Isolation and identification of Trichoderma species from...

183

Turk J Biol35 (2011) 183-194 © TÜBİTAKdoi:10.3906/biy-0905-8

Isolation and identification of Trichoderma species fromdifferent habitats and their use for bioconversion of solid waste

Ahsanur RAHMAN1, Most. Ferdousi BEGUM1, Matiur RAHMAN1, M. A. BARI1,G. N. M. ILIAS2, M. Firoz ALAM1

1Department of Botany, University of Rajshahi, Rajshahi 6205, BANGLADESH2Timber Products and Environment Monitoring Cell, Rural Electrification Board (REB),

Shiromoni, Khulna 9204, BANGLADESH

Received: 20.05.2009

Abstract: The native population of Trichoderma species collected from different habitats was quantified and characterised.The identified strains are T. harzianum (IMI-392432, 392433, 392434); T. pseudokoningii (IMI-392431) and T. virens(IMI-392430). Out of five strains, T. harzianum was the most common in all of the habitats. Colony forming units (cfu’s)of Trichoderma species varied significantly (P = 0.05) in different habitats and were positively correlated with the physico-chemical characteristics of the habitat. Trichoderma was found to have adapted to an acidic habitat. In testing for anefficient bioconversion agent, T. harzianum (IMI-392432) was found to be the most effective in kitchen wastedecomposition. It provided the highest volume (31.80%) and weight (30.80%) losses in waste treated with sporesuspension. Promising results were also noted using a combined treatment with different strains/species of Trichoderma,which resulted in 18% greater decomposition of waste than the control.

Key words: Isolation, distribution, Trichoderma, kitchen waste, bioconversion

Farklı habitatlardan Trichoderma türlerinin izolasyonu ve tanımlanması ve katıatıkların biyolojik dönüşümü için kullanımı

Özet: Farklı habitatlardan toplanan Trichoderma spp.’nin yerli populasyonlar karakterize edilmiş ve miktarlarıbelirlenmiştir. Tanımlanan suşlar, T. harzianum (IMI-392432, 392433, 392434); T. pseudokoningii (IMI-392431) ve T.virens (IMI-392430) olarak isimlendirilmiştir. Beş suş arasından, T. harzianum bütün habitatlarda en yaygın olanıdır.Trichoderma türlerinin koloni oluşturan birimleri (cfu’ler) farklı habitatlarda önemli bir şekilde (P = 0,05) değişmiş veolumlu bir şekilde habitatın fiziko-kimyasal özellikleri ile ilişkilendirilmiştir. Trichoderma’nın asidik bir habitata adapteolduğu bulunmuştur. Etkili biyolojik dönüşüm ajanı denenerek mutfak atığı ayrıştırmasında en etkili türün T. harizanumolduğu bulunmuştur. Spor süspansiyonu ile muamele edilen atıklardaki en yüksek hacim (% 31,80) ve ağırlık kayıpları(% 30,80) bulunmuştur. Gelecek vaadeden sonuçlar, kontrolden %18 daha fazla ayrıştırma ile sonuçlanan Trichoderma’nınfarklı suş ve türleri ile kombine edilmiş bir uygulama kullanılarak da bildirilmiştir.

Anahtar sözcükler: İzolasyon, dağılım, Trichoderma, mutfak atığı, biyolojik dönüşüm

IntroductionTrichoderma species are cosmopolitan fungi,

frequently present in all types of soil, manure anddecaying plant tissues (1). Their dominance in soilmay be attributed to their diverse metabolic capabilityand aggressive competitive nature (2). Thesecharacteristics make them significant decomposers ofwoody and herbaceous material. Trichoderma speciesare also able to degrade domestic waste relativelyquickly without emitting bad odours (3). Among thevarious global environmental hazards, municipalsolid waste (MSW) is one of the greatest modernproblems. Every day large quantities of domestic,kitchen, and municipal waste are generated indeveloped and developing countries (4). Propermanagement and safe disposal of MSW is a major,complex problem that can affect the air, land, andwater. Therefore, attention has been focused on non-hazardous, environmentally friendly, sustainabletechniques involving bioconversion or biologicallybased treatment of domestic waste to overcome thisserious problem (5). Microorganisms from relevantenvironments possess greater degradation capabilitieson related wastes in the biodegradation process (6).Thus, bioconversion or biodegradation might be apotentially effective measure for proper managementof municipal solid waste. Therefore, the presentinvestigation was undertaken to isolate, identify, andquantify Trichoderma species from different habitatsof the Rajshahi metropolitan area and the premises ofthe compost plant of the Natore Development Society(NDS). The physico-chemical characteristics of thesamples were analysed and correlations betweensample characteristics and colony forming units (cfus)were determined. The biodegradative potential ofTrichoderma isolates was also evaluated on kitchensolid waste in vitro.

Materials and methodsSample collection Different types of samples such as soil, humus,

kitchen waste, and compost were collected fromdifferent sites in the Rajshahi metropolitan and itssuburban areas, and on the premises of the NDScompost plant in Natore from April to July 2003. Soilsamples were collected from the different sites (e.g.,

Botanical garden, the 3rd Science building and theAgriculture Research Field of Rajshahi University(RU), Budpara, and agriculture field at Khorkhori,Rajshahi Metropolitan City). Humus was collectedfrom the Rajshahi City Corporation dumping site,Kashiadanga. Kitchen wastes were collected fromdustbins in Rokeya Hall, Nawab Abdul Latif Hall, theTeachers Quarter, the Railway Station, BangabandhuHall and the IBS quarters of RU Campus, and fromthe Binodpur Bazaar, the Kazla Bazaar, the ShahebBazaar and the Science lab quarters of Rajshahi CityCorporation. Compost was collected from thecompost plant and compost heap of NDS, Natore. Thesamples were taken from a 15 cm depth and collectedin sterile polyethylene bags, which were transportedto the laboratory and stored at 4 °C until use.

Isolation, identification, and quantification ofTrichoderma spp.

For isolation of Trichoderma strains, a serialdilution technique was followed and a 103 dilution ofeach sample was prepared. One millilitre of eachsolution was pipetted onto a Rose Bengal Agar (RBA)plate and incubated at 28 °C for 1 week. The cultureplates were examined daily and each colony thatappeared was considered to be one colony formingunit (cfu). After enumeration of cfu, individualcolonies were isolated from the same plates and eachuncommon colony was reisolated onto a fresh PotatoDextrose Agar (PDA) plate. Distinct morphologicalcharacteristics were observed for identification, andthe plates were stored at 4 °C.

Two techniques, visual observation on petri dishesand micro-morphological studies in slide culture,were adopted for identification of Trichodermaspecies. For visual observation, the isolates weregrown on PDA agar for 3-5 days. The mode of myceliagrowth, colour, odour and changes of medium colourfor each isolate were examined every day. For micro-morphological studies, a slide culture technique wasused (6). Examination of the shape, size, arrangementand development of conidiophores or phialidesprovided a tentative identification of Trichoderma spp.Samples were compared to a taxonomic key for thegenus Trichoderma (7) and further verified andconfirmed by CABI, Bioscience, Surrey, UnitedKingdom.

Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste

184

Estimation of moisture content and acidity (pH)of the samples

Freshly collected samples were placed in glasstubes and the initial weights were recorded. Thesamples were then placed inside a hot-air incubatorat 110 °C until constant weights were achieved. Themoisture content was calculated using the followingformula provided by American Wood PreservationAssociation’s Standards (AWPA) (8):

MC (%) = W – w / W × 100 where MC is moisture content, W is the initial weightand w is the constant weight after oven drying.

The acidity of the samples (pH) was measuredusing the method of Black (9).

Chemical analysis of the samplesThe chemical characteristics of all the samples

were analysed by the Soil Analysis Laboratory, SoilResource Development Institute, Regional Office,Shampur, Rajshahi, for the following: organic matter(%), total nitrogen (N) (%), phosphorus (P) (mg/g),and potassium (K) (mL/100 g). Nitrogen wasdetermined by the micro Kjeldhal method. AvailableP was determined using the Modified Olsen method,K was determined using the N NH4OAC method, andorganic matter was determined using the drycombustion method (10).

Preparation of organic solid waste forbioconversion

Kitchen wastes were collected from differentgarbage centres on the RU Campus in polythene bags.Samples were cut into 2-3 mm pieces and 50 g of eachwas aliquotted into a 150 mL bottle, which was thensealed with a cotton plug. The bottles containingkitchen solid waste were then autoclaved at 121 °Cand 1.05 kg/cm2 pressure for 15 min.

Preparation and application of culture discsTrichoderma strains were cultured on potato

dextrose agar (PDA) medium at 30 °C for 48 h. Thena culture disc (5 mm diam.) was cut with a cork borerand 6 culture discs were inoculated with autoclavedkitchen waste. Control treatments were performedwith no inoculation. A sterile thermometer was alsopushed through the cotton plug of each bottle. Thebottles were then sealed with Para film and labelled.

The entire process was completed inside a laminarflow hood.

Preparation and application of sporesuspensions of Trichoderma

Ten millilitres of autoclaved distilled water wasadded to a 4-day-old Trichoderma culture tube andmixed well. Next, 5 mL of this spore suspension wasadded to each autoclaved kitchen waste bottle andmixed well. For the consortium, 1 mL of sporesuspension of each strain (total 5 mL) was used.Control treatments were also performed with noinoculation. As with the culture disc experiment,sterile thermometers were used and the bottles weresealed with Parafilm.

Changes in odour, temperature, volume loss andweight loss of the decomposed organic solid wastewere observed at 10-day intervals up to 30 days. Formeasurement of volume loss (%), the followingformula was used:

Volume loss (%) = V-V1/V × 100 where V is initial volume, V1 is final volume.

Weight loss (%) of decomposed garbage wascalculated employing the following formula:

Weight loss (%) = W-W1/W × 100where W is initial weight, W1 is final weight.

Statistical analysisThe experiment was conducted using a complete

randomised design (CRD) with 3 replications.Correlations were calculated between the sample’sphysico-chemical characteristics and cfus. The datawere statistically analysed with the help of thecomputer package MSTAT-C and also tested byDMRT (11).

Results and discussion Isolation and identification of Trichoderma spp.In this study, a total of 135 isolates of Trichoderma

were isolated from different samples (Table 1). On thebasis of morphological characteristics, these 135isolates of Trichoderma were divided into 5 aggregategroups (Table 2). Representative isolates from eachgroup were sent for identification to CABIBiosciences, Surrey, United Kingdom, and the

A. RAHMAN, M. F. BEGUM, M. RAHMAN, M. A. BARI, G. N. M. ILIAS, M. F. ALAM

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Table 1. Frequency of isolation of Trichoderma species and their sources of origin.

Sources of Trichoderma spp. Sample types Species name and isolate number No. of isolates

BG (Botanical garden, RU) Soil T. virens (RUT-101, RUT-106, RUT-107, RUT-108, RUT-109, RUT-110). 6T. harzianum (RUT-111, RUT-112, RUT-113, RUT-114, RUT-115, RUT-116, RUT-117). 7

RH (Rokeya Hall, RU) Kitchen waste T. harzianum (RUT-118, RUT-119, RUT-120, RUT-121, RUT-122, RUT-123) 6T. pseudokoningii (RUT-102, RUT-124, RUT-125, RUT-126, RUT-127, RUT-135, RUT-136). 7

3SB (3rd science building, RU) Soil T. harzianum (RUT-103, RUT-137, RUT-138, RUT-139, RUT-140, RUT-141, RUT-142, RUT-143. RUT-144). 9T. virens (RUT-145, RUT-146, RUT-147, RUT-148, RUT-149, RUT-150, RUT-151, RUT-152). 8

NALH (Nawab Abdul Latif Hall, RU). Kitchen waste T. harzianum (RUT-153, RUT-154, RUT-155, RUT-156, RUT-157). 5T. pseudokoningii (RUT-158, RUT-159, RUT-160, RUT-161). 4

TQWP (Teachers quarter west para RU). Kitchen waste T. harzianum (RUT-162, RUT-163, RUT-164, RUT-165, RUT-166, RUT-167). 6T. pseudokoningii (RUT-168, RUT-169, RUT-170, RUT-171, RUT-172). 5T. virens (RUT-173, RUT-174, RUT-175, RUT-176). 4

RSRU (Railway station, RU). Kitchen waste T. harzianum (RUT-177, RUT-178, RUT-179). 3BB (Binodpur Bazaar, Rajshahi). Kitchen waste T. harzianum (BBT-180, BBT-181, BBT-182). 3KR (Kazla Bazaar, Rajshahi). Kitchen waste T. harzianum (KJT-183, KJT-184, KJT-185, KJT-186). 4SB (Shaheb Bazaar, Rajshahi). Kitchen waste T. pseudokiningii (SBT-187, SBT-188, SBT-189). 3

T. harzianum (SBT-190, SBT-191, SBT-192. SBT-193, SBT-194). 5BHRU(Bangabandhu Hall, RU). Kitchen waste T. harzianum (RUT-195, RUT-196, RUT-197). 3MHRU (Mannuzan Hall, RU). Kitchen waste T. harzianum (RUT-104, RUT-199, RUT-200, RUT-201). 4

T. pseudokoningii. (RUT-202, RUT-203, RUT-204, RUT-205, RUT-206, RUT-207). 6

ARF (Agriculture research field, RU). Soil T. harzianum (RUT-208, RUT-209, RUT-210). 3IBSQRU (IBS quarter, RU). Kitchen waste T. harzianum (RUT-211, RUT-212, RUT-213, RUT-214). 4BPR, (Budpara, Rajshahi). Soil T. harzianum (BPRT-215, BPRT -216, BPRT -217, BPRT -218). 4TQPRU (Teacher quarter pubapara, RU). Kitchen waste T. harzianum (RUT-219, RUT-220, RUT-221). 3CCDS (City corporation dumping site, Humus T. virens (CCDKT-222, CCDKT -223, CCDKT-224, CCDKT-225). 4Kashiadanga, Rajshahi). T. pseudokoningii (CCDKT-226, CCDKT-227). 2

T. harzianum (CCDKT-228, CCDKT-229, CCDKT-230). 3KR (Agriculture field Khorkhori, Rajshahi.). Soil T. harzianum (AKRT-231, AKRT-232). 2SLQ (Science Lab Quarter, Rajshahi). Kitchen waste T. harzianum (SLQT-233, SLQT-234, SLQT-235). 3NDS (Natore Development Society, Natore) Compost T. harzianum (NDST-236, NDST-237, NDST-238, NDST-239). 4NDS (Natore Development Society, Natore). Compost T. harzianum (NDST-105, NDST-240, NDST-241, NDST-242, NDST-243). 5

Total isolates = 135

number of Trichoderma groups was reduced to 3species. These species of Trichoderma were identifiedas T. harzianum Rifai (IMI -392432, 392433& 392434)(86 isolates), T. pseudokoningii Rifai (IMI-392431) (27isolates), and T. virens (J.H. Mill. Gidden & A.A.Foster) Arx. (IMI-392430) (22 isolates) (Figure 1).The identified Trichoderma species are characterised

in Table 2. T. harzianum was the most frequentlyisolated species, and it was recovered in nearly all thesamples. In a similar study, Zakaria (12) isolated andidentified 5 species of Trichoderma from therhizosphere of rubber trees in Malaysia, namely T.hamatum (Bon). T. harzianum Rifai, T. koningii(Oudemans), T. longibrachiatum Rifai, and T. viride


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Table 2. Characterisation and identification of Trichoderma species isolated from different habitats.

Group and Trichoderma spp. Sources ofStrain with samples Macro-/Microscopic characteristics

IMI No.

Initially the colony colour was observed to be whitish to light green, watery in centre. Next the colony

Group-1 gradually became deep grass green in colour and looked soft T. virens (IMI -392430) Humus, and leathery to the naked eye. The conidiophores were erect,

RUT-101 BG, RU smooth, penicillately branched; asymmetrical branches singly or vertically arranged at different levels, phialides were flask-shaped, coverage toward the main branch, emphasizingthe penicillate branching. Phialospores were sub-globose to elliptical, smooth-walled. (Figure 1, 1a & 1b).

Whitish to pale green, hairy and flappy mycelial mat uniformly Group-2 grown in PDA plate at 3-4 days. Next pale green turned into whitish

T. pseudokoningii (IMI -392431) Kitchen waste, green to dark green colour. Branched conidiophores and dendroid RUT-102 RH, RU conidiophores terminated by phialides carried confused ellipsoids to

subglobose phialospores. Phialospores were pigmented, smooth, elliposide up to a little more than 4 mm long (Figure 1, 2a&2b).

At the early stage whitish to greenish mycelia appeared. Group-3 Next a deep green colour developed in central part and gradually

T. harzianum (IMI -392432) Soil, extended to the periphery. Finally, it appeared a whitish green colour. RUT-103 3SB, RU Mostly globose conidia developed on phialides produced in the

opposite direction in each point. (Figure 1, 3a & 3b).

The colony colour was initially watery white and turned bright green Group-4 to dark green and dull green with compact conidiophores

T. harzianum (IMI -392433) Compost, throughout the petriplates. Reproductive structures were closely RUT-104 MH, RU similar to RUT-103. (Figure 1, 4a& 4b).

The colony was observed to be watery white at the early stage (3-4 days),Group-5 (IMI-392434) Compost heap, bright green and dark green coloured mycelia mat exhibited at the

T. harzianum NDS, Natore central part of PDA plate at the later stage. Reproductive structuresNDST-105 were very similar to RUT-103. (Figure 1, 5a&5b).

BG= Botanical garden; RH= Rokeya Hall; 3SB= 3rd Science building; MH= Mannuzan Hall; NDS= Natore Development Society.


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Figure 1. Photograph shows colonies of Trichoderma strains on PDA plate (dorsal view) andconidiophore with conidia. 1a & 1b. T. virens (IMI-392430), 2a & 2b. T. pseudokoningii(IMI-392431), 3a & 3b. T. harzianum (IMI-392432), 4a &4b. T. harzianum (IMI-392433) and 5a & 5b.T. harzianum (IMI-392434).

Per.ex S.F. Gray. Ilias (13) isolated 4 species ofTrichoderma in healthy and diseased oil palmrhizosphere in Malaysia, namely T. harzianum, T.aureoviride, T. longibrachiatum, and T. virens. Thesewere also identified following the classification ofRifai (7) but verified by IMI, England. It was exhibitedthat T. harzianum was the most abundant species.Okoth et al. (14) isolated 306 Trichoderma strainsfrom 60 soil samples from different places andobserved T. harzianum to be the most frequentlyisolated species, but in coffee farms, T. viride was themost frequently identified species. T. citrinoviride wasthe most common in Napier farms. The present resultis accordance with the above findings.

Quantification and distribution of TrichodermaTrichoderma spp. were recovered from different

habitats. The quantity of the fungi obtained was high,but unequal numbers of the various species werefound in different samples. Colony forming units(cfus) of Trichoderma isolates from the differenthabitats were significantly (P = 0.05) different (Table3 and Figure 2). Populations of Trichoderma speciesranged from 13.31 ± 0.28 to 34.68 ± 2.86 × 103/g inkitchen waste, 17.17 ± 0.39 to 35.32 ± 2.27 × 103/g insoil, 16.68 ± 1.46 × 103/g in humus, and 35.48 ± 2.88× 103 to 36.67 ± 3.66 × 103/g in compost and compostheap. The highest number of cfus (36.67 ± 3.66 ×103/g) was found in compost (NDS) and the lowestnumber of cfus (13.23 ± 0.87 × 103/g) was recorded inthe kitchen waste of Bangabandhu Hall, RajshahiUniversity. The identified Trichoderma species wererandomly distributed in different habitats.

Species of Trichoderma are distributed worldwide.They are generally found in soils, including foresthumus layers and agricultural and orchard soils (15).At the species level, however, individual speciesaggregates may be restricted in their geographicdistribution. Trichoderma populations weresignificantly different for the various sites on RajshahiUniversity campus and in its suburban area. Therewas a significant difference in distribution ofTrichoderma species among the different habitats.Seasonal variation is a factor that affects the numberof Trichoderma species in the samples. Greaterpopulations of Trichoderma species in soils have beenestimated in the spring and summer months ascompared to the autumn and winter (16). Danielson

and Devey (17) found that T. viride and T. polysporumare restricted to areas where low temperatures prevail.On the other hand, T. harzianum is most commonlyfound in warm climate regions. T. hamatum and T.koningii are widely distributed in areas of diverseclimatic conditions (15). In the present study, thesamples were collected in summer (April to July),which is in partial support of the above findings.

Effects of the physical and chemicalcharacteristics of the samples on the occurrence ofTrichoderma species

Soil moisture greatly affected the population (cfus)of Trichoderma species. In the present study, themoisture content of the samples was high, and apositive and significant (P = 0.05) correlation (datanot shown) was observed between the moisturecontent and cfus of almost of the samples. Highmoisture content might be helpful to increase thepopulation of Trichoderma. Populations ofTrichoderma spp. have been reported to be greater inmoist soils or even in soils with excessive moisture ascompared to dry soils (17). Population densities ofTrichoderma were found to be positively associatedwith sample moisture when soil samples werecollected throughout the year in a California alfalfafield (18). Therefore, it may be noted that the densityof the Trichoderma population is dependent on themoisture of habitats.

The results revealed that the pH level in differenthabitats was moderately acidic, and there was apositive correlation between acidity and thepopulation (cfus) of Trichoderma, although this wasnon-significant (P = 0.05). Soil acidity has previouslybeen reported as a factor affecting the presence anddensity of Trichoderma spp. (19). T. viride and T.polysporum Rifai were more prevalent in acidicconiferous forest soils, whereas T. hamatumpredominated in more neutral deciduous forest soils(20), and T. viride predominated in acidic pine forestsoil (21). Therefore, it may be considered that somespecies of Trichoderma may be adapted to acidichabitats.

The sample’s key nutrients, organic matter,nitrogen, phosphorus, and potassium, displayed somevariations with respect to the distribution andabundance of Trichoderma (14). In this study, organic


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190

Tabl

e 3. L

ocat

ion,

sour

ces,

moi

sture

cont

ent,

pH, a

nd ch

emic

al ch

arac

teris

tics o

f diff

eren

t sam

ples

.

Sam

ple n

o.Sa

mpl

e So

urce

s of

Moi

sture

pH

Org

anic

To

tal N

(%)

PK

No.

of c

fu’s

loca

tion

sam

ple

cont

ent (

%)

mat

ter (

%)

μg/g

Ml/1

00 g

(103 /g

of s

ampl

es)

S/1

BG, R

USo

il46

.58

± 5

.44

ab6.

43 ±

0.2

6 bc

de8.

6 ±

0.1

j0.

23 ±

0.0

2 e

55.8

4 ±

5.3

2 k

0.51

± 0

.03

j20

.67

± 0

.45

efg

S/2

RH, R

UKi

tche

n W

aste

44.0

7 ±

5.61

ab6.

35 ±

0.2

2 de

13.4

± 0

.06

ef0.

78 ±

0.0

3 c

214.

67 ±

1.5

1 f

1.58

± 0

.75

f24

.59

± 0.

33 cd

e

S/3

3sb,

RU

Soil

47.5

5 ±

3.48

ab6.

2 ±

0.38

efg

8.22

± 0

.92

j0.

32 ±

0.0

5 de

79.2

3 ±

1.94

ij0.

54 ±

0.0

4 i

17.1

7 ±

0.39

fgh

S/4

NA

LH, R

UKi

tche

n w

aste

44.6

2 ±

0.36

ab5.

9 ±

0.15

fgh

12.2

± 0

.26

fg0.

49 ±

0.0

4 cd

e15

0.5

± 5.

43 g

0.56

± 0

.08

h22

.67

± 0.

97 d

ef

S/5

TQW

P, RU

Kitc

hen

was

te48

.58

± 0.

48 ab

5.7

± 0.

13 h

ij15

.3 ±

0.1

de

0.79

± 0

.02

c13

9.74

± 4

.93

g1.

58 ±

0.1

4 f

19.3

7 ±

0.98

efg

S/6

RSRU

Kitc

hen

was

te41

.47

± 0.

38 ab

6.86

± 0

.04

ab16

.51

± 2.

01 d

1.2

± 0.

06 b

214.

33 ±

6.4

3 f

1.04

± 0

.33

g26

.68

± 1.

28 cd

S/7

BB, R

UKi

tche

n w

aste

43.2

2 ±

4.92

ab5.

76 ±

0.0

8 gh

i11

.61

± 0.

08 fg

h0.

37 ±

0.0

2 de

35.8

6 ±

7.09

l1.

41 ±

0.0

3 f

13.3

1 ±

0.28

h

S/8

KRKi

tche

n w

aste

47.0

8 ±

1.29

ab6.

77 ±

0.0

3 ab

c18

.91

± 0.

13 b

c1.

63 ±

0.3

7 a

261.

42 ±

1.2

6 e

3.96

± 0

.21

ef30

.2 ±

1.2

2 bc

S/9

SBKi

tche

n w

aste

36.9

9 ±

1.26

bcd

5.3

± 0.

05 k

8.91

± 0

.22

ij0.

27 ±

0.0

5 e

60.9

3 ±

1.16

jk0.

46 ±

0.0

2 k

16.3

1 ±

1.06

gh

S/10

BHRU

Kitc

hen

was

te37

.64

± 3.

15 ab

cd5.

33 ±

0.0

8 jk

8.14

± 0

.52

j0.

22 ±

0.1

5 e

62.6

7 ±

2.64

ijk

0.39

± 0

.03

l13

.23

± 0.

87 h

S/11

MH

RUKi

tche

n w

aste

45.1

9 ±

4.26

ab6.

67 ±

0.0

3 ab

cd19

.62

± 0.

28 ab

1.6

± 0.

06 a

1138

± 4

.12

abc

4.04

± 0

.62

ef34

.33

± 2.

87 ab

S/12

ARF

Soil

41.4

7 ±

2.81

ab6.

9 ±

0.12

a21

.82

± 0.

24 a

1.72

± 0

.01

a11

45 ±

9.8

4 ab

c24

.56

± 0.

08 b

35.3

2 ±

2.27

ab

S/13

IBSQ

RUKi

tche

n w

aste

43.5

3 ±

4.62

ab5.

8 ±

0.06

ghi

12.6

1 ±

0.05

fg0.

61 ±

0.0

2 cd

109.

9 ±

6.26

h7.

35 ±

1.5

8 de

24.3

1 ±

2.16

de

S/14

BPR

Soil

48.7

7 ±

2.58

a6.

7 ±

0.12

abcd

17.2

4 ±

0.58

cd1.

21 ±

0.0

6 b

1128

.33

± 4.

09 b

c21

.21

± 3.

46 b

c34

.46

± 2.

62 ab

S/15

TQPU

RKi

tche

n w

aste

49.3

6 ±

4.12

a5.

7 ±

0.11

hijk

10.5

1 ±

0.03

ghi

0.23

± 0

.58

e21

9.76

± 4

.67

f17

.43

± 3.

56 c

27.2

8 ±

3.56

cd

S/16

CCD

SH

umus

28.7

6 ±

5.36

d5.

4 ±

0.12

ijk

9.84

± 0

.15

hij

0.26

± 0

.02

e83

.17

± 3.

18 i

10.9

6 ±

2.29

d16

.68

± 1.

46 g

h

S/17

KRSo

il30

.29

± 3.

19 cd

5.9

± 0.

1 fg

h16

.58

± 0.

49 d

1.32

± 0

.02

b11

00 ±

1.6

3 d

20.7

9 ±

3.23

bc

33.3

2 ±

2.94

ab

S/18

SLQ

Kitc

hen

was

te39

.59

± 4.

88 ab

c6.

5 ±

0.05

abcd

16.6

5 ±

1.02

cd1.

58 ±

0.8

9 a

1127

.33

± 4.

08 c

23.2

7 ±

3.62

b34

.68

± 2.

86 ab

S/19

ND

SC

ompo

st47

.58

± 4.

83 ab

5.9

± 0.

12 fg

h19

.85

± 0.

82 ab

1.72

± 0

.19

a11

58.3

3 ±

0.88

a31

.02

± 2.

93 a

36.6

7 ±

3.66

a(C

ompo

st pl

ant)

S/20

ND

SC

ompo

st45

.34

± 3.

26 ab

6.3

± 0.

27 d

ef19

.96

± 0.

89 ab

1.68

± 0

.04

a11

49.6

7 ±

5.21

bb

35.8

4 ±

2.34

a35

.48

± 2.

88 ab

(Com

post

heap

)

Sam

e let

ters

with

in a

colu

mn

are n

ot si

gnifi

cant

ly d

iffer

ent b

y D

MRT

at th

e 5%

leve

l.BG

=Bot

anic

al g

arde

n, R

U; R

H=R

okey

a H

all,

RU; 3

SB=

3rdsc

ienc

e bu

ildin

g RU

; NA

LH=

Naw

ab A

bdul

Lat

if H

all,

RU; T

QW

P= T

each

ers q

uart

er w

est p

ara,

RU; R

SRU

= Ra

ilway

stat

ion,

RU

; BB=

Bino

dpur

Baz

aar,

Rajsh

ahi;

KR=

Kazla

, Raj

shah

i; SB

= Sh

aheb

Baz

ar, R

ajsh

ahi;

BHRU

= Ba

ngab

andh

u H

all,

RU; M

HRU

= M

annu

zan

Hal

l, RU

; ARF

= A

gric

ultu

re re

sear

ch fi

eld,

RU

; IBS

Q=

IBS

quar

-te

r, RU

; BPR

= Bu

dpar

a, Ra

jshah

i; TQ

PRU

= Te

ache

rs q

uarte

r pur

bapa

ra, R

U; C

CDS=

City

corp

orat

ion

dum

ping

site

, Rajs

hahi

; KR=

Kho

rkho

ri, R

ajsha

hi,S

LQ=S

cien

ce L

ab q

uarte

r, Ra

jshah

i; N

DS=

Na-

tore

Dev

elop

men

t Soc

iety

,Nat

ore.

matter showed a significant (P = 0.05) and positivecorrelation with the population of Trichoderma spp.(data not shown). Nitrogen showed a non-significant(P = 0.05) positive correlation with the occurrence ofTrichoderma species in almost of all the habitats (datanot shown). Trichoderma spp. have been recoveredmore frequently from different sources with highorganic matter content (22). Nitrate-nitrogen is not apreferred nitrogen source for this genus (16). Thepresent result is completely in accordance with thefindings of these studies. The results of the currentstudy further show that P and K had a positive andsignificant (P = 0.05) correlation with Trichodermaspecies (data not shown). Okoth et al. (14) observedthe same results with the most common species ofTrichoderma, i.e. T. harzianum and T. viride.

Bioconversion of solid wasteFungi play an important role in the decomposition

of organic waste and can be important contributorsto optimal agricultural waste bioconversion (23).Among the 5 strains studied, T. harzianum (IMI-392432) showed the highest volume (31.81%) andweight (31.80%) losses using spore suspension (Table4). Using culture discs, the highest volume (22.72%)and weight (28.03%) losses were also observed in T.harzianum (IMI-392432). Zheng and Shetty (24)reported that Trichoderma is capable of producingvarious polysaccharide degrading enzymes, whichmay help to degrade long chain carbon compounds,particularly the bulking materials that normallycomprise sewage sludge. Martin and Dale (25) usedweight loss measurement to estimate the tissuedegradation of Bermuda grass (Cynodon dactylon L.)

stolons and Zoysia grass (Zoysia japonica Steud,‘Mayer’) stolons. Bari et al. (26) reported 31.80% and28.03% weight loss when culture pellets and sporesuspensions of T. harzianum were used, respectively.

Combined treatment with the Trichoderma strainshad promising effects in the decomposition of organicsolid waste, resulting in a greater loss (18%) of theoriginal material than the control. Bari et al. (26)reported that combined treatment was the mosteffective method, promoting 23% greater weight lossthan the control in their experiments. Thus, thecomposition of the microbial culture affects thequality of the compost. In another previous study,mixed microbial inoculants comprised of Trichurusspirals, Aspergillus, and Paccilomyces fusisporusaccelerated the process of decomposition, resulting ina 10% greater loss than the control in 12 weeks ofdecomposition (27).

ConclusionFrom the above findings, it can be concluded that

a significant number of Trichoderma species occupydifferent habitats. Some species of Trichoderma werefound to be adapted to acidic habitats. Thepopulations (cfus) are significantly and positivelycorrelated with moisture, organic matter, phosphorus,and potassium, but non-significantly and positivelycorrelated with pH and nitrogen in almost all of thesamples. The most frequently isolated species was T.harzianum, and the strain T. harzianuum IMI- 392432was identified as an effective agent for solid wastebioconversion using spore suspension. The co-culture


191

BG RH3S

BN

ALH

TQW

P

RSRUBB K

RSB

BHRU

MH

RUARF

IBSQ

RUBP

R

TQPRU

CCD

SKR

SLQ

ND

SCO

M

ND

SCH

Location of Trichoderma isolates

Thic

ho

der

ma

po

pu

lati

on

(C

FU

/10

3g)

4540353025201510

50

cfu’s of Trichoderma spp.

Figure 2. Comparison of Trichoderma population with different habitats.


192

Tabl

e 4. C

hang

es in

tem

pera

ture

, vol

ume l

oss,

and

wei

ght l

osso

f sol

id w

aste

usin

g di

ffere

nt T

richo

derm

a iso

late

s

Stra

inCh

ange

s of c

hara

cter

istic

s of s

olid

was

te af

ter 1

0 da

ys

Chan

ges o

f cha

ract

erist

ics o

f sol

id w

aste

afte

r 20

days

Chan

ges o

f cha

ract

erist

ics o

f sol

id w

aste

afte

r 30

days

Tem

pera

ture

Vo

lum

e W

eigh

tTe

mpe

ratu

reVo

lum

eW

eigh

tTe

mpe

ratu

reVo

lum

eW

eigh

t(°

C)lo

ss· (

%)

loss

• (%

)(°

C)lo

ss• (

%)

loss

· (%

)(°

C)lo

ss• (

%)

loss

· (%

)

CD29

4.72

±0.

094

10.9

2±0.

296

SPS

3014

.81

±0.1

2517

.68

±0.3

17SP

S29

20.7

2 ±1

.320

24.1

4±0.

529

T.vi

rens

(IM

I-39

2430

)SP

S28

6.95

±0.1

4911

.82±

0.28

4CD

3018

.99

±0.1

5420

.88

±0.4

04CD

2828

.32

± 0.

526

27.9

0±0.

264

CD29

4.45

±0.0

5910

.23±

0.21

8SP

S30

13.6

3 ±0

.107

16.5

4 ±0

.581

SPS

2918

.81±

0.49

123

.28±

0.36

0T.

pse

udok

onin

gii.

(IM

I-39

2431

)SP

S28

6.25

±0.5

5412

.58±

0.57

7CD

3018

.72

± 0.

102

22.1

8 ±0

.417

CD28

2830

±0.

644

28.0

2±0.

721

CD29

5.18

±0.1

3912

.44±

0.88

1SP

S30

17.2

7 ±0

.105

21.0

0 ±0

.577

SPS

2922

.72±

0.41

528

.03±

0.79

3T.

har

zian

um (I

MI-

3924

32)

SPS

287.

63±0

.137

14.9

9±0.

577

CD30

20.4

5 ±0

.112

23.8

6 ±0

.404

CD28

31.8

1±0.

515

31.8

0±0.

529

CD29

4.45

±0.1

0611

.66±

0.15

2SP

S30

15.5

4 ±0

.105

19.2

3 ±0

.138

SPS

2922

.45

±0.2

6425

.98±

0.21

4T.

har

zian

um (I

MI-

3924

33)

SPS

285.

63±0

.126

13.2

5±0.

665

CD30

19.7

2 ±0

.122

20.8

8 ±0

.346

CD28

30.0

8 ±0

.567

28.8

8±0.

105

CD29

6.18

±0.1

1511

.24±

0.33

3SP

S30

14.5

4 ±0

.107

19.9

2 ±0

.208

SPS

2921

.73±

0.39

625

.76±

0.43

5T.

har

zian

um (I

MI-

3924

34)

SPS

286.

90±0

.189

13.4

4±0.

305

CD30

20.7

2 ±0

.112

21.4

4 ±0

.321

CD28

29.3

5±0.

366

29.0

4±0.

142

T.h(

IMI-3

9243

2,39

2433

,392

434)

SPS

288.

90±0

.132

15.8

7±0.

120

SPS

3126

.45

±0.1

1524

.82

±0.5

85SP

S29

33.3

5 ±0

.199

36.5

6±0.

624

+ T.

v (I

MI-

3924

30)+

T. p

(IM

I-39

2431

)C

ontro

l-

273.

45±0

.463

8.02

±0.1

11-

278.

18 ±

0.52

315

.06±

0.75

0-

2717

.36±

0.46

618

.56±

0.32

1

•Mea

n of

3 re

plic

atio

ns, S

PS=

Spor

e sus

pens

ion,

CD

= Cu

lture

disc

.T.

h= T

richo

derm

a ha

rzia

num

, T. v

= T

richo

derm

a vi

rens

, T. p

= T

richo

derm

a ps

eudo

koni

ngii.


193

use of different species and strains of Trichodermaresulted higher and quicker ways of solid wastedecomposition than the use of a single species orstrain.

AcknowledgementThe authors express thanks to the Ministry of

Science, Information and CommunicationTechnology, Bangladesh, for financial support, and

are also grateful to CABI Bioscience, U.K., for theidentification of Trichoderma strains.

Corresponding author:Most. Ferdousi BEGUMDepartment of Botany, University of Rajshahi,Rajshahi 6205, BANGLADESHE-mail: ferdrita @yahoo.com

0

5

10

15

20

25

30

35

40

BG RH

3SB

NA

LH

TQ

WP

RSR

UBB K

RSB

BHRU

MH

RU

ARF

IBSQ B

PR

TQ

PRU

CC

DS

KR

SLQ

ND

SC

OM

ND

SCH

Location of Trichoderma isolates

Tri

ch

od

erm

a p

op

ula

tio

n (

CF

U/1

03 g

)

0

200

400

600

800

1000

1200

1400

Ph

ysi

cal

an

d c

hem

ical

ch

ara

cte

rist

ics

of

the s

am

ple

s

cfu N P K pH om

Figure 3. Effect of physico-chemical characteristics on the occurrence of Trichoderma spp.BG=Botanical garden; RH=Rokeya Hall; 3SB= 3rd science building; NALH= Nawab AbdulLatif Hall; TQWP= Teachers quarter west para; RSRU= Railway station; BB= BinodpurBazaar; KR= Kazla Rajshahi; SB= Shaheb Bazaar; BHRU= Bangabandhu Hall; MHRU=Mannuzan Hall; ARF= Agriculture research field; IBSQ= IBS quarter; BPR= BudparaRajshahi; TQPRU= Teachers quarter purbapara, RU; CCDS= City corporation dumping site;KR= Khorkhori Rajshahi; SLQ= Science Lab quarter; NDSCOM= Natore DevelopmentSociety Compost plant;. NDSCH=Natore Development Society Compost Heap.

1. Alexander M. Introduction to soil microbiology. In. Wiley J &Sons. New York and London; 1961.

2. Elad Y. Biological control of foliar pathogens by means ofTrichoderma harzianum and potential modes of action. CropProt 19: 709-714, 2000.

3. Ilias GNM, Rahman MA, Molla AH et al. Composting ofmunicipal garbage by using Trichoderma- a new approach incontext of Bangladesh. Bangladesh J Genet Biotech 6 (1&2): 75-78, 2005.

4. Bartone C. Strategies for improving municipal solid wastemanagement: Lesson forum world bank lending and CWGactivities. Workshop planning for sustainable and integratedsolid waste management Manila: Washington, DC. UrbanManagement Division. World Bank. USA; 2000.

5. Molla AH, Razi FA, Aziz AS et al. A potential resource forbioconversion of domestic waste water sludge. Biores Technol85: 263-272, 2002.

References

6. Leahy JG, Colwell RR. Microbial degradation of hydrocarbonsin the environment. Microbiol Rev 54(3): 305-315, 1990.

7. Rifai MA. A revision of the genus Trichoderma. Mycologicalpapers no. 116. Commonwealth Mycological Institute, Kew,Surrey, England. 1969.

8. AWPA. Book of Standards. American Wood-preserversAssociation Standards. Maryland 21666. USA; 1986.

9. Black GR. Bulk Density. In: Black CA. Ed. Methods of SoilAnalysis Part 1. American Society of Agronomy. Madison, USA;1965: Agronomy 9.

10. Page AL, Miller RH, Keeney (eds.). Methods of soil analysis.Part 2. Chemical and microbiological properties. 2nd ed.Agronomy series 9, ASA, SSSA, Madison, Wis, USA; 1982.

11. Ressel, DF. MSTAT-C. Package Program. Dept. of Crop and SoilSci., Michigan State Univ., USA; 1996.

12. Zakaria MH. Some Aspects of the biology and chemicallyassisted biological control of Ganoderma species in Malaysia,PhD, University of Putra, Malaysia, 1989.

13. Ilias GNM. Trichoderma and its efficacy as a bio-control agentof basal stem rot of oil palms, PhD, University of Putra,Malaysia, 1999.

14. Okoth SA, Roimen H, Mutsotso B et al. Land use systems anddistribution of Trichoderma species in Embu region, Kenya.Trop and Sub trop Agroeco Sys 7: 105-112, 2007.

15. Roiger DJ, Jeffers SN, Caldewell RW. Occurrence ofTrichoderma species in apple orchard and woodland soils. SoilBiol Biochem 23: 353-359, 1991.

16. Widden P, Abitbol JJ. Seasonality of Trichoderma species in aspruce forest soil. Mycologia 72: 775-784, 1980.

17. Danielson RM, Devey CB. The abundance of Trichodermapropagules and the distribution of species in forest soil. Soil BiolBiochem 5: 485-494, 1973.

18. Eastburn DM, Butler EE. Microhabitat characterization ofTrichoderma harzianum in natural soil: evaluation of factorsaffecting population density. Soil Biol Biochem 20: 541-545,1988.

19. Weindling R, Fawcett HS. Experiments in biological control ofRhizoctonia damping off. Phytopathology 24: 1142, 1934.

20. Widden P. Fungal populations from forest soils in southernQuebec. Can J Bot 57: 1324-1331, 1979.

21. Roiger DJ, Jeffers SN. Evaluation of Trichoderma spp. forbiological control of Phytophthora crown and root rot of appleseedlings. Phytopathology 81(8): 910-917, 1991.

22. Novak RO, Whittaingham WF. Soil and litter microfungi of amaple-elm-ash flood plain community. Mycologia 60: 776-787,1968.

23. Raimbault M. General and microbiological aspects of solidsubstrate fermentation. J Biotechnol 1(3): 1-15, 1998.

24. Zheng Z, Shetty K. Cranberry processing waste for solid statefungal inoculant production. Process Biochem 33(3): 323-329,1998.

25. Martin SB, Dale JL. Biodegradation of turf thatch with wood-decay fungi. Phytopathology 70: 297-301, 1980.

26. Bari MA, Begum MF, Sarker KK et al. Mode of action ofTrichoderma spp. on organic solid waste for bioconversion.Plant Environ Dev 1(1): 61-66, 2007.

27. Hajra JN. Enrichment of compost with microbial inoculants.In: Sen SP and Palit P. ed. Biofertilizers Potentialities andProblems; Plant physiology forum, Calcutta: pp.249-254, 1988.


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