Research ArticleProduction and Multiplication of Native CompostFungal Activator by Using Different Substrates andIts Influence on Growth and Development ofCapsicum chinensis Jacq lsquolsquoBhut Jolokiarsquorsquo

Vipin Parkash and Ankur Jyoti Saikia

Mycology and Soil Microbiology Research Laboratory Rain Forest Research Institute (ICFRE)An Autonomous Council of Ministry of Environment amp Forests Government of India Deovan Jorhat Assam 785001 India

Correspondence should be addressed to Vipin Parkash bhardwajvpnparkrediffmailcom

Received 10 July 2014 Revised 12 December 2014 Accepted 12 December 2014

Academic Editor Manuel Canovas

Copyright copy 2015 V Parkash and A J Saikia This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In vitro experiment was carried out to see the effect of saw dusts of Pinus kesiya Shorea robusta and Callicarpa arborea onTrichoderma harzianum isolate TH-13mass production along with its biotization effect onCapsicum chinensis Jacq ldquoBhut JolokiardquoEarly mycelium initiation (2 days) occurred in S robusta followed by P kesiya and C arborea (35 days) The sporulation wasobserved earlier in S robusta (100 after 6 days) than P kesiya (334 after 8 days) and C arborea (167 after 9 days) but nosporulation was observed in control The complete sporulation was also earlier in S robusta (100 after 10 days) than P kesiya(334 after 15 days) and C arborea (164 after 18 days) Higher conidial yield (86 times 106) was also in S robusta than P kesiya(70 times 106) and C arborea (45 times 106) respectively The increase in height (60ndash70 cm) number of leaves (600ndash650) and yield ofchili (120ndash150 fruits) were also more in inoculated C chinensis seedlings than control It is concluded that S robusta saw dust is thebest substrate for mass production of compost fungal activator and can be used in nursery practices for quality stock productionof various cropsplantations

1 Introduction

Compost is a mixture of decayed organic material decom-posed by microorganisms in a warm moist and aerobicenvironment that release nutrients into readily availableforms for plant use Among these microorganisms fungilike Trichoderma spp Pers are important microbes that helpin decomposition of organic material and are known asldquocompost fungal activatorrdquo (CFA) Trichoderma spp producehyphae which are branched creeping forming a tuft orcushion onnatural substrate septate conidiophores are erectindefinite arising from short branched side branches bearingphialides laterally and terminally phialides are surroundedby heads rarely by short chains of slime spores conidia arehyaline or bright coloured one celled and chlamydosporesare terminal or intercalary [1]

Mass production of Trichoderma spp has become afocus of research in the search for alternatives to pollutingpesticides and chemical fertilizers for control of plant diseasesand growth promoter of plantsThemajority of commerciallyavailable biocontrol fungi available in the world are of Tharzianum Rifai origin only and their efficiency is wellestablished Techniques for large-scale biomass generationof this fungus are still in infancy Consequently any mediaused for mass production of Trichoderma spp must beeconomical and able to support production of large quantitiesof biomass and available propagules A variety of mediahave been used by various researchers for production ofTrichoderma spp in stationary flasks [2] shakers [3 4] andliquid fermenters [5] Molasses-brewerrsquos yeast medium isbeing usedwidely for commercial production ofTrichodermaspp by fermentation process [6] In India many commercial

Hindawi Publishing CorporationBiotechnology Research InternationalVolume 2015 Article ID 481363 7 pageshttpdxdoiorg1011552015481363

2 Biotechnology Research International

Trichoderma producers are using molasses-yeast medium formass production Brewerrsquos yeast which serves as nitrogensource is expensive and there is a need to identify inexpensiveand more commonly available alternative to yeast fungi

Jin et al [7] developed amedium frommodified Richardrsquosmedium + V-8 Juice (RM8) to produce high level of des-iccation tolerant conidia of T harzianum strain 1295-22The addition of 9 percent (vv) glycerol to RM8 mediumimproved both biomass production and desiccation toleranceof conidia Several commercial formulations of species of Tri-choderma have beenmade [8] For commercial production itis essential that the product should possess certain qualitiesThe dose requirement should be minimum must have largeshelf life is easy to apply is free from contamination and haseconomic feasibility with positive monetary return

Trichoderma spp are mostly lignocellulose decomposerLignin protects cellulose hemicelluloses and carbohydrate inlignocellulosic materials Lignocellulolytic microorganismsare the key agents in depolymerizing the lignin barrierin organic materials Therefore the selection of effectivelignocellulolytic microbes is a crucial step leading to thesuccess in accelerating composting of lignocellulosic mate-rials Trichoderma spp are widely known as a lignocellulosedecomposer as already explained above because they arefilamentous and have the ability to produce prolific sporeswhich can invade substrates quickly [9] Various studieshave shown that composting of lignocellulosic materialspreinoculated with potential Trichoderma spp can reducethe time of biodegradation [10] T harzianum was used asinoculant to enhance composting of rice straw and weeds[11] However there is no or a little work that has been doneon composting of different saw dusts using Trichoderma spp(CFA) isolated from the related ecological habitats Hence astudy is carried out to analyze the different substrates (sawdusts) suitability for mass production of CFA along withits effect on growth and development of Capsicum chinensisJacq ldquoBhut Jolokiardquo C chinensis Jacq ldquoBhut Jolokiardquo is beingcultivated and consumed in different states ofNortheast Indiaincluding Assam Nagaland Manipur and Mizoram sinceantiquity It is either used as a spice in food or eaten raw alongwith the staple food On account of its refreshing aromapalatability and medicinal properties the populace has beenusing it for pickle preparation flavouring curries apart fromits usage for home remedies of ailments like gastritis arthritisand chronic indigestion problems It is also used as a remedyto summer heat presumably by inducing perspiration [12]To analyze the influence of CFA on growth and developmentldquoBhut Jolokiardquo was selected as it is cultivated by the localpeople in their homestead gardens as a spice and CFAtechnology is extended to Demo village of the Institute atMeleng Grant Jorhat Assam as cited below in nursery trialsin Material and Methods

2 Material and Methods

21 Collection of Soil Samples and Isolation of CompostFungal Activator The soil samples were collected fromNongkhyllem Reserve Forest Nongpoh Meghalaya India

Fungal species Trichoderma harzianum was isolated fromthe soil samples by using serial soil dilution method [13] onpotato dextrose agar (PDA) medium The inoculated plateswere incubated at 30∘C for 4 days The pure fungal colonieswere picked up and purified by streaking on agar slantsand incubated at 30∘C for 7-8 days Green conidia formingfungal bodies were selected and microscopic observationwas done and the fungus was identified to be Trichodermaharzianum (isolateaccession number TH-13) The preservedfungal isolateculture maintained on PDA slants is retainedwith Mycology and Soil Microbiology Laboratory RainForest Research Institute Jorhat Assam India for furtherstudy (Figures 1(a)ndash1(d))

22 Preparation of Solid Substrate Media In this experimentdifferent saw dusts like Pinus kesiya Royle ex Gordon Shorearobusta Gaertn and Callicarpa arborea Roxb were takenfor evaluation The different saw dusts were shade driedThe dried saw dusts were mixed with wheat bran by addingsterilized water in the ratio (wheat bran saw-dust water3 1 4 ww) as explained above The moisture level of themixture was maintained up to 50ndash60 The substrate wassterilized through Autoclave (Labotech BDI-81 make India)at 120∘C and 15 lbsi (Figure 1(e))

23 Mass Multiplication of Trichoderma Inoculum The ino-culum of Trichoderma harzianum was grown on syntheticPDA (Potato Dextrose Agar) medium (SRL India) for 7-8days and incubated at 27ndash30∘ plusmn 1∘C (Figures 1(f)-1(g)) Theinoculum was kept in BOD incubator (Labotech BDI-55make India) for 10ndash12 days for maximum growth and sporu-lation Then the inoculum containing medium was cut intosmall discs and was put in flasks containing wheat bran anddifferent saw-dust medium in the ratio (3 1 4 ww) for massproduction of Trichoderma harzianum Approximately 50 gof substratewas taken in 500mL conical flasks and inoculatedwith 5mm mycelial mat incubated at 28∘C incubator for 7ndash10 days earlier In control set no saw dust component wasadded to the substrate Six replicates of each treatment weretaken The colony forming units were calculated with helpof the following formula through serial dilution of one gramof substrate and the results are expressed as cfu gminus1mLminus1 ofsuspension of each substrate

CFUgmL = Number of colonies per mL platedTotal dilution factor

(1)

24 Nursery ExperimentsTrials Thesubstrate supplementedwith sawdust of Shorea robustawhich proved to be efficient inproduction ofmore cfu gminus1mLminus1 was selected for inoculationexperiment The target bioagent in the form of substrateinoculum was applied at the time of sowing of seedlingsin the nursery of Rain Forest Research Institutersquos Demovillage at Meleng Grant Jorhat Assam located at a distanceof 10 km east of Jorhat city on NH-37 (on Jorhat-TinsukiaHighway Assam India (26∘4610158405310158401015840N 94∘1710158402910158401015840E 107m asl))The annual average precipitation is 500mm and the annualaverage temperature is 26∘C The experiment was conductedusing field soil supplemented with substrate containing the

Biotechnology Research International 3

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q)

Figure 1 (a) Culture of Trichoderma harzianum (isolate TH-13) (b) mycelial mat of T harzianum (c) spores of T harzianum (d)hyphaemycelia of T harzianum ((e) (f)) preparation of substrates and filling of flasks (g) incubation of flasks at 27∘C in BOD incubatorfor 6ndash8 days ((h) (i) (j)) mass production trends of T harzianum on different substrates (k) mass production of T harzianum in a tray (l)variation in treatments on Capsicum chinense ldquoBhut Jolokiardquo after 7DAI ((m) (n) (o)) variation in treatments on C chinense ldquoBhut Jolokiardquoafter 30DAI (p) a control plant after 150DAI (q) a CFA treated plant after 150DAI

target bioagent in the field condition In control rows nobioagent (ie inoculasubstrate) was added The experimentwas designed in a single inoculation treatment Ten replica-tions of each treatment were taken The following design ofthe experiment was adopted1198790= control (no inoculation)1198791= treatmentinoculation of Trichoderma harzia-

num (isolate TH-13 supplemented substrate)Data on plant growth like increase in height (in cm) numberof leaves and total fruit yield (in number) per plant were

observed after 150DAI (days after inoculation) and analyzedto evaluate the efficacy of the preferred bioagent on thegrowth and development of target seedlings The data wereanalyzed statistically for calculating standard error of meanand coefficient of variance (CV) percent by using MS Excelversion 2003 and Gupta [14]

3 Results and Discussion

31 Mycelial Growth and Conidial Yield Different saw dusts(forestryindustry byproduct) were tested as a substrate

4 Biotechnology Research International

Table 1 Effect of substrate (type of saw dust) on Trichoderma harzianummass production

Substrate (3 1)(saw dust wheat bran)

Myceliuminitiation(in days)lowast

Initiation ofsporulation(in days)lowast

Completion ofsporulation(in days)lowast

Number ofreplicatescompletingsporulation

Percentage ofsporulation ()

Colony formingunit

(cfu gminus1mLminus1)lowast

A 3 plusmn 000 8 plusmn 000 15 plusmn 000 26 334 70 times 106 (plusmn001)B 2 plusmn 000 6 plusmn 000 10 plusmn 000 66 100 86 times 106 (plusmn002)C 3 plusmn 000 9 plusmn 000 18 plusmn 000 16 164 45 times 106 (plusmn000)

Control 5 plusmn 000 mdash Nosporulation mdash mdash mdash

CV () mdash mdash mdash mdash mdash 001A Pinus kesiya wheat bran B Shorea robusta wheat bran and C Callicarpa arborea wheat branlowastAverage of six replicates CV = coefficient of variance

constituent along with wheat bran for the mass productionof Trichoderma harzianum (isolate TH-13) micropropagulesas shown in Table 1 After 2 days of incubation at 27∘C plusmn 1the mycelial growth initiation was seen in substrate supple-mented with saw dust of Shorea robusta while in substrateswith Pinus kesiya and Callicarpa arborea mycelial growthappeared after 35 days

In control setmycelia appeared after 5 daysThe initiationof sporulation of bioagent was observed earlier in substrateof S robusta (after 6 days) than P kesiya (after 8 days) andC arborea (after 9 days) The completion of sporulation offungal activator was also observed earlier in substrates of Srobusta after 10 days (100) thanP kesiya after 15 days (334)and C arborea after 18 days (167) (Figures 1(h)ndash1(j))

No sporulationwas observed in control setThe enhancedconidial yield was also higher in S robusta (86 times 106) thanP kesiya (70 times 106) and C arborea (45 times 106) substrates interms of colony forming units (cfu gminus1mLminus1) respectivelyThe screening of various substrates for their potential tosupport T harzianum (as CFA) mass production indicatedthat among the tested saw dusts S robusta saw dust wasfound suitable in terms of production of high conidial yieldand early conversion of substrate into compostThis substratesupplemented with CFA was taken for further inoculationexperiment (Figure 1)

Fungi are one of the major agents of decompositionEstimating fungal biomass in waste is important for under-standing quantitatively their parts in the decompositionprocesses and the nutrient cycling in terrestrial ecosystemsTrichoderma and Gliocladium spp are the mostly workedout antagonists so far the production and delivery systemis concerned [15 16] So their mass production is essentialrequisite for bioinoculating the crops

The substrata used for mass production of inoculum ofTrichoderma spp are wheat straw sorghum grains ligniteand stillage molasses and brewerrsquos yeast and wheat branmedium [15ndash19] Studies have been carried out to evaluatevarious agroindustrial wastes including wheat straw paddystraw shelled maize cob paper waste and sugarcane baggasefor mass multiplication of T harzianum through solid statefermentation technology In order to enhance growth rate

and sporulation various cellulosic residues were also supple-mented with chickpea flour as organic nitrogen supplementat 2 and 4 percent Supplementation of all the substrates withchickpea flour enhanced growth and sporulation However4 chickpea flour supplementation gave maximum responsein all the cases Paddy straw followed by sugarcane baggaseand wheat straw each supplemented with 4 chickpea flourwere found to be promising substrates formassmultiplicationof T harzianum [20] Sterilized oat seeds were found effectivefor large-scale production of T harzianum [21] Dhingra andSinclair [22] also reported that dehulled broken rice grainswere found to be an excellent growth and delivery substratefor T harzianum and T koningii

In this investigation Trichoderma harzianum is culturedonly on wheat bran and different saw dusts which are wastematerials by sawmills for mass production It is reported thatthe sporulation of fungal activator was observed earlier andmore in numbers in substrates consisted of saw dust of Shorearobusta than Pinus kesiya and Callicarpa arborea Howeverthe production of spores of CFA is more in these substratesthan control set where no saw dust was added Noteworthyis the fact that lignin content of test saw dusts which wasobserved to be directly proportional to the conidial yieldof CFA Maximum sporulation (86 times 106 cfu gminus1mLminus1) wasobserved in the media fortified by S robusta where saw dustcontained maximum (53652 plusmn 166mggminus1 dry weight ofwood) lignin content [23] which was followed by P kesiya(70 times 106 cfu gminus1mLminus1) with comparatively less (23543 plusmn247mggminus1 dry weight of wood) lignin composition [24] Onthe other hand least conidial yield (45 times 106 cfu gminus1mLminus1)was observed inC arborea inwhich lignin contentwas 255of cortical dry weight [25]

Addition of T harzianum into organic media like neemcake coir pith farmyard manure and decomposed coffeepulp caused an immediate increase in the population upto three days Soil amendment with organic materials likeneem cake coir compost farmyard manure and Gliricidialeaves showed better growth and survival of antagonist thansoil alone [26] Previous reports suggested that additionof inorganic forms of nitrogen increases the production offungal biomass [27] Serrano-Carreon et al [28] had been

Biotechnology Research International 5

Table 2 Effect of Trichoderma harzianum inoculation on growth and development of Capsicum chinensis ldquoBhut Jolokiardquo after 150 DAI

Treatments Increase in height (in cm)lowast Number of leaveslowast Total fruit yield (in number) per plantlowast

Inoculated 650 plusmn 408 627 plusmn 496 130 plusmn 249Control 253 plusmn 410 426 plusmn 374 55 plusmn 408CV () 1124 083 466lowastAverage of ten replicates CV = coefficient of variance

able to prove that Trichoderma spp prefer the ammoniumform of nitrogen

According to Elad et al [29] biocontrol by Trichodermaviride is dependent on the type of inocula or substrate Differ-ent substrate treatments based on combination of substratelike wheat bran pulse bran sugarcane baggase rice strawwheat straw cow dung poultrymanure groundnut shell andsaw dust with tap water are evaluated for mass multiplicationof T harzianum and Gliocladium virens Many workers usedwheat bran as substrate for mass production of Gliocladiumspp and Trichoderma spp [29ndash33] Sorghum grains were alsoused for mass production of T harzianum These infectedsorghum grains served as bait for growth and multiplicationof T harzianum in soil [16]

A standard preparation of T harzianum and T koningiibyMehrotra [34] was in wheat bran saw-dust medium whichcan be very easily grown in autoclavable plastic bags ofdifferent sizes The preparation is ready for commercial usewithin fifteen days and is stored at room temperature Inthe present investigation also media prepared by combiningwheat bran and saw dusts resulted in maximum yield ofbiomass viable propagules and spores However saw dustas a constituent of substrate was used by previous workersbut through this study it is revealed that the saw dust thathas more lignin content is preferable for mass production ofCFA (Trichoderma spp) In this experiment wheat bran actedas carbohydrate source and saw dust served as medium formoisture for mass production of Trichoderma species

32 Biotization Effect on Growth Parameters The influenceof in vitro mass produced native compost fungal activator(substrate supplemented with CFA) was also observed ongrowth parameters ofCapsicum chinensis Jacq ldquoBhut Jolokiardquoin open field experiments consequent to 150 days afterinoculation

The datum as depicted in Table 2 obviously explains thesignificant increase in the growth parameters The increasein height (60ndash70 cm) number of leaves (600ndash650) andtotal fruit yield (in numbers) (120ndash150 chili fruits) wasalso reported more in inoculated plants with bioagent thannoninoculated control plants (height 20ndash30 cm leaves 400ndash500 and chili fruits 50ndash60) respectively (Figures 1(l)ndash1(q))

Trichoderma harzianum has been reported to promoteplant growth [35] Various strains of Trichoderma have beenfounded to be effective in plant growth characteristics andenhance biomass production [36] These fungi inhabit plantroots and promote plant growth characteristics by increasingevolution and production of some organic acids in therhizosphere such as gluconic citric andor fumaric acids by

Trichoderma which decrease soil pH and lead to increasedsolubility of the insoluble compound and an availability ofmicronutrient as well as an increase in plant nutrient uptake[37 38] Improvement of plant nutrient uptake and its trans-port from root to aerial parts together with the producedplant stimulators might result in higher photosynthetic rates[39] required for producing enough energy used to derivethe enhanced growth response [38] The CFA inoculationis observed to increase shoot height number of leaves andtotal fruit yield (in number) per plant in the present study inaccordance with the results which were obtained by previousworkers on various plants [38 40]

It would be beneficial to prepare a composted wheat branand sawdust (thatmust contain high lignin content) substratethat may serve as a nutritionally rich biodegradable productto be effective in improving the growth and development ofchili plants possibly by improving the organic content of soilThe composting of organic wastes with the help of microbialinoculants not only helps in recycling of waste but alsoresults in the preparation of economic and environmentalfriendly organic biofertilizer that could provide benefits toagriculturehorticultureforestry crops This technology ofbioinoculants should also be extended to other economicallyand medicinally important plants to check their potential toincrease the bioactive constituents and metabolites which isa future prospective

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The senior author Vipin Parkash is thankful to the IndianCouncil of Forestry Research and Education Dehradun forthe financial assistance in the Project no RFRI-422008-09SFM to carry out this research work

References

[1] M N Khare and H K Jharia ldquoTrichoderma a potentialbiocontrol agent against seed and soil borne pathogens forsustainable crop productionrdquo in Plant Pest Management P CTiwari Ed pp 263ndash276 Aavishkar Publishers amp DistributersJaipur India 2002

[2] S T Patel and A Mishra ldquoProduction of Trichoderma in sta-tionary flasksrdquoGujarat Agricultural University Research Journalvol 19 pp 53ndash56 1994

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Biotechnology Research International

Trichoderma producers are using molasses-yeast medium formass production Brewerrsquos yeast which serves as nitrogensource is expensive and there is a need to identify inexpensiveand more commonly available alternative to yeast fungi

Jin et al [7] developed amedium frommodified Richardrsquosmedium + V-8 Juice (RM8) to produce high level of des-iccation tolerant conidia of T harzianum strain 1295-22The addition of 9 percent (vv) glycerol to RM8 mediumimproved both biomass production and desiccation toleranceof conidia Several commercial formulations of species of Tri-choderma have beenmade [8] For commercial production itis essential that the product should possess certain qualitiesThe dose requirement should be minimum must have largeshelf life is easy to apply is free from contamination and haseconomic feasibility with positive monetary return

Trichoderma spp are mostly lignocellulose decomposerLignin protects cellulose hemicelluloses and carbohydrate inlignocellulosic materials Lignocellulolytic microorganismsare the key agents in depolymerizing the lignin barrierin organic materials Therefore the selection of effectivelignocellulolytic microbes is a crucial step leading to thesuccess in accelerating composting of lignocellulosic mate-rials Trichoderma spp are widely known as a lignocellulosedecomposer as already explained above because they arefilamentous and have the ability to produce prolific sporeswhich can invade substrates quickly [9] Various studieshave shown that composting of lignocellulosic materialspreinoculated with potential Trichoderma spp can reducethe time of biodegradation [10] T harzianum was used asinoculant to enhance composting of rice straw and weeds[11] However there is no or a little work that has been doneon composting of different saw dusts using Trichoderma spp(CFA) isolated from the related ecological habitats Hence astudy is carried out to analyze the different substrates (sawdusts) suitability for mass production of CFA along withits effect on growth and development of Capsicum chinensisJacq ldquoBhut Jolokiardquo C chinensis Jacq ldquoBhut Jolokiardquo is beingcultivated and consumed in different states ofNortheast Indiaincluding Assam Nagaland Manipur and Mizoram sinceantiquity It is either used as a spice in food or eaten raw alongwith the staple food On account of its refreshing aromapalatability and medicinal properties the populace has beenusing it for pickle preparation flavouring curries apart fromits usage for home remedies of ailments like gastritis arthritisand chronic indigestion problems It is also used as a remedyto summer heat presumably by inducing perspiration [12]To analyze the influence of CFA on growth and developmentldquoBhut Jolokiardquo was selected as it is cultivated by the localpeople in their homestead gardens as a spice and CFAtechnology is extended to Demo village of the Institute atMeleng Grant Jorhat Assam as cited below in nursery trialsin Material and Methods

2 Material and Methods

21 Collection of Soil Samples and Isolation of CompostFungal Activator The soil samples were collected fromNongkhyllem Reserve Forest Nongpoh Meghalaya India

Fungal species Trichoderma harzianum was isolated fromthe soil samples by using serial soil dilution method [13] onpotato dextrose agar (PDA) medium The inoculated plateswere incubated at 30∘C for 4 days The pure fungal colonieswere picked up and purified by streaking on agar slantsand incubated at 30∘C for 7-8 days Green conidia formingfungal bodies were selected and microscopic observationwas done and the fungus was identified to be Trichodermaharzianum (isolateaccession number TH-13) The preservedfungal isolateculture maintained on PDA slants is retainedwith Mycology and Soil Microbiology Laboratory RainForest Research Institute Jorhat Assam India for furtherstudy (Figures 1(a)ndash1(d))

22 Preparation of Solid Substrate Media In this experimentdifferent saw dusts like Pinus kesiya Royle ex Gordon Shorearobusta Gaertn and Callicarpa arborea Roxb were takenfor evaluation The different saw dusts were shade driedThe dried saw dusts were mixed with wheat bran by addingsterilized water in the ratio (wheat bran saw-dust water3 1 4 ww) as explained above The moisture level of themixture was maintained up to 50ndash60 The substrate wassterilized through Autoclave (Labotech BDI-81 make India)at 120∘C and 15 lbsi (Figure 1(e))

23 Mass Multiplication of Trichoderma Inoculum The ino-culum of Trichoderma harzianum was grown on syntheticPDA (Potato Dextrose Agar) medium (SRL India) for 7-8days and incubated at 27ndash30∘ plusmn 1∘C (Figures 1(f)-1(g)) Theinoculum was kept in BOD incubator (Labotech BDI-55make India) for 10ndash12 days for maximum growth and sporu-lation Then the inoculum containing medium was cut intosmall discs and was put in flasks containing wheat bran anddifferent saw-dust medium in the ratio (3 1 4 ww) for massproduction of Trichoderma harzianum Approximately 50 gof substratewas taken in 500mL conical flasks and inoculatedwith 5mm mycelial mat incubated at 28∘C incubator for 7ndash10 days earlier In control set no saw dust component wasadded to the substrate Six replicates of each treatment weretaken The colony forming units were calculated with helpof the following formula through serial dilution of one gramof substrate and the results are expressed as cfu gminus1mLminus1 ofsuspension of each substrate

CFUgmL = Number of colonies per mL platedTotal dilution factor

(1)

24 Nursery ExperimentsTrials Thesubstrate supplementedwith sawdust of Shorea robustawhich proved to be efficient inproduction ofmore cfu gminus1mLminus1 was selected for inoculationexperiment The target bioagent in the form of substrateinoculum was applied at the time of sowing of seedlingsin the nursery of Rain Forest Research Institutersquos Demovillage at Meleng Grant Jorhat Assam located at a distanceof 10 km east of Jorhat city on NH-37 (on Jorhat-TinsukiaHighway Assam India (26∘4610158405310158401015840N 94∘1710158402910158401015840E 107m asl))The annual average precipitation is 500mm and the annualaverage temperature is 26∘C The experiment was conductedusing field soil supplemented with substrate containing the

Biotechnology Research International 3

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q)

Figure 1 (a) Culture of Trichoderma harzianum (isolate TH-13) (b) mycelial mat of T harzianum (c) spores of T harzianum (d)hyphaemycelia of T harzianum ((e) (f)) preparation of substrates and filling of flasks (g) incubation of flasks at 27∘C in BOD incubatorfor 6ndash8 days ((h) (i) (j)) mass production trends of T harzianum on different substrates (k) mass production of T harzianum in a tray (l)variation in treatments on Capsicum chinense ldquoBhut Jolokiardquo after 7DAI ((m) (n) (o)) variation in treatments on C chinense ldquoBhut Jolokiardquoafter 30DAI (p) a control plant after 150DAI (q) a CFA treated plant after 150DAI

target bioagent in the field condition In control rows nobioagent (ie inoculasubstrate) was added The experimentwas designed in a single inoculation treatment Ten replica-tions of each treatment were taken The following design ofthe experiment was adopted1198790= control (no inoculation)1198791= treatmentinoculation of Trichoderma harzia-

num (isolate TH-13 supplemented substrate)Data on plant growth like increase in height (in cm) numberof leaves and total fruit yield (in number) per plant were

observed after 150DAI (days after inoculation) and analyzedto evaluate the efficacy of the preferred bioagent on thegrowth and development of target seedlings The data wereanalyzed statistically for calculating standard error of meanand coefficient of variance (CV) percent by using MS Excelversion 2003 and Gupta [14]

3 Results and Discussion

31 Mycelial Growth and Conidial Yield Different saw dusts(forestryindustry byproduct) were tested as a substrate

4 Biotechnology Research International

Table 1 Effect of substrate (type of saw dust) on Trichoderma harzianummass production

Substrate (3 1)(saw dust wheat bran)

Myceliuminitiation(in days)lowast

Initiation ofsporulation(in days)lowast

Completion ofsporulation(in days)lowast

Number ofreplicatescompletingsporulation

Percentage ofsporulation ()

Colony formingunit

(cfu gminus1mLminus1)lowast

A 3 plusmn 000 8 plusmn 000 15 plusmn 000 26 334 70 times 106 (plusmn001)B 2 plusmn 000 6 plusmn 000 10 plusmn 000 66 100 86 times 106 (plusmn002)C 3 plusmn 000 9 plusmn 000 18 plusmn 000 16 164 45 times 106 (plusmn000)

Control 5 plusmn 000 mdash Nosporulation mdash mdash mdash

CV () mdash mdash mdash mdash mdash 001A Pinus kesiya wheat bran B Shorea robusta wheat bran and C Callicarpa arborea wheat branlowastAverage of six replicates CV = coefficient of variance

constituent along with wheat bran for the mass productionof Trichoderma harzianum (isolate TH-13) micropropagulesas shown in Table 1 After 2 days of incubation at 27∘C plusmn 1the mycelial growth initiation was seen in substrate supple-mented with saw dust of Shorea robusta while in substrateswith Pinus kesiya and Callicarpa arborea mycelial growthappeared after 35 days

In control setmycelia appeared after 5 daysThe initiationof sporulation of bioagent was observed earlier in substrateof S robusta (after 6 days) than P kesiya (after 8 days) andC arborea (after 9 days) The completion of sporulation offungal activator was also observed earlier in substrates of Srobusta after 10 days (100) thanP kesiya after 15 days (334)and C arborea after 18 days (167) (Figures 1(h)ndash1(j))

No sporulationwas observed in control setThe enhancedconidial yield was also higher in S robusta (86 times 106) thanP kesiya (70 times 106) and C arborea (45 times 106) substrates interms of colony forming units (cfu gminus1mLminus1) respectivelyThe screening of various substrates for their potential tosupport T harzianum (as CFA) mass production indicatedthat among the tested saw dusts S robusta saw dust wasfound suitable in terms of production of high conidial yieldand early conversion of substrate into compostThis substratesupplemented with CFA was taken for further inoculationexperiment (Figure 1)

Fungi are one of the major agents of decompositionEstimating fungal biomass in waste is important for under-standing quantitatively their parts in the decompositionprocesses and the nutrient cycling in terrestrial ecosystemsTrichoderma and Gliocladium spp are the mostly workedout antagonists so far the production and delivery systemis concerned [15 16] So their mass production is essentialrequisite for bioinoculating the crops

The substrata used for mass production of inoculum ofTrichoderma spp are wheat straw sorghum grains ligniteand stillage molasses and brewerrsquos yeast and wheat branmedium [15ndash19] Studies have been carried out to evaluatevarious agroindustrial wastes including wheat straw paddystraw shelled maize cob paper waste and sugarcane baggasefor mass multiplication of T harzianum through solid statefermentation technology In order to enhance growth rate

and sporulation various cellulosic residues were also supple-mented with chickpea flour as organic nitrogen supplementat 2 and 4 percent Supplementation of all the substrates withchickpea flour enhanced growth and sporulation However4 chickpea flour supplementation gave maximum responsein all the cases Paddy straw followed by sugarcane baggaseand wheat straw each supplemented with 4 chickpea flourwere found to be promising substrates formassmultiplicationof T harzianum [20] Sterilized oat seeds were found effectivefor large-scale production of T harzianum [21] Dhingra andSinclair [22] also reported that dehulled broken rice grainswere found to be an excellent growth and delivery substratefor T harzianum and T koningii

In this investigation Trichoderma harzianum is culturedonly on wheat bran and different saw dusts which are wastematerials by sawmills for mass production It is reported thatthe sporulation of fungal activator was observed earlier andmore in numbers in substrates consisted of saw dust of Shorearobusta than Pinus kesiya and Callicarpa arborea Howeverthe production of spores of CFA is more in these substratesthan control set where no saw dust was added Noteworthyis the fact that lignin content of test saw dusts which wasobserved to be directly proportional to the conidial yieldof CFA Maximum sporulation (86 times 106 cfu gminus1mLminus1) wasobserved in the media fortified by S robusta where saw dustcontained maximum (53652 plusmn 166mggminus1 dry weight ofwood) lignin content [23] which was followed by P kesiya(70 times 106 cfu gminus1mLminus1) with comparatively less (23543 plusmn247mggminus1 dry weight of wood) lignin composition [24] Onthe other hand least conidial yield (45 times 106 cfu gminus1mLminus1)was observed inC arborea inwhich lignin contentwas 255of cortical dry weight [25]

Addition of T harzianum into organic media like neemcake coir pith farmyard manure and decomposed coffeepulp caused an immediate increase in the population upto three days Soil amendment with organic materials likeneem cake coir compost farmyard manure and Gliricidialeaves showed better growth and survival of antagonist thansoil alone [26] Previous reports suggested that additionof inorganic forms of nitrogen increases the production offungal biomass [27] Serrano-Carreon et al [28] had been

Biotechnology Research International 5

Table 2 Effect of Trichoderma harzianum inoculation on growth and development of Capsicum chinensis ldquoBhut Jolokiardquo after 150 DAI

Treatments Increase in height (in cm)lowast Number of leaveslowast Total fruit yield (in number) per plantlowast

Inoculated 650 plusmn 408 627 plusmn 496 130 plusmn 249Control 253 plusmn 410 426 plusmn 374 55 plusmn 408CV () 1124 083 466lowastAverage of ten replicates CV = coefficient of variance

able to prove that Trichoderma spp prefer the ammoniumform of nitrogen

According to Elad et al [29] biocontrol by Trichodermaviride is dependent on the type of inocula or substrate Differ-ent substrate treatments based on combination of substratelike wheat bran pulse bran sugarcane baggase rice strawwheat straw cow dung poultrymanure groundnut shell andsaw dust with tap water are evaluated for mass multiplicationof T harzianum and Gliocladium virens Many workers usedwheat bran as substrate for mass production of Gliocladiumspp and Trichoderma spp [29ndash33] Sorghum grains were alsoused for mass production of T harzianum These infectedsorghum grains served as bait for growth and multiplicationof T harzianum in soil [16]

A standard preparation of T harzianum and T koningiibyMehrotra [34] was in wheat bran saw-dust medium whichcan be very easily grown in autoclavable plastic bags ofdifferent sizes The preparation is ready for commercial usewithin fifteen days and is stored at room temperature Inthe present investigation also media prepared by combiningwheat bran and saw dusts resulted in maximum yield ofbiomass viable propagules and spores However saw dustas a constituent of substrate was used by previous workersbut through this study it is revealed that the saw dust thathas more lignin content is preferable for mass production ofCFA (Trichoderma spp) In this experiment wheat bran actedas carbohydrate source and saw dust served as medium formoisture for mass production of Trichoderma species

32 Biotization Effect on Growth Parameters The influenceof in vitro mass produced native compost fungal activator(substrate supplemented with CFA) was also observed ongrowth parameters ofCapsicum chinensis Jacq ldquoBhut Jolokiardquoin open field experiments consequent to 150 days afterinoculation

The datum as depicted in Table 2 obviously explains thesignificant increase in the growth parameters The increasein height (60ndash70 cm) number of leaves (600ndash650) andtotal fruit yield (in numbers) (120ndash150 chili fruits) wasalso reported more in inoculated plants with bioagent thannoninoculated control plants (height 20ndash30 cm leaves 400ndash500 and chili fruits 50ndash60) respectively (Figures 1(l)ndash1(q))

Trichoderma harzianum has been reported to promoteplant growth [35] Various strains of Trichoderma have beenfounded to be effective in plant growth characteristics andenhance biomass production [36] These fungi inhabit plantroots and promote plant growth characteristics by increasingevolution and production of some organic acids in therhizosphere such as gluconic citric andor fumaric acids by

Trichoderma which decrease soil pH and lead to increasedsolubility of the insoluble compound and an availability ofmicronutrient as well as an increase in plant nutrient uptake[37 38] Improvement of plant nutrient uptake and its trans-port from root to aerial parts together with the producedplant stimulators might result in higher photosynthetic rates[39] required for producing enough energy used to derivethe enhanced growth response [38] The CFA inoculationis observed to increase shoot height number of leaves andtotal fruit yield (in number) per plant in the present study inaccordance with the results which were obtained by previousworkers on various plants [38 40]

It would be beneficial to prepare a composted wheat branand sawdust (thatmust contain high lignin content) substratethat may serve as a nutritionally rich biodegradable productto be effective in improving the growth and development ofchili plants possibly by improving the organic content of soilThe composting of organic wastes with the help of microbialinoculants not only helps in recycling of waste but alsoresults in the preparation of economic and environmentalfriendly organic biofertilizer that could provide benefits toagriculturehorticultureforestry crops This technology ofbioinoculants should also be extended to other economicallyand medicinally important plants to check their potential toincrease the bioactive constituents and metabolites which isa future prospective

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The senior author Vipin Parkash is thankful to the IndianCouncil of Forestry Research and Education Dehradun forthe financial assistance in the Project no RFRI-422008-09SFM to carry out this research work

References

[1] M N Khare and H K Jharia ldquoTrichoderma a potentialbiocontrol agent against seed and soil borne pathogens forsustainable crop productionrdquo in Plant Pest Management P CTiwari Ed pp 263ndash276 Aavishkar Publishers amp DistributersJaipur India 2002

[2] S T Patel and A Mishra ldquoProduction of Trichoderma in sta-tionary flasksrdquoGujarat Agricultural University Research Journalvol 19 pp 53ndash56 1994

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Biotechnology Research International 3

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

(k) (l) (m) (n) (o)

(p) (q)

Figure 1 (a) Culture of Trichoderma harzianum (isolate TH-13) (b) mycelial mat of T harzianum (c) spores of T harzianum (d)hyphaemycelia of T harzianum ((e) (f)) preparation of substrates and filling of flasks (g) incubation of flasks at 27∘C in BOD incubatorfor 6ndash8 days ((h) (i) (j)) mass production trends of T harzianum on different substrates (k) mass production of T harzianum in a tray (l)variation in treatments on Capsicum chinense ldquoBhut Jolokiardquo after 7DAI ((m) (n) (o)) variation in treatments on C chinense ldquoBhut Jolokiardquoafter 30DAI (p) a control plant after 150DAI (q) a CFA treated plant after 150DAI

target bioagent in the field condition In control rows nobioagent (ie inoculasubstrate) was added The experimentwas designed in a single inoculation treatment Ten replica-tions of each treatment were taken The following design ofthe experiment was adopted1198790= control (no inoculation)1198791= treatmentinoculation of Trichoderma harzia-

num (isolate TH-13 supplemented substrate)Data on plant growth like increase in height (in cm) numberof leaves and total fruit yield (in number) per plant were

observed after 150DAI (days after inoculation) and analyzedto evaluate the efficacy of the preferred bioagent on thegrowth and development of target seedlings The data wereanalyzed statistically for calculating standard error of meanand coefficient of variance (CV) percent by using MS Excelversion 2003 and Gupta [14]

3 Results and Discussion

31 Mycelial Growth and Conidial Yield Different saw dusts(forestryindustry byproduct) were tested as a substrate

4 Biotechnology Research International

Table 1 Effect of substrate (type of saw dust) on Trichoderma harzianummass production

Substrate (3 1)(saw dust wheat bran)

Myceliuminitiation(in days)lowast

Initiation ofsporulation(in days)lowast

Completion ofsporulation(in days)lowast

Number ofreplicatescompletingsporulation

Percentage ofsporulation ()

Colony formingunit

(cfu gminus1mLminus1)lowast

A 3 plusmn 000 8 plusmn 000 15 plusmn 000 26 334 70 times 106 (plusmn001)B 2 plusmn 000 6 plusmn 000 10 plusmn 000 66 100 86 times 106 (plusmn002)C 3 plusmn 000 9 plusmn 000 18 plusmn 000 16 164 45 times 106 (plusmn000)

Control 5 plusmn 000 mdash Nosporulation mdash mdash mdash

CV () mdash mdash mdash mdash mdash 001A Pinus kesiya wheat bran B Shorea robusta wheat bran and C Callicarpa arborea wheat branlowastAverage of six replicates CV = coefficient of variance

constituent along with wheat bran for the mass productionof Trichoderma harzianum (isolate TH-13) micropropagulesas shown in Table 1 After 2 days of incubation at 27∘C plusmn 1the mycelial growth initiation was seen in substrate supple-mented with saw dust of Shorea robusta while in substrateswith Pinus kesiya and Callicarpa arborea mycelial growthappeared after 35 days

In control setmycelia appeared after 5 daysThe initiationof sporulation of bioagent was observed earlier in substrateof S robusta (after 6 days) than P kesiya (after 8 days) andC arborea (after 9 days) The completion of sporulation offungal activator was also observed earlier in substrates of Srobusta after 10 days (100) thanP kesiya after 15 days (334)and C arborea after 18 days (167) (Figures 1(h)ndash1(j))

No sporulationwas observed in control setThe enhancedconidial yield was also higher in S robusta (86 times 106) thanP kesiya (70 times 106) and C arborea (45 times 106) substrates interms of colony forming units (cfu gminus1mLminus1) respectivelyThe screening of various substrates for their potential tosupport T harzianum (as CFA) mass production indicatedthat among the tested saw dusts S robusta saw dust wasfound suitable in terms of production of high conidial yieldand early conversion of substrate into compostThis substratesupplemented with CFA was taken for further inoculationexperiment (Figure 1)

Fungi are one of the major agents of decompositionEstimating fungal biomass in waste is important for under-standing quantitatively their parts in the decompositionprocesses and the nutrient cycling in terrestrial ecosystemsTrichoderma and Gliocladium spp are the mostly workedout antagonists so far the production and delivery systemis concerned [15 16] So their mass production is essentialrequisite for bioinoculating the crops

The substrata used for mass production of inoculum ofTrichoderma spp are wheat straw sorghum grains ligniteand stillage molasses and brewerrsquos yeast and wheat branmedium [15ndash19] Studies have been carried out to evaluatevarious agroindustrial wastes including wheat straw paddystraw shelled maize cob paper waste and sugarcane baggasefor mass multiplication of T harzianum through solid statefermentation technology In order to enhance growth rate

and sporulation various cellulosic residues were also supple-mented with chickpea flour as organic nitrogen supplementat 2 and 4 percent Supplementation of all the substrates withchickpea flour enhanced growth and sporulation However4 chickpea flour supplementation gave maximum responsein all the cases Paddy straw followed by sugarcane baggaseand wheat straw each supplemented with 4 chickpea flourwere found to be promising substrates formassmultiplicationof T harzianum [20] Sterilized oat seeds were found effectivefor large-scale production of T harzianum [21] Dhingra andSinclair [22] also reported that dehulled broken rice grainswere found to be an excellent growth and delivery substratefor T harzianum and T koningii

In this investigation Trichoderma harzianum is culturedonly on wheat bran and different saw dusts which are wastematerials by sawmills for mass production It is reported thatthe sporulation of fungal activator was observed earlier andmore in numbers in substrates consisted of saw dust of Shorearobusta than Pinus kesiya and Callicarpa arborea Howeverthe production of spores of CFA is more in these substratesthan control set where no saw dust was added Noteworthyis the fact that lignin content of test saw dusts which wasobserved to be directly proportional to the conidial yieldof CFA Maximum sporulation (86 times 106 cfu gminus1mLminus1) wasobserved in the media fortified by S robusta where saw dustcontained maximum (53652 plusmn 166mggminus1 dry weight ofwood) lignin content [23] which was followed by P kesiya(70 times 106 cfu gminus1mLminus1) with comparatively less (23543 plusmn247mggminus1 dry weight of wood) lignin composition [24] Onthe other hand least conidial yield (45 times 106 cfu gminus1mLminus1)was observed inC arborea inwhich lignin contentwas 255of cortical dry weight [25]

Addition of T harzianum into organic media like neemcake coir pith farmyard manure and decomposed coffeepulp caused an immediate increase in the population upto three days Soil amendment with organic materials likeneem cake coir compost farmyard manure and Gliricidialeaves showed better growth and survival of antagonist thansoil alone [26] Previous reports suggested that additionof inorganic forms of nitrogen increases the production offungal biomass [27] Serrano-Carreon et al [28] had been

Biotechnology Research International 5

Table 2 Effect of Trichoderma harzianum inoculation on growth and development of Capsicum chinensis ldquoBhut Jolokiardquo after 150 DAI

Treatments Increase in height (in cm)lowast Number of leaveslowast Total fruit yield (in number) per plantlowast

Inoculated 650 plusmn 408 627 plusmn 496 130 plusmn 249Control 253 plusmn 410 426 plusmn 374 55 plusmn 408CV () 1124 083 466lowastAverage of ten replicates CV = coefficient of variance

able to prove that Trichoderma spp prefer the ammoniumform of nitrogen

According to Elad et al [29] biocontrol by Trichodermaviride is dependent on the type of inocula or substrate Differ-ent substrate treatments based on combination of substratelike wheat bran pulse bran sugarcane baggase rice strawwheat straw cow dung poultrymanure groundnut shell andsaw dust with tap water are evaluated for mass multiplicationof T harzianum and Gliocladium virens Many workers usedwheat bran as substrate for mass production of Gliocladiumspp and Trichoderma spp [29ndash33] Sorghum grains were alsoused for mass production of T harzianum These infectedsorghum grains served as bait for growth and multiplicationof T harzianum in soil [16]

A standard preparation of T harzianum and T koningiibyMehrotra [34] was in wheat bran saw-dust medium whichcan be very easily grown in autoclavable plastic bags ofdifferent sizes The preparation is ready for commercial usewithin fifteen days and is stored at room temperature Inthe present investigation also media prepared by combiningwheat bran and saw dusts resulted in maximum yield ofbiomass viable propagules and spores However saw dustas a constituent of substrate was used by previous workersbut through this study it is revealed that the saw dust thathas more lignin content is preferable for mass production ofCFA (Trichoderma spp) In this experiment wheat bran actedas carbohydrate source and saw dust served as medium formoisture for mass production of Trichoderma species

32 Biotization Effect on Growth Parameters The influenceof in vitro mass produced native compost fungal activator(substrate supplemented with CFA) was also observed ongrowth parameters ofCapsicum chinensis Jacq ldquoBhut Jolokiardquoin open field experiments consequent to 150 days afterinoculation

The datum as depicted in Table 2 obviously explains thesignificant increase in the growth parameters The increasein height (60ndash70 cm) number of leaves (600ndash650) andtotal fruit yield (in numbers) (120ndash150 chili fruits) wasalso reported more in inoculated plants with bioagent thannoninoculated control plants (height 20ndash30 cm leaves 400ndash500 and chili fruits 50ndash60) respectively (Figures 1(l)ndash1(q))

Trichoderma harzianum has been reported to promoteplant growth [35] Various strains of Trichoderma have beenfounded to be effective in plant growth characteristics andenhance biomass production [36] These fungi inhabit plantroots and promote plant growth characteristics by increasingevolution and production of some organic acids in therhizosphere such as gluconic citric andor fumaric acids by

Trichoderma which decrease soil pH and lead to increasedsolubility of the insoluble compound and an availability ofmicronutrient as well as an increase in plant nutrient uptake[37 38] Improvement of plant nutrient uptake and its trans-port from root to aerial parts together with the producedplant stimulators might result in higher photosynthetic rates[39] required for producing enough energy used to derivethe enhanced growth response [38] The CFA inoculationis observed to increase shoot height number of leaves andtotal fruit yield (in number) per plant in the present study inaccordance with the results which were obtained by previousworkers on various plants [38 40]

It would be beneficial to prepare a composted wheat branand sawdust (thatmust contain high lignin content) substratethat may serve as a nutritionally rich biodegradable productto be effective in improving the growth and development ofchili plants possibly by improving the organic content of soilThe composting of organic wastes with the help of microbialinoculants not only helps in recycling of waste but alsoresults in the preparation of economic and environmentalfriendly organic biofertilizer that could provide benefits toagriculturehorticultureforestry crops This technology ofbioinoculants should also be extended to other economicallyand medicinally important plants to check their potential toincrease the bioactive constituents and metabolites which isa future prospective

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The senior author Vipin Parkash is thankful to the IndianCouncil of Forestry Research and Education Dehradun forthe financial assistance in the Project no RFRI-422008-09SFM to carry out this research work

References

[1] M N Khare and H K Jharia ldquoTrichoderma a potentialbiocontrol agent against seed and soil borne pathogens forsustainable crop productionrdquo in Plant Pest Management P CTiwari Ed pp 263ndash276 Aavishkar Publishers amp DistributersJaipur India 2002

[2] S T Patel and A Mishra ldquoProduction of Trichoderma in sta-tionary flasksrdquoGujarat Agricultural University Research Journalvol 19 pp 53ndash56 1994

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Biotechnology Research International

Table 1 Effect of substrate (type of saw dust) on Trichoderma harzianummass production

Substrate (3 1)(saw dust wheat bran)

Myceliuminitiation(in days)lowast

Initiation ofsporulation(in days)lowast

Completion ofsporulation(in days)lowast

Number ofreplicatescompletingsporulation

Percentage ofsporulation ()

Colony formingunit

(cfu gminus1mLminus1)lowast

A 3 plusmn 000 8 plusmn 000 15 plusmn 000 26 334 70 times 106 (plusmn001)B 2 plusmn 000 6 plusmn 000 10 plusmn 000 66 100 86 times 106 (plusmn002)C 3 plusmn 000 9 plusmn 000 18 plusmn 000 16 164 45 times 106 (plusmn000)

Control 5 plusmn 000 mdash Nosporulation mdash mdash mdash

CV () mdash mdash mdash mdash mdash 001A Pinus kesiya wheat bran B Shorea robusta wheat bran and C Callicarpa arborea wheat branlowastAverage of six replicates CV = coefficient of variance

constituent along with wheat bran for the mass productionof Trichoderma harzianum (isolate TH-13) micropropagulesas shown in Table 1 After 2 days of incubation at 27∘C plusmn 1the mycelial growth initiation was seen in substrate supple-mented with saw dust of Shorea robusta while in substrateswith Pinus kesiya and Callicarpa arborea mycelial growthappeared after 35 days

In control setmycelia appeared after 5 daysThe initiationof sporulation of bioagent was observed earlier in substrateof S robusta (after 6 days) than P kesiya (after 8 days) andC arborea (after 9 days) The completion of sporulation offungal activator was also observed earlier in substrates of Srobusta after 10 days (100) thanP kesiya after 15 days (334)and C arborea after 18 days (167) (Figures 1(h)ndash1(j))

No sporulationwas observed in control setThe enhancedconidial yield was also higher in S robusta (86 times 106) thanP kesiya (70 times 106) and C arborea (45 times 106) substrates interms of colony forming units (cfu gminus1mLminus1) respectivelyThe screening of various substrates for their potential tosupport T harzianum (as CFA) mass production indicatedthat among the tested saw dusts S robusta saw dust wasfound suitable in terms of production of high conidial yieldand early conversion of substrate into compostThis substratesupplemented with CFA was taken for further inoculationexperiment (Figure 1)

Fungi are one of the major agents of decompositionEstimating fungal biomass in waste is important for under-standing quantitatively their parts in the decompositionprocesses and the nutrient cycling in terrestrial ecosystemsTrichoderma and Gliocladium spp are the mostly workedout antagonists so far the production and delivery systemis concerned [15 16] So their mass production is essentialrequisite for bioinoculating the crops

The substrata used for mass production of inoculum ofTrichoderma spp are wheat straw sorghum grains ligniteand stillage molasses and brewerrsquos yeast and wheat branmedium [15ndash19] Studies have been carried out to evaluatevarious agroindustrial wastes including wheat straw paddystraw shelled maize cob paper waste and sugarcane baggasefor mass multiplication of T harzianum through solid statefermentation technology In order to enhance growth rate

and sporulation various cellulosic residues were also supple-mented with chickpea flour as organic nitrogen supplementat 2 and 4 percent Supplementation of all the substrates withchickpea flour enhanced growth and sporulation However4 chickpea flour supplementation gave maximum responsein all the cases Paddy straw followed by sugarcane baggaseand wheat straw each supplemented with 4 chickpea flourwere found to be promising substrates formassmultiplicationof T harzianum [20] Sterilized oat seeds were found effectivefor large-scale production of T harzianum [21] Dhingra andSinclair [22] also reported that dehulled broken rice grainswere found to be an excellent growth and delivery substratefor T harzianum and T koningii

In this investigation Trichoderma harzianum is culturedonly on wheat bran and different saw dusts which are wastematerials by sawmills for mass production It is reported thatthe sporulation of fungal activator was observed earlier andmore in numbers in substrates consisted of saw dust of Shorearobusta than Pinus kesiya and Callicarpa arborea Howeverthe production of spores of CFA is more in these substratesthan control set where no saw dust was added Noteworthyis the fact that lignin content of test saw dusts which wasobserved to be directly proportional to the conidial yieldof CFA Maximum sporulation (86 times 106 cfu gminus1mLminus1) wasobserved in the media fortified by S robusta where saw dustcontained maximum (53652 plusmn 166mggminus1 dry weight ofwood) lignin content [23] which was followed by P kesiya(70 times 106 cfu gminus1mLminus1) with comparatively less (23543 plusmn247mggminus1 dry weight of wood) lignin composition [24] Onthe other hand least conidial yield (45 times 106 cfu gminus1mLminus1)was observed inC arborea inwhich lignin contentwas 255of cortical dry weight [25]

Addition of T harzianum into organic media like neemcake coir pith farmyard manure and decomposed coffeepulp caused an immediate increase in the population upto three days Soil amendment with organic materials likeneem cake coir compost farmyard manure and Gliricidialeaves showed better growth and survival of antagonist thansoil alone [26] Previous reports suggested that additionof inorganic forms of nitrogen increases the production offungal biomass [27] Serrano-Carreon et al [28] had been

Biotechnology Research International 5

Table 2 Effect of Trichoderma harzianum inoculation on growth and development of Capsicum chinensis ldquoBhut Jolokiardquo after 150 DAI

Treatments Increase in height (in cm)lowast Number of leaveslowast Total fruit yield (in number) per plantlowast

Inoculated 650 plusmn 408 627 plusmn 496 130 plusmn 249Control 253 plusmn 410 426 plusmn 374 55 plusmn 408CV () 1124 083 466lowastAverage of ten replicates CV = coefficient of variance

able to prove that Trichoderma spp prefer the ammoniumform of nitrogen

According to Elad et al [29] biocontrol by Trichodermaviride is dependent on the type of inocula or substrate Differ-ent substrate treatments based on combination of substratelike wheat bran pulse bran sugarcane baggase rice strawwheat straw cow dung poultrymanure groundnut shell andsaw dust with tap water are evaluated for mass multiplicationof T harzianum and Gliocladium virens Many workers usedwheat bran as substrate for mass production of Gliocladiumspp and Trichoderma spp [29ndash33] Sorghum grains were alsoused for mass production of T harzianum These infectedsorghum grains served as bait for growth and multiplicationof T harzianum in soil [16]

A standard preparation of T harzianum and T koningiibyMehrotra [34] was in wheat bran saw-dust medium whichcan be very easily grown in autoclavable plastic bags ofdifferent sizes The preparation is ready for commercial usewithin fifteen days and is stored at room temperature Inthe present investigation also media prepared by combiningwheat bran and saw dusts resulted in maximum yield ofbiomass viable propagules and spores However saw dustas a constituent of substrate was used by previous workersbut through this study it is revealed that the saw dust thathas more lignin content is preferable for mass production ofCFA (Trichoderma spp) In this experiment wheat bran actedas carbohydrate source and saw dust served as medium formoisture for mass production of Trichoderma species

32 Biotization Effect on Growth Parameters The influenceof in vitro mass produced native compost fungal activator(substrate supplemented with CFA) was also observed ongrowth parameters ofCapsicum chinensis Jacq ldquoBhut Jolokiardquoin open field experiments consequent to 150 days afterinoculation

The datum as depicted in Table 2 obviously explains thesignificant increase in the growth parameters The increasein height (60ndash70 cm) number of leaves (600ndash650) andtotal fruit yield (in numbers) (120ndash150 chili fruits) wasalso reported more in inoculated plants with bioagent thannoninoculated control plants (height 20ndash30 cm leaves 400ndash500 and chili fruits 50ndash60) respectively (Figures 1(l)ndash1(q))

Trichoderma harzianum has been reported to promoteplant growth [35] Various strains of Trichoderma have beenfounded to be effective in plant growth characteristics andenhance biomass production [36] These fungi inhabit plantroots and promote plant growth characteristics by increasingevolution and production of some organic acids in therhizosphere such as gluconic citric andor fumaric acids by

Trichoderma which decrease soil pH and lead to increasedsolubility of the insoluble compound and an availability ofmicronutrient as well as an increase in plant nutrient uptake[37 38] Improvement of plant nutrient uptake and its trans-port from root to aerial parts together with the producedplant stimulators might result in higher photosynthetic rates[39] required for producing enough energy used to derivethe enhanced growth response [38] The CFA inoculationis observed to increase shoot height number of leaves andtotal fruit yield (in number) per plant in the present study inaccordance with the results which were obtained by previousworkers on various plants [38 40]

It would be beneficial to prepare a composted wheat branand sawdust (thatmust contain high lignin content) substratethat may serve as a nutritionally rich biodegradable productto be effective in improving the growth and development ofchili plants possibly by improving the organic content of soilThe composting of organic wastes with the help of microbialinoculants not only helps in recycling of waste but alsoresults in the preparation of economic and environmentalfriendly organic biofertilizer that could provide benefits toagriculturehorticultureforestry crops This technology ofbioinoculants should also be extended to other economicallyand medicinally important plants to check their potential toincrease the bioactive constituents and metabolites which isa future prospective

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The senior author Vipin Parkash is thankful to the IndianCouncil of Forestry Research and Education Dehradun forthe financial assistance in the Project no RFRI-422008-09SFM to carry out this research work

References

[1] M N Khare and H K Jharia ldquoTrichoderma a potentialbiocontrol agent against seed and soil borne pathogens forsustainable crop productionrdquo in Plant Pest Management P CTiwari Ed pp 263ndash276 Aavishkar Publishers amp DistributersJaipur India 2002

[2] S T Patel and A Mishra ldquoProduction of Trichoderma in sta-tionary flasksrdquoGujarat Agricultural University Research Journalvol 19 pp 53ndash56 1994

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Biotechnology Research International 5

Table 2 Effect of Trichoderma harzianum inoculation on growth and development of Capsicum chinensis ldquoBhut Jolokiardquo after 150 DAI

Treatments Increase in height (in cm)lowast Number of leaveslowast Total fruit yield (in number) per plantlowast

Inoculated 650 plusmn 408 627 plusmn 496 130 plusmn 249Control 253 plusmn 410 426 plusmn 374 55 plusmn 408CV () 1124 083 466lowastAverage of ten replicates CV = coefficient of variance

able to prove that Trichoderma spp prefer the ammoniumform of nitrogen

According to Elad et al [29] biocontrol by Trichodermaviride is dependent on the type of inocula or substrate Differ-ent substrate treatments based on combination of substratelike wheat bran pulse bran sugarcane baggase rice strawwheat straw cow dung poultrymanure groundnut shell andsaw dust with tap water are evaluated for mass multiplicationof T harzianum and Gliocladium virens Many workers usedwheat bran as substrate for mass production of Gliocladiumspp and Trichoderma spp [29ndash33] Sorghum grains were alsoused for mass production of T harzianum These infectedsorghum grains served as bait for growth and multiplicationof T harzianum in soil [16]

A standard preparation of T harzianum and T koningiibyMehrotra [34] was in wheat bran saw-dust medium whichcan be very easily grown in autoclavable plastic bags ofdifferent sizes The preparation is ready for commercial usewithin fifteen days and is stored at room temperature Inthe present investigation also media prepared by combiningwheat bran and saw dusts resulted in maximum yield ofbiomass viable propagules and spores However saw dustas a constituent of substrate was used by previous workersbut through this study it is revealed that the saw dust thathas more lignin content is preferable for mass production ofCFA (Trichoderma spp) In this experiment wheat bran actedas carbohydrate source and saw dust served as medium formoisture for mass production of Trichoderma species

32 Biotization Effect on Growth Parameters The influenceof in vitro mass produced native compost fungal activator(substrate supplemented with CFA) was also observed ongrowth parameters ofCapsicum chinensis Jacq ldquoBhut Jolokiardquoin open field experiments consequent to 150 days afterinoculation

The datum as depicted in Table 2 obviously explains thesignificant increase in the growth parameters The increasein height (60ndash70 cm) number of leaves (600ndash650) andtotal fruit yield (in numbers) (120ndash150 chili fruits) wasalso reported more in inoculated plants with bioagent thannoninoculated control plants (height 20ndash30 cm leaves 400ndash500 and chili fruits 50ndash60) respectively (Figures 1(l)ndash1(q))

Trichoderma harzianum has been reported to promoteplant growth [35] Various strains of Trichoderma have beenfounded to be effective in plant growth characteristics andenhance biomass production [36] These fungi inhabit plantroots and promote plant growth characteristics by increasingevolution and production of some organic acids in therhizosphere such as gluconic citric andor fumaric acids by

Trichoderma which decrease soil pH and lead to increasedsolubility of the insoluble compound and an availability ofmicronutrient as well as an increase in plant nutrient uptake[37 38] Improvement of plant nutrient uptake and its trans-port from root to aerial parts together with the producedplant stimulators might result in higher photosynthetic rates[39] required for producing enough energy used to derivethe enhanced growth response [38] The CFA inoculationis observed to increase shoot height number of leaves andtotal fruit yield (in number) per plant in the present study inaccordance with the results which were obtained by previousworkers on various plants [38 40]

It would be beneficial to prepare a composted wheat branand sawdust (thatmust contain high lignin content) substratethat may serve as a nutritionally rich biodegradable productto be effective in improving the growth and development ofchili plants possibly by improving the organic content of soilThe composting of organic wastes with the help of microbialinoculants not only helps in recycling of waste but alsoresults in the preparation of economic and environmentalfriendly organic biofertilizer that could provide benefits toagriculturehorticultureforestry crops This technology ofbioinoculants should also be extended to other economicallyand medicinally important plants to check their potential toincrease the bioactive constituents and metabolites which isa future prospective

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The senior author Vipin Parkash is thankful to the IndianCouncil of Forestry Research and Education Dehradun forthe financial assistance in the Project no RFRI-422008-09SFM to carry out this research work

References

[1] M N Khare and H K Jharia ldquoTrichoderma a potentialbiocontrol agent against seed and soil borne pathogens forsustainable crop productionrdquo in Plant Pest Management P CTiwari Ed pp 263ndash276 Aavishkar Publishers amp DistributersJaipur India 2002

[2] S T Patel and A Mishra ldquoProduction of Trichoderma in sta-tionary flasksrdquoGujarat Agricultural University Research Journalvol 19 pp 53ndash56 1994

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Biotechnology Research International

[3] X Jin G E Harman and A G Taylor ldquoConidial biomass anddesiccation tolerance of Trichoderma harzianum produced atdifferentmediumwater potentialsrdquoBiological Control vol 1 no3 pp 237ndash243 1991

[4] G E Harman X Jin T E Stasz G Peruzzotti A C Leopoldand A G Taylor ldquoProduction of conidial biomass of Tricho-derma harzianum for biological controlrdquo Biological Control vol1 no 1 pp 23ndash28 1991

[5] G C Papavizas M T Dunn J A Lewis and J Beagle-RistainoldquoLiquid fermentation technology for experimental productionof biocontrol fungirdquo Phytopathology vol 74 no 10 pp 1171ndash1175 1984

[6] R D Lumsden J A Lewis and R D Fravel ldquoBioradiationalpest control agents formulations and deliveryrdquo in ACS Sympo-sium series 595 R Franklin W Hall A John and C S BarryEds p 306 American Chemicals Society Washington DCUSA 1995

[7] X Jin A G Taylor and G E Harman ldquoDevelopment ofmedia and automated liquid fermentation methods to producedesiccation-tolerant propagules of Trichoderma harzianumrdquoBiological Control vol 7 no 3 pp 267ndash274 1996

[8] J M Whipps ldquoStatus of biological disease control in horticul-turerdquo Biocontrol Science and Technology vol 2 no 1 pp 3ndash241992

[9] R P Tengerdy and G Szakacs ldquoBioconversion of lignocellulosein solid substrate fermentationrdquo Biochemical Engineering Jour-nal vol 13 no 2-3 pp 169ndash179 2003

[10] N Mohammad M Z Alam N A Kabbashi and A AhsanldquoEffective composting of oil palm industrial waste by filamen-tous fungi a reviewrdquo Resources Conservation and Recycling vol58 pp 69ndash78 2012

[11] V C Cuevas S N Samulde and P G Pajaro ldquoTrichodermaharzianum Rifai as activator for rapid composting of agricul-tural wastesrdquoThe Philippine Agriculturist vol 71 no 4 pp 461ndash469 1988

[12] J Purkayastha S I Alam H K Gogoi L Singh and V VeerldquoMolecular characterization of ldquoBhut Jolokiardquo the hottest chillirdquoJournal of Biosciences vol 37 no 4 pp 757ndash768 2012

[13] S A Waksman Principle of Soil Microbiology Williams ampWilkins Baltimore Md USA 1927

[14] B N Gupta Statistics (Theory amp Practice) Sahitya BhawanPublishers amp Distributors Agra India 2001

[15] G C Papavizas ldquoTrichoderma and Gliocladium biology eco-logy and potential for biocontrolrdquo Annual Review of Phy-topathology vol 23 no 1 pp 23ndash54 1985

[16] J P Upadhyay and A N Mukhopadhyay ldquoBiological controlof Sclerotium rolfsii by Trichoderma harzianum in sugar beetrdquoTropical Pest Management vol 32 no 3 pp 215ndash220 1986

[17] J Kumar Studies on root-rot of sunflower with special referenceto rhizosphere includingmycorrhizae [PhD thesis] KurukshetraUniversity Kurukshetra Haryana India 1997

[18] R D Prasad and R Rangeshwarn ldquoAn improved mediumfor mass production of the biocontrol fungus Trichodermaharzianumrdquo Journal of Mycology and Plant Pathology vol 30no 2 pp 233ndash235 2000

[19] L Tewari and C Bhanu ldquoScreening of various substrates forsporulation and mass multiplication of biocontrol agent Tri-choderma harzianum through solid state fermentationrdquo IndianPhytopathology vol 56 no 4 pp 476ndash478 2003

[20] L Tewari and C Bhanu ldquoEvaluation of agro industrial wastesfor mass production of Trichoderma harzianummdasha biofungi-cide for sustainable agriculturerdquo Journal of Mycology and PlantPathology vol 32 no 1 pp 138ndash139 2002

[21] E Mirkova ldquoIn vitro study of antagonistic activity of Tricho-derma sp towards some soil pathogensrdquoGardinarrkai hozarrkaNauka vol 19 pp 75ndash81 1982

[22] O D Dhingra and J B Sinclair Basic Plant Pathology MethodsCRC Press Florida Fla USA 1985

[23] S Mukhopadhyay S N Roy and V C Joy ldquoEnhancementof soil enzyme activities by the feeding impact of detritivorearthropods on tropical forest leaf littersrdquo Tropical Ecology vol55 no 1 pp 93ndash108 2014

[24] T Jelonek W Pazdrowski M Arasimowicz-Jelonek J GzylA Tomczak and J Floryszak-Wieczorek ldquoThe relationshipbetween the form of dead bark and lignin content in Scots pine(Pinus sylvestris L)rdquo Turkish Journal of Agriculture and Forestryvol 33 no 5 pp 455ndash462 2009

[25] J Wu K Fukazawa and J Ohtani ldquoLignin analysis in sometropical hardwoods using ultraviolet microscopyrdquo ResearchBulletins of the College Experiment Forests vol 47 no 2 pp 353ndash366 1990

[26] K A Saju M Anandraj and Y R Sarma ldquoOn farm productionof Trichoderma harzianum using organic matterrdquo Indian Phy-topathology vol 55 no 3 pp 277ndash281 2002

[27] J Jayaraj and R Ramabadran ldquoEffect of certain nitrogenoussources on the in vitro growth sporulation and production ofantifungal substances by Trichoderma harzianumrdquo Journal ofMycology and Plant Pathology vol 28 pp 23ndash25 1998

[28] L Serrano-Carreon Y Hathout M Bensoussan and J-MBelin ldquoLipid accumulation in Trichoderma speciesrdquo FEMSMicrobiology Letters vol 93 no 2 pp 181ndash187 1992

[29] Y Elad I Chat and J Katan ldquoTrichoderma harzianum a bio-control agent effective against Sclerotium rolfsii and Rhizoctoniasolanirdquo Phytopathology vol 70 no 2 pp 119ndash121 1980

[30] A Sivan Y Elad and I Chat ldquoBiological control of plant patho-gensrdquo inMicrobiology of Aerial Plant Surfaces C H Dickinsonand T F Preece Eds Academic Press London UK 1984

[31] D Maiti and C Sen ldquoIntegrated biocontrol of Sclerotiumrolfsiiwith nitrogenous fertilizers andTrichoderma harzianumrdquoIndian Journal of Agricultural Sciences vol 55 pp 464ndash4681985

[32] M G Parkash K Vinayagopal M Anandraj and Y R SarmaldquoEvaluation of substrates for mass multiplication of fungal bio-control agents Trichoderma harzianum and T virensrdquo Journal ofSpices and Aromatic Crops vol 8 pp 207ndash210 1999

[33] S C Dubey and B Patel ldquoMass multiplication of antagonistsand standardization of effective dose for management of webblight of urd and mung beanrdquo Indian Phytopathology vol 55no 3 pp 338ndash341 2002

[34] R S Mehrotra ldquoOn certain aspects of Trichodermmdashtaxonomyecology biology and biocontrolrdquo Kavaka vol 25 pp 1ndash10 1997

[35] M Shoresh G E Harman and F Mastouri ldquoInduced systemicresistance and plant responses to fungal biocontrol agentsrdquoAnnual Review of Phytopathology vol 48 pp 21ndash43 2010

[36] I Yedidia N Benhamou and I Chet ldquoInduction of defenseresponses in cucumber plants (Cucumis sativus L) by theBiocontrol agent Trichoderma harzianumrdquo Applied and Envi-ronmental Microbiology vol 65 no 3 pp 1061ndash1070 1999

[37] M Entesari F Sharifzadeh M Ahmadzadeh and M Farhang-far ldquoSeed biopriming with Trichoderma species and Pseu-domonas fluorescent on growth parameters enzymes activity

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Biotechnology Research International 7

and nutritional status of soybeanrdquo International Journal ofAgronomy and Plant Production vol 4 no 4 pp 610ndash619 2013

[38] G E Harman ldquoMyths and dogmas of biocontrolmdashchanges inperceptions derived from research on Trichoderma harzianumT-22rdquo Plant Disease vol 84 no 4 pp 377ndash393 2000

[39] R Azarmi BHajieghrari andAGiglou ldquoEffect of trichodermaisolates on tomato seedling growth response and nutrientuptakerdquo African Journal of Biotechnology vol 10 no 31 pp5850ndash5855 2011

[40] U Bal and S Altintas ldquoEffects of Trichoderma harzianum onlettuce in protected cultivationrdquo Journal of Central EuropeanAgriculture vol 9 no 1 pp 63ndash70 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology