and this is conducted by the bacterial community residing in casing

layer. The change in nutritional status of the casing is highly correlated with microbial flora. Therefore, an attempt was

made to characterize the bacterial flora in casing layer, i.e., Farm Yard Manure and Spent Mushroom Substrate (FYM+SMS,

3:1) and Farm Yard Manure and Vermi Compost (FYM+VC, 3:1), employing phenetic approaches. Morphotypically

different and functionally characterized bacterial isolates were identified by partial 16S rDNA gene fragment sequencing.

Available data showed a significant variety of organisms that included Acinetobacter and Pseudomonas of the γ-

proteobacteria, that were most frequently encountered genera. Amongst Gram-positive bacteria, Bacillus was the most

highly represented genus that was derived from the agaric fruit bodies. In addition, FYM+SMS was found to be a high

yielding casing mixture, which took minimum case run period together with superior fruit body quality as compared to

FYM+VC.

Keywords: Acinetobacter, Bacillus, casing, endotrophs, phenetic approach, Pseudomonas

Introduction

Mushroom science is a discipline concerned with

the principles and practices of mushroom cultivation.

Consistent production of successful mushroom crops

is built upon scientific knowledge and practical

experience. Agaricus bisporus (Lange) Imbach is the

most widely cultivated species of edible mushroom

and it is the most popular cultivar among the

artificially grown fungi of the world that contributes

about 31.8% to the global mushroom cultivation and

85% of the total produce in India1. The triple

advantage of growing mushroom is that mushroom

culture is a biotechnological process that recycles

lignino-cellulosic wastes, mushrooms are soma of

health food for human consumption and the spent

mushroom substrates can be utilized profitably in

different ways2. A. bisporus requires two different

substrates to form the fruit bodies, i.e., the compost

for nutrition on which it grows vegetatively and the

nutrient deficient casing soil in which the suitable

physicochemical/biological conditions stimulate the

initiation process of pin head formation for fruit body

production3. Inspite of being nutritionally deficient

medium, casing layer plays an important role in the

productivity of button mushroom.

The casing layer is one of the important growing

parameter and source of variation in production,

quality and uniformity of commercial cropping. A

variety of casing materials have been used the world

over. Among these, use of farm yard manure (FYM)

as a casing medium for mushroom cultivation has

been in vogue in Indian subcontinent because of its

easy availability and non-availability of peat moss

generally used for casing in Europe and USA. The

advantages of using FYM as a casing material over

other agro-industrial wastes have been highlighted in

a recent report4,5

.

Spent mushroom substrate (SMS) is the remainant

of compost from which mushrooms have been

produced and for which several important roles have

been described6. Despite evaluation of large number

of agro-industrial wastes for their use as casing

material in A. bisporus cultivation, scant attention has

been given to the importance of biological properties

of the casing layer7. Bacteria present in casing layer

considerably influence the growth and morphogenesis

____________________

*Author for correspondence:

Tel: 91-755-4005202/03 ext. 4280

Email: [email protected]

Present Address: Immunology Laboratory (IL), Centre for

Scientific Research & Development (CSRD), People’s Group,

By-Pass Road, Bhanpur, Bhopal 462 010, India

INDIAN J BIOTECHNOL, JANUARY 2009

98

of A. bisporus production. It supports beneficial

microbial populations that release growth stimulating

substances, which are reportedly involved in

stimulating the initiation of pin heads. Several reports

are available on the beneficial effects of casing soil

microbes, especially Pseudomonas putida and

Alcaligenes faecalis, on A. bisporus8. Not much

information is available in the literature about the role

of associated microflora in the casing layer and how

the resident microflora in casing layer interacts with

the vegetative mycelium of A. bisporus. There are two

production related issues requiring attention of

researchers in casing soil microbiology, viz., (i) the

composition of microorganisms present in casing soil,

and (ii) their influence on mushroom production in

situ. Several methods and approaches are now

available to generate information on microorganisms

that reside in casing layer, which allow better

assessment of microbial flora, wherein molecular

tools for the identification of microorganisms are now

in common use, and 16S rRNA gene analysis is

intensively used in phylogenetic investigations.

Among the 16S rRNA gene analysis method,

amplified ribosomal DNA restriction analysis

(ARDRA) is commonly used to estimate phylogenetic

relationship among different microbial isolates or

rDNA clones recovered from the environment9,10

.

Furthermore, 16S rDNA sequence typing approach

now permits identification of the surviving and

culturable bacterial species, based on employing

highly conserved 16S rDNA oligonucleotide primers

for the eubacteria with an intervening hypervariable

gene sequence, which could be used as signature

sequence to aid in species identification11,12

.

The aim of the present study was to investigate the

cultivable bacterial flora of casing layer and its impact

on quality of casing amendments.

Materials and Methods Sampling Site

Casing substrates for present study included Spent

Mushroom Substrate (SMS), and Vermi Compost

(VC). Both the substrates in the stages were amended

with Farm Yard Manure (FYM) in the ratio of 3:1.

These casing substrates were collected from

Mushroom Research and Training Centre (MRTC),

GB Pant University of Agriculture and Technology,

Pantnagar, India. Samples were drawn from

successive stages of each casing, namely, 0 day

casing (when casing was applied over the mycelium

impregnated compost, i.e., at the time of casing),

mycelium impregnated stage (MIS) of casing, casing

at primordial stage (PS), and harvesting stage (HS)

casing.

Physico-chemical and Nutrient Analysis of Casing Mixtures

Processed casings (formalin treated, 2% v/v) were

used for physico-chemical analysis using methods

suggested by various workers: Bulk density13

, water

holding capacity14

, porosity15

, electrical conductivity

and pH16

(TPS Smartchem Laboratory Analyzer),

total organic matter and C17

, available N18

, available

P19

and available K20

. For macro and micronutrients

(Ca, Mg, Cu, Zn, Mn & Fe) analysis procedure of

Tandon21

was employed.

Isolation of Bacterial Isolates

Casing sample (10 g) was suspended in 90 mL of

0.85% normal saline (pH 7.0) and shaken vigorously

at 150 rpm at 18°C for 1 h. The resulting slurry was

serially diluted (100 µL) to 900 µL of 0.85% normal

saline in each Eppendorf tube and appropriate dilution

(10-4

) of this suspension (0.1 mL or 100 µL) was

spread plated in triplicate on King’s B medium22

.

Cultures were incubated at 20°C±2 for 2 d. For

experimental use, isolates were transferred when

needed to King’s B medium that was stored at 4°C.

Phenetic Characterization

Recovered bacterial isolates were phenotypically

(morphotypic, physiological & functional) and

genotypically characterized.

Morphological

Isolates were checked for fluorescence under UV

light (312 nm) (Geldocmega, Biosystematica) and

distinct colonies were picked up. A total of 50 isolates

were thus randomly selected morphologically from all

the four successive stages of two types of casing soil,

namely, 0 day casing, mycelium-impregnated casing,

casing with initials (primordial) and harvesting stage

(cropping). Colony morphology of isolates was

studied under a stereoscope microscope (Leica). This

included shape, edge, elevation, surface and

pigmentation. Cellular morphology was based upon

cell shape and Gram staining (Leica fluorescent

microscope).

Physiological

Morphotypically different bacterial isolates isolated

from various stages of two type of casing mixtures

were further characterized based on their substrate

utilization and enzyme secretion. These included:

catalase and oxidase enzyme substrates, gelatin

CHOUDHARY et al: FUNCTIONAL ASSESSMENT OF MUSHROOM CASING MIXTURES

99

hydrolysis, citrate utilization test, nitrate reductase

(NO3– → NO2

– →N2 )/denitrification test and trehalose

utilization in substrate utilization tests.

Functional

Assessment of functional diversity among isolates

was studied by qualitative plate assay for amylolytic,

proteolytic, cellulolytic, xylanolytic, pectinolytic and

laminarinolytic activity, phosphorus solubilization

(inorganic phosphates, and organic phosphates), and

siderophore production. Minimal medium was used

for plate assay wherein isolates were spot-inoculated

with sterile tooth-pick on solid medium and incubated

at 20±2°C. The zone of diameter of clearing, if any, of

all +ve isolates were measured.

Amylase (Starch Diastase)All isolates were spot

inoculated on functional minimal medium amended

with 1% (w/v) starch and incubated for 24-72 h at

20±2°C for growth. Plates were then flooded with

Lugol’s iodine for 10 min. Then iodine was drained

off and +ve isolates exhibit a zone of clearance

against dark blue background.

Protease—The minimal medium amended with

skimmed milk (20 mL L–1

) was spot inoculated with

isolates. Plates were incubated for 24-72 h at 20± 2°C

for growth. Formation of a clear halo around the

bacterial colony was considered as +ve result for this

test.

Cellulase and XylanaseThe minimal medium was

supplemented with 1% (w/v) birch wood xylan and

carboxymethyl cellulose (CMC) and spot inoculated

with bacterial isolates. Plates were incubated in an

incubator for 72 h at 20±2°C for growth and flooded

with Congo red solution (0.2% w/v) for 30 min.

Excess reagent was discarded after destaining with 1

M NaCl solution for 30 min. Zone of clearance

around bacterial colonies was considered as +ve result

for this test.

PectinaseAll isolates were spot inoculated on minimal

medium amended with 1% (w/v) pectin; plates were

incubated for 72 h 20±2°C for growth. After

incubation, plates were flooded with hexadecyl (cetyl)

trimethyl ammonium bromide (CTAB, 1% w/v) for 30

min and destained with 1 M NaCl for 30 min. A halo

around bacterial colonies was considered as +ve result.

Laminarinase (ββββ-1,3-Glucanase)Bacterial isolates were

spot inoculated on the minimal medium amended with

1% (w/v) laminarin and plates were incubated for 3-5

d at 20±2°C for growth. Activity of β-1,3-glucanase

was determined by the ability of bacteria to grow on

laminarin as the sole carbon source.

Siderophore ProductionChromeazurol ‘S’ agar plates23

were spot inoculated with bacterial isolates, and

incubated for 48-72 h at 20±2°C for growth.

Formation of an orange halo around bacterial colony

was considered as +ve result.

Inorganic ‘P’ SolubilizationBacterial isolates were spot

inoculated on Pikovaskya’s agar and incubated for 3-5

d at 20±2°C for growth. Formation of a clear zone

around bacterial colonies was considered as +ve

result.

Organic ‘P’ Solubilization (Phosphatase)Bacterial

isolates were spot inoculated on tryptose phosphate

agar supplemented with methyl green (0.05 mg mL–1

)

as indicator dye. Plates were incubated for 3-5 d at

20± 2°C for growth. Development of green colour by

bacterial colonies was considered as +ve result.

Genotypic Characterization

Recovery of Genomic DNA

Total DNA from bacterial isolates was prepared

following the procedure outlined by Bazzicalupo and

Fani24

with the exception that for Gram-negative

bacteria, no lysozyme was used. Quantification and Detection of Purity of Extracted DNA

Extracted genomic DNA was run in 0.8% agarose

gel at 80 V for 45 min with quantitative marker in one

lane (Low DNA Mass Ladder, MBI Fermentas). Gel

was visualized under UV transilluminator. DNA was

quantified spectrophotometrically by measuring OD

at 260 nm and 280 nm. Purity of DNA was checked

measuring the extinction at A260/A280 on a DU 640 B

Beckman spectrophotometer. The concentration of

DNA was calculated as:

[DNA] = A260 × 100 × Dilution factor (µg mL-1

)

REP-PCR (Box Element)

REP-PCR fingerprints were obtained using the

following BOX-AIR sequence as primer25

: 5′ GAT

CGG CAA GGC GAC GCT GAC G 3′. The

amplification was carried out in 30 µL reaction

mixture containing following working concentration:

Milli Q water, Taq DNA Pol buffer + MgCl2 15 mM,

1.0 ×; dNTP (10 mM), 0.3 mM; Box Primer (10 µm),

0.25 µM; Taq DNA Polymerase (3 U), 1.0 U/Rxn;


100

and Target DNA, 20-100 ng. One positive control

with standard DNA template and one negative control

without DNA were maintained to check reaction

mixture preparation and contamination. The reaction

conditions were: Initial denaturation at 95°C for 5

min, followed by 40 cycles of denaturation at 95°C

for 1 min, annealing at 60°C for 1 min, extension at

72°C for 1 min, and final annealing at 60°C for 5 min

and extension at 72°C for 5 min. The reaction was

performed in a PTC-100 Thermal cycler (MJ

Research). Amplified product was analyzed by

agarose gel (2.0%) electrophoresis in 1× TAE buffer

at 70 V for 4 h. The amplification profile was

analyzed using NTSYS pc version 2.02i. The

clustering was done using Jaccard’s similarity

coefficient based on presence and absence of band

ignoring their intensities. Only sharp and consistent

bands were considered for analysis.

PCR Amplification of 16S rDNA

The amplified 16S rRNA gene was obtained from

each bacterial isolate by PCR amplification employing

the eubacterial universal primers26

fDI (5′-

AGAGTTTGATCCTGG -3′) and rP2 (5′-

TACCTTGTTACGACTT -3′) which were targeted at

universally conserved regions and permitted

amplification of ~1,500-bp fragment. PCR

amplification was carried out in a PTC-100

thermocycler (MJ Research). Reaction tubes contained

25 ng (5 µL) of DNA extract, 1 U of Taq polymerase

(Genei), 1× buffer (10 mM Tris-Chloride [pH 9.0], 1.5

mM MgCl2, 500 mM KCl) (Genei), 10 mM dNTPs

(Genei) and 0.25 mM of each primer (Genei). Initial

DNA-denaturation and enzyme activation steps were

performed at 95°C for 7 min, followed by 25 cycles of

denaturation at 94°C for 1 min, annealing at 51°C for 1

min and extension at 72°C for 1 min, and a final

extraction at 72°C for 10 min. The presence and yield

of specific PCR product (16S rRNA gene) was

monitored on 0.8% agarose (w/v) (Life Technologies

Inc.); gel electrophoresis was carried out at 100 V for

30 min in 1× Tris-acetate-EDTA buffer and

visualization by ethidium bromide staining and

viewing on a UV transilluminator (Biosystematica).

Amplified rDNA Reaction Analysis (ARDRA)

16S rDNA amplification product of Pseudomonas

standards (Departmental culture collection at GB Pant

University of Agriculture & Technology, Pantnagar)

and recovered isolates was digested with three

restriction endonucleases; Msp1, Alu1, and Taq1 that

were tetracutters. 15 µL of (120 µg) of 16S rDNA

amplification product was digested with restriction

endonuclease in the reaction mixture. For 30 µL

reaction mixture, the following were added:

Restriction enzyme (10 U), 1.0 U (MBI Fermentas);

Restriction buffer (10 ×), 1.0 × (MBI Fermentas); 16S

rDNA amplicon 120 µg, and Milli Q water. The

reaction mixture was incubated at 37°C for Alu1 and

Msp1, and at 65°C for Taq1 enzyme for 4 h.

Restriction product was resolved on 2.5% agarose gel

in 1× TBE at 60 V for 5 h and visualized on a UV

transilluminator (GelDocMega, Biosystematica). The

restriction profile was analyzed using NTSYS pc

version 2.02i. The clustering was done using

Jaccard’s similarity coefficient based on presence and

absence of band ignoring their intensities.

Partial Sequencing of 16S rDNA

PCR products obtained from 33 bacterial strains

were purified with an EXO-SAP. Components were

supplemented with gold buffer (Applied Biosystem)

and sequenced on an Applied Biosystem 310 Genetic

analyzer (ABI Prism 310 Genetic analyzer), using big

dye terminator cycle sequencing Ready Kit (Lab

India). The partial sequences amplified by the f DI

primer were used to determine the similarities.

Homology tree for the data sets were inferred from

the Fast Alignment method by employing software

DNAMAN version 4.0, Lynnon Biosoft, USA.

Several accessions (borrowed from NCBI database)

were employed for making relationship among

recovered bacterial isolates (present study) from

different stages of two casing materials under

evaluation.

Nucleotide Sequence Accession Numbers

The sequences determined in this study have been

deposited in the GenBank database of NCBI and

discussed under the accession numbers: AY961042-

AY961047, AY961049-AY961053, AY961055-

AY961059, AY961061, AY967718-AY967722,

AY967724, DQ074746, DQ074751-DQ074752,

DQ074754-DQ074757.

Cultivation of Button Mushroom

The experimental trial was conducted seasonally

for the year (2003-04) under controlled conditions

(temperature 18-25°C and relative humidity 70-80%)

in crop room at the Mushroom Research and Training

Centre, Pantnagar. FYM (2-yr-old), SMS and VC

(1-yr-old) based (3:1 v/v), chemically treated (4%

formalin) casing material was used for cultivation of

button mushroom. Mushroom cultivation was carried


101

out in polybags of 45 × 60 cm2 size each containing

10 kg of wheat straw-chicken manure based

pasteurized compost. The spawn of strain X-13 of A.

bisporus was mixed with the compost @ 0.75% and

spawn run done at 22-25°C. After complete spawn

run, the bags were cased uniformly with above casing

mixtures (4.0 cm thickness). Five replicates of each

treatment were arranged in completely randomized

design (CRD). The yield data were obtained for six

weeks of cropping and were subjected to Analysis of

Variance (ANOVA) in terms of kg mushrooms/100

kg compost. The quality parameters of the

mushrooms harvested from casing, FYM+SMS (3:1)

and FYM+VC (3:1) were assessed from first flush,

which included: protein content (mg g-1

of fresh wt),

whole mushroom wt (g), whole mushroom length

(cm), pileus diameter (cm), pileus thickness (cm),

stipe length (cm), stipe diameter (cm), pileus wt (g),

stipe wt (g), whiteness (%) and dry weight of

mushroom (g).

Results Physico-chemical Properties of Casing Samples

The casing sample FYM+SMS (3:1) had minimum

bulk density (BD) (0.60 g/cm3), while the casing

FYM+VC (3:1) showed a BD value of 0.68 g/cm3.

The porosity was significantly high (92.0%) for

FYM+SMS. Water holding capacity appears directly

related to porosity and bulk density and may directly

affect microbial build up and yield of A. bisporus. It

was high (191.19%) for FYM+SMS. A significant

increase in electrical conductivity (EC) was observed

for FYM+VC (570.33 deci-simen-1

) whereas low

value (398.00 deci-simen-1

) was obtained for

FYM+SMS (Table 1). The high value of EC is

harmful for fructification of button mushroom.

Macro and Micronutrients Analysis of Casing

A. bisporus was grown on wheat straw based

mushroom compost (pasteurized/long method). Two

casing mixtures prepared from three casing materials,

FYM, SMS and VC were evaluated for their

nutritional status. In order to assess the nutritional

status of casing mixtures (FYM + SMS, FYM + VC)

during various stages of cropping, status of

availability macro and micronutrients were analyzed

(Table 2). Maximum level of organic matter was

recorded from the two casing mixtures (28.91% ± 0.2

& 26.42% ± 0.08) collected at mycelium-impregnated

stage, followed by the pinning stage (27.76% ± 0.2 &

25.82 % ± 0.3). Minimum organic matter for

FYM+SMS and FYM+VC was recorded in samples

collected at the harvesting stage (26.06 % ± 0.08 &

25.02 % ± 0.6); 0 day stage casing soil mixtures

showed comparatively lesser organic matter content,

i.e., 25.38 % ± 0.11 and 24.81 % ± 0.2. A significant

variation was recorded in FYM+SMS casing

preparation, which showed different nutritional status

than FYM+VC. Micronutrients estimation (ppm)

included Cu, Zn, Mn and Fe. The relative order of

nutritional status (macro and micronutrients) for

various stages of casing mixture was, mycelium-

impregnated stage > pinning state > harvesting stage

> 0 day stage (Table 2).

Population Dynamics of Recovered Isolates

There was little variation (6.02 ± 0.81 to 5.87 ±

0.81) in total bacterial population counts (log10 CFU)

among casing layers FYM+SMS and FYM+VC tried

at successive stages of cropping (Fig. 1). The casing

layer FYM+VC from the mycelium-impregnated

stage showed slightly higher population (6.02 ± 0.81)

compared to FYM+SMS (5.92 ± 1.69). The lowest

population was observed at pinning stage in FYM +

SC (5.87 ± 0.81).

The endotrophic mesophilic bacterial population

recovered from tissues at primordial stage and button

stage showed little variation. Bacterial population was

higher at button stage (5.52±1.69) compared to

primordial stage (5.34±1.24) (Fig. 2).

Physiological and Functional Characterization

Considerable differences were observed among

isolates recovered from successive stages of casing

soils, including endotrophs, in their

nutritional/physiological characteristics. Most of the

isolates utilized citrate and exhibited NO3– reductase

Table 1—Physico-chemical properties of casing mixtures

Properties No. Casing soil

Bulk density

(g/cm3)

Porosity

(%)

Water holding

capacity(%)

pH Electrical conductivity

(deci-simen-1)

1 FYM+SC; 3:1 0.60 92.00 191.19 7.21 398.00

2 FYM+VC; 3:1 0.68 82.00 95.97 7.13 570.33

FYM= Farm Yard Manure, SMS= Spent Mushroom Substrate, VC= Vermi Compost


102

and catalase activity. Bacteria recovered from

primordial and button stages showed oxidase, catalase

activity and utilized trehalose as sole carbon-source.

None of the isolates hydrolyzed gelatin (Table 3).

Bacterial morphotypes isolated from FYM+VC at

mycelium-impregnated stage showed protease,

cellulase, siderophore and ‘P’-solubilizing activity.

However, none of the isolates recovered from this

casing mixture at 0 d, pinning and harvesting stages

exhibited protease, cellulase, amylase, pectinase,

xylanase and siderophore activity; but exhibited ‘P’-

solubilization activity which was maximum for 0 day

stage morphotypes. Endotrophic bacterial

morphotypes isolated from primordial and button

stages of cropping showed good amylase activity.

Amylase +ve diversity was much higher for

primordial stage morphotypes compared to button

stage.

Table 2—Chemical characteristics with respect to macro and micronutrients for casing mixtures recovered during the successive stages of

button mushroom

FYM + SC (3 :1) FYM + VC (3 :1)

Characteristics 0 d stage Mycelium-

impregnated

stage

Pinning

stage

Harvesting

stage

0 d stage Mycelium-

impregnated

stage

Pinning

stage

Harvesting

stage

Organic matter

(%) 25.38 ± 0.11 28.91 ± 0.20 27.76 ± 0.20 26.06 ± 0.08 24.81 ± 0.20 26.42 ± 0.08 25.82 ± 0.30 25.02 ± 0.16

Macronutrients

Organic C (%) 14.10 ± 0.14 16.06 ± 0.16 15.42 ± 0.14 14.47 ± 0.23 13.78 ± 0.07 14.67 ± 0.01 14.34 ± 0.01 13.9 ± 0.2

N (%) 0.92 ± 0.02 1.41 ± 0.05 1.32 ± 0.04 1.09 ± 0.09 1.02 ± 0.04 1.33 ± 0.08 1.21 ± 0.02 1.15 ± 0.40

P (%) 0.78 ± 0.01 1.20 ± 0.09 1.05 ± 0.01 0.98 ± 0.04 0.8 ± 0.05 1.08 ± 0.02 0.94 ± 0.04 0.90 ± 0.02

K (%) 0.90 ± 0.02 1.21 ± 0.01 1.01 ± 0.02 1.06 ± 0.19 0.83 ± 0.12 1.16 ± 0.01 1.04 ± 0.06 0.94 ± 0.11

Ca (ppm) 11.51 ± 0.18 13.72 ± 0.08 13.8 ± 0.16 12.91 ± 0.07 7.99 ± 0.14 11.76 ± 0.04 10.16 ± 0.01 9.69 ± 0.16

Mg (ppm) 5.23 ± 0.02 5.01 ± 0.02 4.98 ± 0.02 4.84 ± 0.02 6.33 ± 0.01 7.89 ± 0.18 7.56 ± 0.05 5.8 ± 0.01

Micronutrients

Cu (ppm) 0.025 ± 1.65 0.037 ± 1.03 0.032 ± 0.97 0.028 ±1.04 0.016 ± 0.01 0.023 ± 0.96 0.019 ± 1.43 0.017 ± 1.36

Zn (ppm) 0.128 ± 0.04 0.416 ± 0.01 0.329 ± 0.01 0.282 ± 0.01 0.067 ± 0.11 0.269 ± 0.08 0.188 ± 0.01 0.221 ± 0.01

Mn (ppm) 1.01 ± 0.01 1.2 ± 0.02 1.16 ± 0.03 1.10 ± 0.01 0.86 ± 0.02 1.04 ± 0.16 0.98 ± 0.05 0.91 ± 0.02

Fe (ppm) 0.059 ± 0.01 0.088 ± 0.02 0.076 ± 0.03 0.063 ± 0.01 0.034 ± 0.02 0.056 ± 0.01 0.048 ± 0.01 0.041 ± 0.02

FYM= Farm Yard Manure, SMD= Spent Mushroom Snbstrate, VC= Vermi Comprost

Fig. 1—Total population count log10 cfu of recovered mesophilic

bacteria on KBA from successive stages of casing soils. 1- 0 d

stage, 2- Mycelium-impregnated stage (MIS), 3- Pinning stage

(PS), 4- Harvesting stage (HS)

Fig. 2—Total endotrophic population count log10 cfu on KBA for

primordial (1) and button stage (2)


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104

Morphotypes isolated from FYM+SMS at

mycelium-impregnated stage showed higher diversity

of protease and cellulase. Low potential of cellulolytic

bacteria was observed in FYM+SMS casing

morphotypes from the harvesting stage. Morphotypes

isolated from different stages of cropping from FYM

+ SMS casing layer also showed siderophore and ‘P’-

solubilization activity; harvesting stage morphotypes

were an exception where none of the isolates showed

‘P’-solubilization. A significant variation in

siderophore secreting diversity was observed in

FYM+SMS morphotypes from mycelium-

impregnated stage. Maximum ‘P’-solubilization

diversity was observed in FYM+SMS layer

morphotypes derived from the mycelium-impregnated

stage and least in those recovered at the pinning stage

of FYM+SMS (3:1); none of the isolates recovered

from successive stages of cropping exhibited amylase,

xylanase and pectinase producing morphotypes

(Table 3).

Genotypic Characterization

REP-PCR (Box Element)

Distinctive banding pattern was observed in the

Box element. The dendrogram of banding patterns

based on UPGMA showed significant discriminatory

relationship among the isolates. Isolates placed within

8 clusters showed 100% similarity within any single

group. Isolates CVC2, TVC1 and TVC2 exhibited

100% similarity with each other and made single

group. Likewise, isolates USC11-USC14, UVC2-

UVC4 made another single group with 100%

similarity. Isolates USC12, USC15 and USC25-

USC32 made single largest group wherein 10 isolates

showed 100% similarity with each other, whereas

smallest groups made by isolates UVC5, UVC8 and

PH4, FB1 wherein 2 isolates exhibited 100%

similarity with each other (Fig. 3).

ARDRA Analysis of Bacterial Isolates

Fifty casing soils isolates, representating different

cropping stages/environmental niche (source location)

including endotrophs, were selected based on colony

morphology, and were subjected to ARDRA analysis

by digestion of the amplified 16S rRNA gene with

Taq1, Msp1, and Alu1. The dendrogram of banding

patterns was obtained after combination of the three

independent digestions. The similarity value ranged

widely between 50-100%. The first group was

represented by reference strains, viz., biovar of P.

fluorescens I-V that showed 100% similarity, whereas

reference strain P. chlororaphis showed, ~60%

similarity with biovars of P. fluorescens. Most groups

showed 90-100% similarity with some groups

delineated at 80-90% similarity level. Isolates USC5

and USC7-USC10 made single largest group and

exhibited 100% similarity with each other, whereas

isolates USC3-USC4 and USC13-USC14 made two

smallest groups with 100% similarity. Other isolates

exhibited discriminatory relationship with each other

(Fig. 4). 16S rRNA Gene Partial Sequencing

Partial 16S rDNA sequences were determined for

33 isolates and were compared with the corresponding

sequences of standard strains obtained from the

GenBank database to evaluate the phylogenetic

diversity. Most strains were grouped together and

were close to the genus Pseudomonas and

Acinetobacter of the family Pseudomonadaceae and

Moraxellaceae, respectively. Four strains recovered

from basidiocarp of A. bisporus, i.e., FB1

(DQ074754), PH4 (DQ074755), PH1 (DQ074756),

and PH3 (DQ 074757), exhibited high level of

similarity with reference strain Bacillus (AY842872)

and they showed 55% similarity with reference strain

Sphingobacterium (AY881645) (Fig. 5).

Family PseudomonadaceaeOf 33 strains, 11 exhibited

close proximity with the genus Pseudomonas. All the

11 strains showed 95-99% similarity with reference

strain Psedomonas putida (AY785244).

Family MoraxellaceaeLarge numbers of strains were

distributed in family Moraxellaceae and they

exhibited close proximity to genus Acinetobacter.

Largest group of this family compromised of 5 strains

(UVC4, TSC2, UVC3, USC2 & USC29), which

exhibited close proximity (95-100%) with reference

strains Acinetobacter sp. (AY362002) and

A. calcoaceticus (AJ888983). Yield and Quality of Fruit Body of Button Mushroom

The data on effect of casing treatments on fruit

body yield and quality are presented in Table 4. The

bags cased with casing mixture of FYM+SMS (3:1)

took a minimum of 11 d for case run during cropping,

while FYM and VC (3:1) took a maximum of 14 d.

The yield data for 6 wk of cropping showed that

FYM+SMS favoured development of a higher

number (1202.3) of sporophores. On the basis of yield

performance, it was concluded that the casing

material prepared by FYM+SMS in was the superior

over FYM+VC (Table 4).


105

Discussion Considering the significance of mushroom

production in the country and yet limited availability

of information on casing soil microflora, an exhibitive

analysis of bacterial diversity of this unique material

was undertaken. A significant variation in bacterial

flora was observed at various stages of cropping from

the casing soils. The different morphotypes were

screened and quantified based on colony and cellular

morphology. Microbial populations and communities

have been described in terms of a wide range of

phenotypic traits, many of which are related to the

practical interest in the habitat studied27

.

The casing material was so selected that its nutritional

status was very low compared to that of compost

and was thus expected to create conditions of nutritional

Fig. 3—Composite UPGMA dendrogram of bacterial isolates based on REP-PCR (Box). 1-50 in dendrogram represent strains USC1-

USC15, USC23-USC32, UVC1-UVC5, UVC8, CSC1-CSC2, TSC1-TSC2, CVC1-CVC2, TVC1-TVC3, SFB, TFB, TPH, PH1-PH4 and

FB1-FB3, respectively.

Table 4—Effect of casing treatments on mushroom yield and fruit body quality

Yield Fruit body quality*

Casing

treatment

Case

run

period

(d)

No. wt

(kg/100kg

compost)

Soluble

protein

(mg g-1)

Mushroom

wt

(g)

Mushroom

length

(cm)

Pileus

diam

(cm)

Pileusthi

ckness

(cm)

Stipe

length

(cm)

Stipe

diam

(cm)

Pileus

wt

(g)

Stipe

wt

(g)

Whiteness

(%)

Dry

wt

(g)

FYM+SMS

(3:1)

11 1202.3 14.70 8.12 14.98 4.8 6.8 2.3 2.6 1.8 6.68 2.68 83 9.89

FYM+VC

(3:1)

14 1089.6 12.28 6.98 12.56 3.3 4.3 1.7 2.1 1.4 5.33 1.96 74 7.36

CD at 5% 78.2 0.75

FYM= Farm Yard Manure, SMS= Spent Mushroom Substrate, VC= Vermi Compost *Fruit body quality: All readings are mean of 10 values


106

stress. If the casing material is rich in organic matter, it

is likely to disturb the selectivity of compost and thus

provide continued support to the vegetative growth of

the fungus and thus discourage fruiting.

Characteristically, microorganisms bring about physical

and chemical changes to their habitats28

. Water holding

capacity appears directly related to porosity and bulk

density. These factors are directly affecting microbial

build up and yield of A. bisporus and was maximum

(191.19%) for FYM+SMS (3:1). In general, increase in

electrical conductivity (EC) is almost proportional to

decrease in number of pinheads16

. Data of the present

investigation on EC (Table 1) and mushroom yield

(Table 4) exhibited that the higher EC of the casing

FYM+VC (3:1) resulted in lower yield. The results

indicate that the EC plays an important role in the

production of button mushroom but it is not the sole

controlling factor. These findings are in agreement with

the finding of Bhatt et al29

. Carbon/nitrogen content

seemed to be directly affecting the mushroom yield30

. In

addition, physico-chemical properties of Indian casing

materials have also been reported by Singh et al31

. The

macro and micronutrients levels increased and decreased

correspondingly.

Molecular tools for the identification of casing soil

bacteria were used and 16S rRNA gene analysis was

intensively used to understand the phylogenetic

relationships. Among the 16S rRNA gene analysis,

amplified ribosomal DNA restriction analysis (ARDRA)

was performed. This molecular technique has been

successfully used for bacterial community analysis in

a great variety of environments32

. This study

examined the cultivable bacterial community

comprising successive stages of casing soils and

endotrophs using ARDRA and 16S rRNA

gene fragment sequencing. Results show considerable

Fig. 4—Composite UPGMA dendrogram of Pseudomonas standards together with 50 casing layer bacterial isolates including endotrophs

based on 16S rDNA restriction profile. 1-56 in dendrogram represent strains P. fluorescens bv. 1, P. fluorescens bv. 2, P. fluorescens bv.

3, P. fluorescens bv. 4, P. fluorescens bv. 5, P. chlororaphis, USC1-USC15, USC23-USC32, UVC1-UVC5, UVC8, CSC1-CSC2, TSC1-

TSC2, CVC1-CVC2, TVC1-TVC3, SFB,TFB, TPH, PH1-PH4 and FB1-FB3, respectively.


107

diversity of bacterial community in casing soils based

on the large number of ARDRA patterns obtained.

Phylogenetic analysis revealed that 85% of the

bacterial isolates belonged to γ-proteobacteria group

and other isolates were bacilli. A single isolate in this

study was found to belong to the genus

Fig. 5—Homology tree of 33 bacterial isolates recovered from casing soils including endotrophs and their comparison with sequences

(AB020205, AY842872, AY881645, AJ888983, AY362002, and AY785244) borrowed from the GenBank. The numbers at the

branching points are the percentage of occurrence of in 1000 bootstrapped tree. Tree was made by Fast Alignment Method of Software

DNAMAN ver 4.0 of Lynnon Biosoft.


108

Sphingobacterium. Two genera, Acinetobacter and

Pseudomonas were dominant and were sole

representative of γ-proteobacteria. Dominance of the

genus Acinetobacter was of significance since this has

not earlier been reported from the mushroom casing

ecosystem.

Bacterial community of casing layers used in

mushroom production and bacteria recovered from

pinheads and fruiting bodies of A. bisporus were

assessed in this study. Morphotypes recovered at various

stages of cropping and those from the fruit bodies were

different compared to reference strains, P. fluorescens

and P. chlororaphis. Box element (rep-PCR) enabled

discrimination amongst bacterial isolates recovered from

successive stages of casing soils and endotrophic

population. Most isolates showed 100% similarity

among themselves based on the presence of palindromic

unit sequence. To our knowledge, no 16S rDNA

sequences from microorganisms in the casing layer at

successive stages of cropping including endotrophs of A.

bisporus have been reported. Partial sequencing of

bacterial isolates showed a 62-100% overlap with

several other bacterial isolates. However, comparison of

the 16S rDNA region could be used to build up more

complicated phylogenetic affiliations, e.g., our sequence

analysis indicated that there is a cluster that incorporates

only endotrophs and another cluster incorporates

bacterial sequences recovered from mycelium-

impregnated stage of FYM-VC (Fig. 5). In this

preliminary study, 16S rDNA PCR and direct automatic

sequencing of the amplicons was only carried out on

culturable organisms isolated on the nonselective King’s

medium B (KMB). In conclusion, this is the first

preliminary report on the microbial diversity of casing

soils and demonstrates the presence of Acinetobacter sp.

that has not been previously described in casing

material.

Acknowledgement The financial support from the Ministry of

Environment and Forest, Government of India, New

Delhi to BNJ is greatfully appreciated. Authors are

grateful to Dr R P Singh (Joint Director), Dr R C

Verma and Dr K K Mishra of Mushroom Research

and Training Centre, G B Pant University of

Agriculture and Technology, Pantnagar for providing

necessary facilities and technical assistance in

preparation of casing materials.

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characterization of functional activity in composted...

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