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Phytoparasitica ISSN 0334-2123 PhytoparasiticaDOI 10.1007/s12600-011-0188-y

Plant defense activation and managementof tomato root rot by a chitin-fortifiedTrichoderma/Hypocrea formulation

Manoj Kumar Solanki, Nidhi Singh,Rajesh Kumar Singh, Pratiksha Singh,Alok K. Srivastava, Sudheer Kumar, PremL. Kashyap & Dilip K. Arora

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Plant defense activation and management of tomato root rotby a chitin-fortified Trichoderma/Hypocrea formulation

Manoj Kumar Solanki & Nidhi Singh & Rajesh Kumar Singh & Pratiksha Singh &

Alok K. Srivastava & Sudheer Kumar & Prem L. Kashyap & Dilip K. Arora

Received: 12 September 2010 /Accepted: 27 July 2011# Springer Science+Business Media B.V. 2011

Abstract Tomato root rot caused by Rhizoctoniasolani is a major soilborne disease resulting insignificant yield loss. The culture filtrates of sixisolates of Trichoderma/Hypocrea species were eval-uated for in vitro production of hydrolytic enzymes.Results demonstrated that all the six isolates wereable to produce chitinase, β-1, 3 glucanase andprotease in the range of 76–235 μmol GlcNAc min-1

mg-1 protein, 31.90–37.72 nmol glucose min-1 mg-1

proteins and 63.05–86.22 μmol min-1 mg-1 proteins,respectively. Trichoderma/Hypocrea-based formula-tion(s) were prepared with chitin (1% v:v) and CMC(0.5% w:v) for root rot management in a greenhouse.Root dip application with bioformulation(s) resultedin a significant reduction of the root rot index. Inaddition, bioformulations increased plant growthattributing traits significantly relative to untreatedcontrol. Accumulation of total phenols, peroxidase,polyphenoloxidase and phenylalanine ammonia lyaseincreased in chitin-supplemented Trichoderma/Hypo-crea formulation-treated plants challenged with R.solani. The results suggest that chitin-fortified bio-

formulation(s) could be an effective system to controlroot rot of tomato in an eco-compatible manner.

Keywords Biocontrol . Chitinase . Dual culture .

Rhizoctonia solani

Introduction

Tomato (Lycopersicon esculentum L.) root rot causedby Rhizoctonia solani is one of the major constraintsfor successful cultivation of tomato (Montealegre etal. 2010; Morsy et al. 2009). Existing commercialcultivars of tomato do not provide sufficient diseaseresistance. Effective fungicides are available tomanage the pathogen, but the increased environmen-tal concern over their use has necessitated a largeupsurge in biological disease control. The emergenceof fungicide resistance among the pathogens, pollu-tion of ground water and food, and development ofoncogenic risks have further encouraged the exploi-tation of potential antagonistic microflora in diseasemanagement. Among various antagonists used for themanagement of plant diseases, plant growth-promoting fungi play a vital role (Contreras-Cornejoet al. 2009).

Trichoderma spp. have revolutionized the field ofbiological control of soilborne plant pathogens(Radjacommare et al. 2010). Trichoderma/Hypocreacan improve plant growth and development throughmycoparasitism, antibiosis, competition, induced

PhytoparasiticaDOI 10.1007/s12600-011-0188-y

M. K. Solanki :N. Singh :R. K. Singh : P. Singh :A. K. Srivastava : S. Kumar : P. L. Kashyap :D. K. Arora (*)National Bureau of Agriculturally ImportantMicroorganisms,Kusmaur,Mau, Uttar Pradesh 275101, Indiae-mail: nbaimicar@gmail.com

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plant defense and act as avirulent symbionts (DeSouza et al. 2008; Harman et al. 2004). Moreover,application of Trichoderma spp. restricts the growthof soilborne pathogens by secreting various extra-cellular enzymes such as chitinases (Almeida et al.2007), β-glucanases (Haran et al. 1995) and protei-nases (Geremia et al. 1993). Chitinase production byT. harzianum was found to be induced using wheatbran-based solid medium containing 1% colloidalchitin and the chitinase led to the lysis of thephytopathogenic fungus Colletotrichum gloeospor-ioides (Sandhya et al. 2005). However, applicationof these Trichoderma formulations has been difficult,mainly against soilborne pathogens like R. solani,because their efficacy not only requires an exces-sively large amount of inocula but also meets withresistance under different types of soil conditions(Kolombet et al. 2008).

Chitin is a copious renewable natural resourceobtained from marine invertebrates, insects, fungi andalgae. More than 80,000 Mt of chitin is obtained peryear from marine waste (Loganathan et al. 2010). Theapplication of a biocontrol agent along with chitin hasgreat importance in plant disease management (Kishoreet al. 2005), as such agents are expected to inhibit thegrowth of phytopathogenic fungi in the rhizosphere(Bade and Wick 1988; Bell et al. 1998; Benhamou andTheriault 1992; Sultana et al. 2000) as well as on plantsurfaces (Radjacommare et al. 2010; Sahebani andHadavi 2008). Chitin amendment alone can drasti-cally reduce the population of stubby root nemat-odes (Trichodorus spp.), Collembola (Protaphoruraspp.), millipedes (Brachydesmus spp.) and wireworm(Agrioles spp.), as reported by Ellis et al. (1998).The combined application of a formulation mixtureof Trichoderma spp. along with chitin reduced theincidence of the complex of diseases incited by thecombined action of Sclerotinia sclerotiorum andMeloidogyne incognita and resulted in a significantincrease in the activities of phenylalanine ammonialyase (PAL), peroxidase (POX), polyphenoloxidase(PPO) and chitinase in cabbage (Loganathan et al.2010). Kishore et al. (2005) also reported thatsupplementation of Bacillus circulans GRS 243 andSerratia marcescens GPS5 with 1% colloidal chitinin the greenhouse reduced lesion frequency of lateleaf spot of groundnut caused by Phaeoisariopsispersonata by 60%, when compared with applicationof bacterial cells alone.

However, there is little information concerningthe influence of chitin supplementation on theefficacy of antagonistic Trichoderma/Hypocrea spp.in controlling root rot caused by R. solani in tomato.Hence, the present study was carried out with thefollowing objectives: (i) in vitro screening ofTrichoderma/Hypocrea isolates for hydrolytic en-zyme production; (ii) developing a bio-formulationcontaining Trichoderma spp. along with colloidalchitin and evaluating their potential to manage rootrot of tomato under greenhouse conditions; and (iii)deciphering the possible biochemical basis of plantdefense activation during tri-trophic interactions ofTrichoderma/Hypocrea spp. and R. solani withtomato.

Materials and methods

Biocontrol agents and pathogen Six isolates ofTrichoderma/Hypocrea , namely, T. hamatum(NAIMCC-F-01721), H. lixii (NAIMCC-F-01735),H. lixii (NAIMCC-F-01760), T. asperellum(NAIMCC-F-01763), H. virens (NAIMCC-F-01775)and H. lixii (NAIMCC-F-01809) and one virulentculture of Rhizoctonia solani (NAIMCC- F-01971)causing root rot of tomato were obtained from theNBAIM culture collection, India. Molecular identifi-cation of Trichoderma/Hypocrea isolates was per-formed using sequences of the translation-elongationfactor 1-alpha and submitted to NCBI GenBank withaccession no. JN228189-194. These fungi werecultured at 28±2°C in potato dextrose agar (PDA)(HiMedia, Mumbai, India).

Screening of Trichoderma/Hypocrea isolates for en-zyme production All the six isolates of Trichodermaspp. were characterized by performing enzymaticassays (chitinase, β-1, 3-glucanase and protease assay)and were grown in the minimal medium (MM)(MgSO4.7H2O, 0.2 g; K2HPO4, 0.9 g; KCl, 0.2 g;NH4NO3, 1.0 g; FeSO4.7H2O, 0.002 g; MnSO4,0.002 g; ZnSO4, 0.002 g; distilled water, 1.0 l; pH6.3) supplemented with the appropriate carbonsource (0.5% w:v): laminarin for glucanase, chitinfor chitinase and casein for protease, and incubatedfor 7 days at 28±2°C. The experiment wasconducted in five replicates and repeated threetimes.

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Estimation of chitinases (E.C.3.2.1.14) The colori-metric estimation of chitinase (E.C.3.2.1.14) wascarried out as per the method described by Bollerand Mauch (1988). One ml of colloidal chitin (1% v:v) in citrate phosphate buffer (0.1 M, pH 6.5) wasincubated with 1.0 ml of culture filtrate of Tricho-derma/Hypocrea strains at 37°C for 2 h. The resultingchitin oligomers were treated with 2 ml of 4-dimethylaminobenzaldehyde at 37°C for 20 min andabsorbance was measured at 585 nm. GlcNAc servedas the standard. One unit of enzyme activity is definedas the amount of enzyme required to produce0.5 μM ml-1 of N-acetylglucosamine per hour.Specific activity is expressed as μmol GlcNAc min-1

mg-1 protein. The experiment was conducted in fivereplicates and repeated three times.

Estimation of β-1,3-glucanase (E.C.3.2.1.6) β-1,3glucanase (E.C.3.2.1.6) was estimated by incubating1.0 ml of laminarin (0.5% w:v) in sodium phosphatebuffer (0.1 M, pH 4.0) with 1.0 ml of culture filtrateof Trichoderma/Hypocrea strains at 55°C for 5 min.The reaction was stopped in an ice bath and 3 ml of3,5 dinitrosalicylic acid (DNS) reagent (Miller 1959)was added, and the mixture heated in a boiling waterbath for 12 min. As a control, 1.0 ml of laminarinsolution was incubated and cooled, then 1 ml ofenzyme solution together with 3 ml of DNS reagentwas added to correct the reducing sugars in thesubstrate and the enzyme solution. Reducing sugarequivalents were measured in both the original andcontrol solutions by the colorimetric method of Pan etal. (1991), with glucose as standard at 450 nm. Theexperiment was conducted in five replicates andrepeated three times.

Estimation of protease A reaction mixture containing1.0 ml soluble casein (1% v:v) in citrate phosphatebuffer (0.05 M, pH 6.5) and 1.0 ml culture filtrate ofTrichoderma/Hypocrea spp. was used. The reactionmixture was incubated for 1 h at 37°C and reactionwas stopped by adding trichloroacetic acid (TCA,10% v:v), kept for another 20 min at the sametemperature, followed by centrifugation (4000 rpm,20 min). Samples (75 μl) were removed and theamount of tyrosine was determined according toLowry et al. (1951). The absorbance of the superna-tant was measured at 280 nm. One unit of the enzymeactivity is defined as the amount required for the

formation of 1.0 μmol min-1 mg-1 protein of thereaction, under the standard assay conditions. Theexperiment was conducted in five replicates andrepeated three times.

Bio-control studies: Greenhouse experiment

Mass multiplication of pathogenic fungi and biocon-trol agent The mass inoculum of R. solani was raisedin Erlenmeyer flasks (250 ml) containing pearl milletseeds (80 g) and bean meal (20 g) in distilled water(30 ml) and autoclaved for two consecutive days for1 h at 121°C. Each flask was inoculated separately byone disk (5 mm diam) of the fungal strain andincubated at 28±2°C for 5 days. The mycelium wascollected by filtration and dried to a constant weightand all mycelia and sclerotia were mixed thoroughlyin sterile potting soil for greenhouse experiments.

For the propagation of a biocontrol agent, differentisolates of Trichoderma/Hypocrea spp. were multi-plied in a broth containing molasses (30 ml), KH2PO4

(2 g), MgSO4 (0.2 g), sucrose (10 g) and 1 l distilledwater (pH 5.8). After incubation (10 days), conidia ofthe Trichoderma/Hypocrea were separated from my-celia by filtering though sterile glass wool andquantified using a hemocytometer and the populationset to 5×106 spores ml-1 to be used for the seedlingtreatments.

Plant material and experimental design Tomato (cv.‘Navratan’) seeds were surface sterilized with ethanol(70% v:v) for 5 min, followed by sodium hypochlo-rite (1%) for 1 min and rinsed thrice with steriledistilled water. Sterilized seeds were sown in seedlingtrays (70 plugs), with each plug measuring 4×4.5×2.5 cm. Prior to transplanting, inoculum of R. solaniwas mixed into sterilized soil in plastic pots (0.35 m×0.50 m×0.04 m) containing 400 g mixture ofsterilized sand: soil (2:1, w:w) at the rate of 30 g kg-1.Three-week-old tomato seedlings were dipped inchitin-amended liquid suspension containing Tricho-derma spp. (5×106 spores ml-1), CMC (0.5% w:v) andchitin (1% v:v) and transplanted into pots (3 seedlingspot-1) and maintained in the greenhouse at 28±2°Cwith 70–90% r.h. Treatments in the in vivo biocontrolexperiment were: tomato seedlings dipped in liquidsuspension containing chitin-supplemented Tricho-

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derma/Hypocrea spp., liquid suspension of Tricho-derma/Hypocrea spp. without chitin (control A) andseedlings without inoculation (control B). Theplants were irrigated twice daily with tap water.Experiments were arranged in a completely ran-domized design. The experiment was conducted infive replicates and repeated three times. Theobservations involved various plant growth param-eters, namely, root length (cm), shoot length (cm),root and shoot dry weight (g plant-1) for differenttreatments. Vigor index (VI) was calculated as:

VI ¼ Root lengthþ Shoot lengthð Þ� Percent seedling survivability

Disease assessment To assess the efficacy of chitin-supplemented Trichoderma/Hypocrea formulations insuppression of root rot, 4-week-old tomato trans-planted plants were observed for root rot symptoms inthe greenhouse. Ten plants selected at random werecarefully excised from each replication. After rinsingfor 20 min under running tap water, roots were ratedseparately for disease symptoms. Root rot index wasdetermined according to a linear scale of 0–4, where 0 =healthy root, 1 = 25% infected, 2 = 50% infected, 3 =primary root exists, secondary roots do not exist,4 = primary root does not exist (dead plant)(Moataza 2006). The root rot index (DI %) andsurvival percentage (S %) were estimated as follows:DI %ð Þ ¼ P0

di¼4 n� dið Þ= N � 4ð Þh i

� 100 and Sð%Þ ¼ðN � di4=NÞ �100, where di = disease index, n =number of diseased plants in index, N = total numberof plants investigated, di4 = total number of deadplants.

Biochemical basis of plant defense activation

Sample collection for biochemical analysis The effectof colloidal chitin supplemented liquid formulation(s)of Trichoderma/Hypocrea spp. on the induction ofplant defense system were evaluated by estimation ofPOX, PPO, PAL and total phenols (TP). The leaves oftomato plants from various treatments were collectedafter 4 weeks. Leaf samples (5 g f.wt.) were washedunder running tap water, dried gently and ground with

a mortar and pestle under liquid nitrogen andextracted in 10 ml of extraction buffer (50 mM Trisat pH 8.5 containing 14.4 mmol-1 2-mercaptoethanoland insoluble polyvinyl polypyrrorolidone, 1% w:v)and centrifuged at 8000 rpm (10 min, 4°C). Thesupernatant was used as leaf extract to assay totalPOX and PPO. All assays were performed in fivereplicates and repeated three times along with control.

Estimation of peroxidase (PO) Plant leaves (1 g) werehomogenized in 2 ml of 0.1 M phosphate buffer (pH7.0) at 4°C. The homogenate was centrifuged at16000 rpm (4°C for 15 min) and the supernatant wasused as the enzyme source. The reaction mixturecontained 1.5 ml of 0.05 M pyrogallol (HiMedia),0.5 ml of enzyme extract and 0.5 ml of 1% H2O2

(HiMedia). The reaction mixture was incubated atroom temperature (28±2°C). The changes inabsorbance at 420 nm were recorded at 30-sintervals for 3 min. The enzyme activity isexpressed as the changes in absorbance min−1

mg−1 protein (Hammerschmidt et al. 1982)

Estimation of polyphenol oxidase (PPO) For PPOactivity, the reaction mixture contained 0.2 ml leafextract in 1.5 ml of 0.1 M sodium phosphate buffer(pH 6.5), and 0.2 ml of 0.01 M catechol (Sigma) wasadded to start the reaction. The PPO activity isexpressed as the change in absorbance at495 nm min-1 g–1 f.wt. (Mayer et al. 1965).

Estimation of total phenol (TP) Plant leaves (1 g)were homogenized in 10 ml of 80% methanol (v:v)and shaken for 15 min at 70°C. One ml of methanolicextract was added to 5 ml distilled water and 250 μlFolin-Ciocalteau reagent (1N) and kept at 25°C. Theabsorbance of the developed blue color was measuredat 725 nm with catechol as the standard. The amountof phenolics is expressed as catechol μg-1 g–1 f.wt.(Zieslin and Ben-Zaken 1993).

Estimation of phenylalanine ammonia lyase (PAL)Leaf samples (1 g) were homogenized in 2 ml ofchilled 0.1 M sodium borate buffer (pH 7.0) contain-ing 1.4 mM of 2-mercaptoethanol and 0.1 g insolublepolyvinylpyrrolidone. The extract was centrifuged(16000 rpm, 15 min) and the supernatant was usedas the enzyme source. Enzyme extract (0.4 ml) with0.5 ml of 0.1 M borate buffer (pH 8.8) containing

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0.5 ml of 12 mM L-phenylalanine (HiMedia) wasincubated for 30 min at 30°C. PAL activity wasdetermined as the rate of conversion of L-phenylalanine to cinnamic acid at 290 nm. Theamount of cinnamic acid synthesized was calculated(Dickerson et al. 1984). Enzyme activity is expressedas μg cinnamic acid min-1 g-1 f.wt.

Statistical analysis All experiments and treatmentswere arranged in a completely randomized blockdesign with five replications for each treatment. Thedata were statistically evaluated using one-wayANOVA for in vitro quantification of hydrolyticenzymes of Trichoderma/Hypocrea and means werecompared by using Tukey–Kramer post hoc test (P≤0.05), using SPSS 16.0 software (SPSS Inc., Chicago,IL, USA). Two-way ANOVA was used to detect thepresence of a significant Trichoderma/Hypocrea–chitin interaction in the biocontrol experiment con-ducted in a greenhouse (Field 2009).

Results

In vitro quantification of hydrolytic enzymes ofTrichoderma/Hypocrea All Trichoderma/Hypocreacultures used in the present study produced variouscell wall-degrading enzymes (chitinase, β-1,3 gluca-nase and protease), which may be involved in thecell’s lysis of R. solani. The mycoparasitic activities

of these cultures were observed on liquid mediacontaining different carbon sources (laminarin forglucanase, chitin for chitinase and casein for prote-ase), along with the production of lytic enzymes. Datapresented in Table 1 show that all the mycoparasiticstrains produced chitinase, β-1,3 glucanase andprotease in the range of 76–235 μmol GlcNAc min-1

mg-1 protein, 31.90–37.72 nmol glucose min-1 mg-1

protein, and 63.05–86.22 μmol min-1 mg-1 protein,respectively. Maximum chitinase was produced by T.asperellum NAIMCC-F-01763 followed by H. lixiiNAIMCC-F-01760 and, least, by T. hamatumNAIMCC-F-01721.

Of six Trichoderma/Hypocrea isolates, only two(H. lixii NAIMCC-F-01760 and H. virens NAIMCC-F001775) showed high β-1,3 glucanase activity,although all the isolates also produced β-1,3 gluca-nase (31–37 nmol glucose min-1 mg-1 protein).Similarly, protease production by the strains wasrecorded in the range of 1.58–3.17 μmol min-1 mg-1

protein with the maximum by T. hamatumNAIMCC-F-01721 and H. lixii NAIMCC-F-01735(Table 1).

In vivo evaluation of chitin-supplemented Tricho-derma/Hypocrea spp. for control of R. solani Theeffect of chitin-amended formulations of Tricho-derma/Hypocrea spp. on the root rot index (DI),seedling survival, root length, shoot length, vigorindex, root and shoot dry weight of tomato plants wasstudied and the data are presented in Table 2.

Table 1 In vitro quantification of hydrolytic enzymes produced by Trichoderma / Hypocrea isolates (For each experiment, valuesrepresent the means of three independent experiments; each experiment was conducted in five replicates)

Treatments Cell wall-degrading enzymesz

Chitinasey Glucanasex Proteasew

T. hamatum NAIMCC-F-01721 76.74±8.77a 32.39±0.60a 83.23±4.07b

H. lixii NAIMCC-F-01735 199.87±2.68c 33.40±0.88a 86.22±1.18b

H. lixii NAIMCC-F-01760 225.89±5.62cd 36.72±0.44b 67.64±1.50a

T. asperellum NAIMCC-F-01763 235.53±12.91d 31.24±0.44a 63.05±1.68a

H. virens NAIMCC-F-01775 153.80±7.10b 37.72±0.44b 65.21±0.97a

H. lixii NAIMCC-F-01809 182.74±6.48b 31.90±0.88a 69.51±0.93a

zWithin columns, means ± SE values with a common letter do not differ significantly (P<0.05), according to Tukey’s testyμmol of GlcNAc min-1 mg-1 proteinx nmol of glucose min-1 mg-1 proteinwμmol min-1 mg-1 protein

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Table

2Effectof

Trichoderm

a/Hypocreatreatm

entwith

(C+)andwith

out(C

- )chitinsupplementatio

non

root

rotindexandgrow

thattributes

oftomatoin

agreenhouse

(For

each

experiment,values

representthemeans

ofthreeindependentexperiments;each

experimentw

asconductedin

five

replicates.V

aluesin

parenthesesrepresentstandard

errorof

themeans)

Treatment(s)

DI(%

)zPercent

reduction

over

control

Rootlength

(cm)

Shoot

length

(cm)

Survival(%

)ySeedlingvigorx

Rootdrywt.(g

plant-1)

Shoot

drywt.(g

plant-1)

C-

C+

C-

C+

C-

C+

C-

C+

C-

C+

C-

C+

C-

C+

C-

C+

T.hamatum

NAIM

CC-

F-01721

+R.solani

33.33(5.46)

30.90(2.96)

54.25(4.79)

53.26(5.39)

8.11

(0.10)

11.37(1.73)

16.62(2.04)

18.72(2.37)

88.89(1.91)

93.33(4.54)

2199.42(200.80)

2800.74(193.18)

0.94

(0.04)

1.05

(0.54)

1.42

(0.34)

1.65

(0.23)

H.lixii

NAIM

CC-

F-01735

+R.solani

31.12(4.52)

27.78(1.67)

57.20(4.63)

50.85(6.30)

8.87

(1.16)

10.47(2.52)

19.52(1.06)

22.22(2.31)

86.67(6.37)

93.33(3.00)

2464.12(224.51)

3065.96(412.71)

0.96

(0.03)

1.12

(0.58)

1.79

(0.22)

2.16

(0.58)

H.lixiistrain

NAIM

CC-

F-01760

+R.solani

19.44(3.00)

13.89(2.52)

73.67(5.82)

76.76(4.39)

10.93(0.59)

12.92(1.53)

19.47(3.02)

23.54(2.03)

88.89(4.60)

95.56(2.31)

2691.60(220.43)

3488.25(171.05)

1.13

(0.37)

1.60

(0.27)

2.08

(0.58)

2.72

(0.42)

T.asperellu

mNAIM

CC-

F-01763

+R.solani

23.33(3.76)

21.11(3.61)

68.23(3.22)

65.14(7.26)

8.58

(0.88)

10.32(2.21)

16.23(1.80)

20.62(1.53)

93.33(3.73)

92.33(1.00)

2300.80(176.52)

2862.99(354.13)

0.86

(0.14)

1.45

(0.33)

1.77

(0.40)

1.87

(0.55)

H.virens

NAIM

CC-

F-01775

+R.solani

26.11(3.95)

23.89(3.01)

64.45(2.68)

59.63(2.46)

10.56(0.67)

12.28(2.35)

18.36(0.98)

20.29(2.00)

91.11(4.87)

89.89(4.65)

2632.09(175.20)

2923.45(121.08)

0.93

(0.11)

1.15

(0.47)

1.46

(0.40)

2.27

(0.49)

H.lixii

NAIM

CC-

F-01809

+R.solani

28.89(2.52)

22.78(2.08)

60.09(4.93)

61.78(5.01)

8.34

(0.46)

9.18

(2.08)

18.51(2.96)

22.50(1.16)

91.11(4.40)

93.33(2.85)

2442.11(240.27)

2960.80(236.20)

1.06

(0.08)

1.29

(0.37)

1.87

(0.50)

2.72

(0.20)

R.solani

72.78(5.14)

60.00(6.57)

5.14

(0.81)

3.57

(0.82)

14.21(1.73)

9.45

(2.04)

46.67(5.28)

60.00(5.78)

915.42

(165.66)

1328.47(213.02)

0.51

(0.05)

0.62

(0.15)

0.90

(0.06)

1.11

(0.35)

Control

6.43

(0.31)

7.37

(0.94)

15.43(1.39)

17.53(1.16)

98.67(0.39)

98.67(1.00)

2156.16(105.07)

2457.25(113.07)

0.78

(0.12)

0.85

(0.13)

0.98

(0.45)

1.21

(0.15)

CD

(P≤0

.05)

Trichoderm

a/Hypocrea

0.022

0.541

0.034

0.012

0.063

0.002

0.101

0.041

Chitin

0.001

0.001

0.001

0.023

0.001

0.001

0.261

0.010

Trichoderm

a/Hypocrea×

Chitin

0.821

0.922

0.991

0.991

0.601

0.953

0.982

0.961

zDIð%

Þ¼½P

0 di¼4

ðn�diÞ=ðN

�4��

100,where

di=diseaseindex,

n=nu

mberof

diseased

plantsin

indexandN

=totalnu

mberof

plantsinvestigated

ySurvivalð%

Þ¼ðN

�di

4=N�

100,where

N=totalnu

mberof

plantsinvestigated,anddi4=totalnu

mberof

dead

plants

xSeedlingvigorSV

ðÞ¼

RootlengthþShootlength

ðÞ�

Percentseedlin

gsurvivability

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Among the various liquid formulations of Tricho-derma/Hypocrea spp., 54–68% reduction in the DI wasobserved in Trichoderma/Hypocrea that was lackingchitin amendments. The disease reduction was furtherenhanced up to 76% with colloidal chitin (1% v:v)amendments in Trichoderma/Hypocrea formulations(Table 2). The maximum control of DI was observedin tomato seedlings that received H. lixii NAIMCC-F-01760 + CMC (0.5% w:v) mixture with and withoutchitin amendment, followed by T. asperellumNAIMCC-F-01763 (71.07%) and T. lixii NAIMCC-F-01809 (68.65%). T. hamatum NAIMCC-F-01721 wasleast effective, as the DI could be reduced to 54.25%.Control treatments had 73% DI with the majority ofplants left completely stunted or dead.

Chitin-fortified Trichoderma/Hypocrea formula-tions significantly (P≤0.05) enhanced plant growth-attributing parameters, viz., seedlings survival per-centage, vigor, shoot and root length, root and shootdry weight, etc. Appreciable extents of plant growth-attributing parameters were recorded from the seed-lings treated with Trichoderma/Hypocrea spp. (withand without chitin amendment) compared with

untreated controls. Maximum plant growth-attributing parameters, viz., root length (99%), shootlength (53%), root fresh weight (84%), root dryweight (86%), shoot fresh weight (66%) and shootdry weight (99%) were observed in tomato rootsdipped in chitin-supplemented H. lixii NAIMCC-F-01809 followed by H. lixii NAIMCC-F-01760 +colloidal chitin and H. virens NAIMCC-F-01775 +colloidal chitin. The same treatment also resulted inthe maximum vigor index (3484.59) followed by H.lixii NAIMCC-F-02735 + colloidal chitin (3050.96).Over all, tomato seedlings treated with chitin-fortifiedbioformulations recorded significant (P≤0.05) enhance-ment in plant growth-promoting attributes comparedwith those treated with bioformulations lacking chitinamendments (Table 2). Furthermore, ANOVA resultsshowed that there was no significant (P≤0.05) effect ofTrichoderma and chitin interaction on root rot index,root length and dry weight, shoot length and dryweight and seedling vigor (Table 2).

Biochemical basis of plant defense induction inresponse to bioformulation application To assess the

Table 3 Induction of defense enzymes by Trichoderma/Hypocrea species in combination with (C+) or without (C-)chitin against root-rot complex in tomato plants (For each

experiment, values represent the means of three independentexperiments; each experiment was conducted in five replicates.Values in parentheses represent standard error of the means)

Treatment(s) Peroxidase (ΔOD min-1

g-1fresh wt.)Polyphenol oxidase(ΔOD min-1 g-1

fresh wt.)

Phenylalanine ammonialyase (Cinnamic acidμg-1 g-1 fresh wt.)

Total phenol (Catecholμg-1 g-1 fresh wt.)

C- C+ C- C+ C- C+ C- C+

T. hamatum NAIMCC-F-01721 + R. solani

0.91 (0.18) 0.95 (0.45) 1.74 (0.58) 2.19 (0.53) 23.67 (1.45) 30.78 (5.78) 18.69 (5.04) 24.72 (2.52)

H. lixii NAIMCC-F-01735 + R. solani

1.04 (0.24) 1.18 (0.10) 2.32 (0.75) 2.76 (0.29) 24.60 (1.80) 29.43 (5.08) 17.89 (2.89) 27.47 (1.46)

H. lixii NAIMCC-F-01760 + R. solani

1.23 (0.12) 1.72 (0.45) 1.69 (0.51) 2.70 (0.58) 26.17 (1.97) 39.10 (3.96) 19.93 (2.31) 26.79 (2.52)

T. asperellum NAIMCC-F-01763 + R. solani

1.07 (0.20) 1.31 (0.30) 1.48 (0.45) 2.49 (0.50) 26.55 (2.08) 28.15 (3.08) 16.79 (3.47) 22.82 (2.31)

H. virens NAIMCC-F-01775 + R. solani

0.94 (0.22) 1.23 (0.20) 2.06 (0.57) 2.25 (0.53) 22.85 (3.47) 31.32 (1.88) 22.14 (2.33) 24.31 (1.53)

H. lixii NAIMCC-F-01809 + R. solani

1.09 (0.35) 1.36 (0.35) 1.50 (0.51) 2.61 (0.51) 20.43 (2.55) 31.50 (4.59) 15.93 (1.52) 24.10 (2.08)

R. solani 0.40 (0.12) 0.50 (0.25) 0.56 (0.20) 0.63 (0.12) 17.16 (2.05) 17.54 (3.82) 11.81 (2.52) 12.90 (1.00)

Control 0.17 (0.05) 0.21 (0.10) 0.37 (0.12) 0.48 (0.23) 11.48 (1.32) 13.63 (2.60) 11.05 (2.19) 14.21 (2.89)

CD (P≤0.05)Trichoderma/Hypocrea 0.132 0.033 0.001 0.001

Chitin 0.001 0.001 0.001 0.001

Trichoderma/Hypocrea × Chitin

0.992 0.880 0.440 0.700

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biochemical basis of plant defense induction inresponse to bioformulation applications, experimentswere conducted under greenhouse conditions tostudy the possible role of defense enzymes, viz.,PO, PPO, PAL and TP in leaf samples of tomatoseedlings (Table 3). The assays revealed that chitin-supplemented Trichoderma/Hypocrea formulationsinduced a greater amount of enzymes in tomatoleaves than the untreated sets (positive control) andR. solani-treated control (negative control). Theinduction of the PO enzyme was significantly (P≤0.05) higher than the untreated control (0.17 ΔODmin-1 g-1 f. wt.), with the maximum in tomato rootstreated with H. lixii NAIMCC-F-01760 + chitin(1.72 ΔOD min-1 g-1 f.wt.) (Table 3). Similarly,there was higher induction of PPO in tomato leavestreated with H. lixii NAIMCC-F-01735 + chitin(2.76 ΔOD min-1 g-1 f.wt.) followed by H. lixiiNAIMCC-F-01760 + chitin (2.7072 ΔOD min-1 g-1

f.wt.) and H. lixii NAIMCC-F-01809 + chitin(2.6172 ΔOD min-1 g-1 f.wt.). A combination ofchitin with H. lixii NAIMCC-F-01760 (39.10 cin-namic acid μg-1 g-1 f.wt.) followed by chitin + H.virens NAIMCC-F-01775 (31.92 cinnamic acid μg-1

g-1 f.wt.) induced high accumulation of PAL enzymein tomato leaves relative to untreated sets (11.48cinnamic acid μg-1 g-1 f.wt.) and R. solani-treatedpositive control (17.16 cinnamic acid μg-1 g-1 f.wt.).Accumulation of total phenols in leaves treatedwith H. lixii NAIMCC-F-1735 + chitin bioformula-tion (27.2027 catechol μg-1 g-1 f.wt.) was signifi-cantly higher than all other treatments (Table 3).Two-way ANOVA results showed highly significant(P≤0.005) plant defense activation by PPO, PAL andTP when leaves were treated with chitin-fortifiedTrichoderma/Hypocrea formulation. There was nosignificant (P≤0.05) effect of Trichoderma/Hypocreaand chitin interaction on the activity of defenseenzymes (Table 3).

Discussion

Inducing the plant’s own defense mechanisms byprior application of biotic and abiotic inducers is anovel plant protection strategy (Kashyap and Dhiman2009). Fungi belonging to Trichoderma genera are thewell known biological inducers and antagonistic

toward a number of plant pathogenic fungi (Jyotsanaet al. 2008). There are several mechanisms involvedin Trichoderma antagonism, namely, antibiosis –whereby the antagonist fungus produces antibiot-ics, competes for nutrients and mycoparasitism,whereas Trichoderma directly attacks the plantpathogen by excreting lytic enzymes such as chiti-nases, ß-1,3 glucanases and proteases (Almeida et al.2007; Lorito et al. 1993; Radjacommare et al. 2010).Such hydrolytic enzymes partially degrade thepathogen cell wall that leads to parasitization(Kubicek et al. 2001). The present investigationdemonstrated significant enhancement in levels ofhydrolytic enzymes (β-1,3 glucanase, chitinase andprotease) in comparison with control, which causedthe lysis of R. solani hyphae. Similar observationswere made by Cortes et al. (1998) and Howell(2003) wherein the activity of lytic enzymes (β-1,3glucanses, chitinases and proteases) was responsiblefor lysis of R. solani hyphae through digestion ofmajor cell wall components.

Several attempts have been made to utilizechitinase-producing micro-organisms to manage fun-gal diseases through inhibition in growth of phyto-pathogenic fungi on plant surfaces (Kishore et al.2005; Yu et al. 2008). However, interest in theutilization of chitinolytic antagonist fungi has grownas chitin supplementations increased the attainablelevel of disease control. Chitin amendments drastical-ly reduced populations of Ph. personata (Kishore etal. 2005) and stubby root nematodes (Trichodorusspp.), collembola (Protaphorura spp.), millipedes(Brachydesmus spp.) and wireworm (Agrioles spp.),as reported by Ellis et al. (1998). The application ofchitin alone or in combination with the biocontrolagents in managing plant diseases has been demon-strated in only a few crops so far (Kishore et al. 2005;Nandakumar et al. 2001; Radjacommare et al. 2010;Rajkumar et al. 2008). Unique biological properties ofchitin oligomers including their antifungal potentialagainst various plant pathogenic fungi such asFusarium oxysporum f.sp. radicis-lycopersici andPythium aphanidermatum are well documented(Manjula and Podile 2001). Moreover, chitin haswide applications against various pathogens (Kishore etal. 2005; Yu et al. 2008) as the monomer is the inducerfor chitinase production by Trichoderma/Hypocrea spp.which, in turn, enhances the mycoparasitic potential ofTrichoderma/Hypocrea; this perhaps justifies its use as

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an amendment in bioformulation mixtures in thepresent study.

The root treatment with liquid bioformulations ofTrichoderma/Hypocrea alone and in combination withchitin (1% v:v) was evaluated for management oftomato root rot. The results revealed that the chitinamendment considerably increased biocontrol potentialof Trichoderma/Hypocrea spp. against the root rot. Thechitin either alone or in combination with Trichoderma/Hypocrea spp. may also induce resistance in plantsagainst fungal pathogens (Kishore et al. 2005; Radja-commare et al. 2010). Sandhya et al. (2005) reportedthat wheat bran-based solid medium formulations of T.harzianum containing 1% colloidal chitin inducedresistance against C. gloeosporioides. The resultsobtained by Yu et al. (2008) also strengthened the ideaof chitin supplementation in biocontrol formulations asyeast dextrose broth amended with chitin at 0.5–1.0%greatly improved the antagonistic potential of Crypto-coccus laurentii against Penicillium expansum in pearfruits. Our results indicated that Trichoderma/Hypocreaspp. were capable of attacking the chitinaceous hyphalwall of the target pathogens in response to chitinamendments. Trichoderma/Hypocrea formulationsamended with chitin reduced vanilla disease under fieldconditions and led to enhanced biocontrol efficacy of theantagonist owing to chitinolytic activity (Radjacommareet al. 2010; Rajkumar et al. 2008). Our results are alsoin harmony with those of Kokalis-Burelle et al. (1991)as the insoluble polymer chitin, selectively enhancedgrowth and antagonism of chitinolytic Trichoderma/Hypocrea spp. The effect of chitin in inducing systemicresistance either alone or in combination with thebiocontrol agents, has been reported in various host–pathogen combinations (Radjacommare et al. 2010;Sid Ahmed et al. 2003). The observations of Anithaand Rabeeth (2009) also confirm our results onsupplementation of chitin in the antagonistic formula-tions of Trichoderma/Hypocrea spp. The latter inves-tigators observed that root dipping of tomato plantswith chitin-amended Serratia griseus suspension,significantly reduced the disease severity of fusarialwilt in the plant.

The improved control of tomato root rot by chitin-supplemented applications of Trichoderma/Hypocreaspp. is reflected in the enhanced activities of defense-related enzymes in tomato. The phenolic compoundsmay contribute to enhanced mechanical strength of thehost cell wall, and thus may also inhibit the fungal

growth (Senthilraja et al. 2010). Recently, reports ofwork conducted in various laboratories suggested thatTrichoderma/Hypocrea isolates might stimulate theproduction of biochemical compounds of phenolicnature associated with the host defense (Kavino et al.2008; Radjacommare et al. 2010). Of these, the earlyinduction of PAL is more important, since it is the firstenzyme in the phenylpropanoid pathway that leads toproduction of phytoalexin and phenolic substancesleading to the formation of lignin with peroxidases.Conspicuously, the present study also recorded thehigher activity of PAL, PPO and PO in plants treatedwith Trichoderma spp. + chitin formulations and thusgenerating the speculations of induced defenseresponses in plants treated with biocontrol agents. Thus,enhanced accumulation of phenolic compounds intomato plants treated with chitin-fortified Trichodermaformulations in this study, indicates the involvement ofreduced infection by R. solani and enhanced plantgrowth attributes. Moreover, the current study offers thescope for effective management of tomato root rot in asustainable way.

In conclusion, our results showed that the antago-nistic activity of Trichoderma spp. could be enhancedby chitin (1% v:v) supplementation and thus offerenormous practical potential in reducing the tomatoroot rot menace. The combination of Trichoderma/Hypocrea with CMC (0.5% w:v) and chitin (1% v:v)offered cost-effective biocontrol of R. solani undergreenhouse conditions. The efficacy of the bioformu-lated product for the control of Rhizoctonia root rot intomato could be further enhanced by combinationswith crop rotation and/or soil sterilization by solari-zation, and the timing of application.

Acknowledgment This work was funded by the Indian Councilof Agriculture Research (ICAR) by a network project “Applicationof Microorganisms in Agriculture and Allied Sectors” (AMAAS).The help of the culture collection unit of NBAIM is highlyappreciated for providing cultures for this study.

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