T. asperellum ZJSX5003 had the best antagonistic activity. Seed coating with chemical fungicides were not effective against CSR due to its non-lasting preventative effects after the seedling stage. Hydrolytic enzymes and secondary metabolites significantly contributed to antagonistic activity of Trichoderma spp. against FG. Trichoderma spp., have an active metabolism and produce large amounts of enzymes. In vivo greenhouse test, confirmed that T. asperellum ZJSX5003 is an effective potential strain against FG.

1. Antagonistic and biocontrol potential of Trichoderma
asperellum ZJSX5003 against the maize stalk rot pathogen
Fusarium graminearum.
NAME: ANAMIKA
ID NO.: 49672

2. ABSTRACT
The efficacy of seven strains of Trichoderma asperellum collected from the fields
in Southern China was assessed against Fusarium graminearum (FG) the causal
agent of corn stalk rot of maize. These were assessed in vitro for their
antagonistic properties followed by statistical model of principal component
analysis to identify the beneficial antagonist T. asperellum strain. The key factors
of antagonist activity were attributed to a total of 13 factors including cell wall
degrading enzymes (chitinase, protease and β-1,3-glucanases), secondary
metabolites and peptaibols.These were analyzed from eight strains of
Trichoderma. A linear regression model demonstrated that interaction of enzymes
and secondary metabolites of T. asperellum strain ZJSX5003 enhanced the
antagonist activity against FG. Further, this strain displayed a disease reduction of
71 % in maize plants inoculated with FG compared to negative control. Pointing
out that the T. asperellum strain ZJSX5003 is a potential source for the
development of a biocontrol agent against corn stalk rot.

4. What is Biocontrol?
 Biocontrol is mechanism by which live organism or a molecule inhibit or
suppress the growth of disease causing organism or leading to its suppression.
 Biological control is the use of natural enemies to reduce the numbers of
damaging organism.
 Damaging organisms can be anything from a bacterium to a bird.

5. BIOCONTROL AGENT

7. Why we need biocontrol?
Most farmers use chemical methods to control their pest problems, there are a number of
disadvantages to this method:
 Chemicals may be non-specific and kill beneficial insects.
 Pest may develop resistance to the pesticide.
 Pesticides may enter the food chains, accumulate and harm other organisms.

8. • KINGDOM - Plantae
• DIVISION - Magnoliophyta
• CLASS - Liliopsida
• ORDER - Poales
• FAMILY - Poaceae
• GENUS - Zea
• SPECIES - mays
MAIZE

9.  Important cereal crop.
 First introduced in Mexico.
 Its 3rd largest consumed cereal crop.
 Cultivated in 4 % agriculture land.
 Production (2016)- 24500 MT.
 3.2 to 39.5% loss of maize production due to CSR.
 Products include corn starch, maltodextrins, corn oil, corn syrup and products of
fermentation and distillation industries.

12. Fusarium graminearum Schwabe
Kingdom : Fungi
Phylum : Ascomycota
Class : Sordariomycetes
Subclass : Hypocreomycetidae
Order : Hypocreales
Family : Nectriaceae
Genus : Fusarium
Species : graminearum

13.  Commonly found on cereal grains, most commonly on wheat and barley.
 Major economic impacts in the agriculture industry.
 Reduction of 18.7% in cob weight and 11.2% in 1000- grain weight
in the infected plants.
 Fusarium graminearum is best known for the detrimental interactions with
various grains.

14. Trichoderma asperellum
KINGDOM : Fungi
PHYLUM : Ascomycota
CLASS : Sordariomycetes
SUBCLASS : Hypocreomycetidae
ORDER : Hypocreales
FAMILY : Hypocreaceae
GENUS : Trichoderma
SPECIES : asperellum

15.  Hypocrea (teleomorph)
 Considerable biofertiliser effect – solubilises phosphate –
enhancing strong growth and branching of roots.
 Promotion of acquired and induced systemic resistance of the
plant.
 Produces an enzyme that destroys cuticle of nematode eggs and
protects roots from attack from Meloidogyne spp.
 Rapid colonisation of roots to compete with most soil borne
diseases invading the roots.

16. Trichoderma spp. :Biocontrol attributes
Trichoderma
AntibioticMycoparasitismCompetition
Effective antagonist
Growth promotion
Rapid substrate colonization
SAR
ISR
Enzyme

17. Systemic Acquired Resistance
 After plant infected by one pathogen has recovered, it can show remarkable resistance
to future infections by the same or other pathogens. Something similar to immunity in
animals.
 Resultant of increased level of PR proteins.
 Salicylic acid is thought to establish SAR in other parts of the plant.

19. Induced Systemic Resistance
 Activated by non pathogenic micro-organisms.
 Selected plant growth-promoting bacteria and fungi in the rhizosphere prime the whole plant
body for enhanced resistance.
 Selected strains of plant growth-promoting rhizobacteria (PGPR) suppress diseases by
antagonism between the bacteria and soil-borne pathogens.

21. Multi-farious role of Trichoderma secondary metabolites
Anti-
aging

22. •Corn stalk rot - Fusarium graminearum
•Banded leaf and sheath blight - Rhizoctonia solani
•Brown spot - Physoderma maydis
•Ear rots - Aspergillus niger
•Eyespot - Aureobasidium zeae
•Late wilt - Cephalosporium maydis

23. Symptoms
•Inner stalk show light pink discoloration.
•Ears may be small and lower nodes may be shredded or broken.
Pathogens Involved
•Fusarium graminearum Schwabe
•Teleomorph, Giberella zeal (Schwein).
Time of Occurrence
•Occurs after corn pollination.
Conditions Favouring Disease
•Warm, dry weather.
•Insect injury.
•Environmental stress.
Corn Stalk Rot

24. PEPTAIBOLS
Peptaibols are biologically active peptides containing between 7 and 20 amino
acid residues, some of which are non-proteinogenic amino acids.
Examples:-
•Alamethicins or trichorzians
•Harzianins or zervamicins
•TrichoginA IV

26. Strains Soil type Plant District ITS(GI) Tefla(GI)
T. harzianum SH2303 Heilu corn ShangHai KJ755187 KJ855089
T.asperellum GDFS5001 Irrigation-silting farmland Guangdong KJ820409 KJ855088
T.asperellum GDFS1009 Irrigation-silting farmland Guangdong 385282606 385282623
T.asperellum GDZQ1008 Heilu vegetables Guangdong 385282607 385282621
T.asperellum HNCS4002 Heilu horticulture Hunan 385282608 385282625
T.asperellum HNLY1002 Aeolian sandy horticulture Henan 385282614 385282629
T.asperellum ZJSX5002 Heilu vegetables Zhejiang 383792277 383792481
T.asperellum ZJSX5003 Heilu vegetables Zhejiang 385282613 385282617
Trichoderma strains

27. Trichoderma strains were maintained and
cultured in PDA medium.
Stored in glycerol stocks at -80°C
Incubated at 28°C

28. In vitro Antagonist Assay
Determination of Cell Wall-
Degrading Enzyme Activity
Extraction and Purification
of Peptaibols
UPLC-QTOF-MS/MS
Analysis of
Trichoderma Peptaibols
Extraction and
GC–MS Analysis
of Secondary
Metabolites
In vivo
Antagonistic
Activity
APPROACH

30. In vitro Antagonist Assay
Dual culture technique was used.
Mycelial disc (5mm) was removed from cultured Trichoderma and FG plate.
Placed on Potato Dextrose Agar plate at equal distance.
For control, pathogenic fungus was placed on the PDA plate.
Incubated at 28 ± 2°C for 5 days.
Diameter of mycelial growth was measured.

31. Determination of Percentage of inhibition :
I = (C - T)
C × 100
I – inhibition percentage
C - radial growth (mm) of pathogen alone control
T - radial growth (mm) of pathogen in the presence of
Trichoderma strains.

32. Fig. 1. In vitro antagonist activity;
(a) Fusarium graminearum on PDA petri dish,
(b) antagonism of T. asperellum strain ZJSX5003 (T) against Fusarium
graminearum (FG).

33. Fig. 2. In vitro antagonistic activity of different T. asperellum strains and positive control of
T. harzianum SH2303 against F. graminearum. Results shown are mean ± SEM (n = 3), bars
with a same letters are not statistically different among the antagonistic activity of Trichoderma
strains following Duncan’s test (p0.05).

34. Determination of Cell Wall-Degrading Enzyme Activity
Trichoderma strain was cultured in liquid production medium.
Incubate for 72 hours at 180 rpm.
Centrifuged at 3000 rpm for 10 min and the supernatant were collected and used for enzyme
assay.
.
1 ml supernatant + 2 ml
pachyman (1% w/v)
1ml supernatant + 2 ml chitin (1%
w/v)
1ml supernatant + 2 ml gelatin
(1% w/v)

35. Fig. 3. Cell wall-degrading enzyme activity of Trichoderma strains, results shown are mean ± SEM (n = 3),
bars with a same letters are not statistically different among the enzyme activity of Trichoderma strains
following Duncan’s test (p0.05).

36. Extraction and Purification of Peptaibols
Strain were inoculated in 50 ml mineral medium flask.
Incubate at 28°C for 20 days.
Culture fluid was extracted twice with a mixture of butanol (3:1).
Centrifuged at 4000 rpm for 15 min.
Supernatant was fully evaporated under vacuum.
Dissolved in 80 ml of methanol and dichloromethane (1:1) mixture.

37. Filtered with polytetrafluoroethylene membrane.
Evaporated in 5 ml of an 85:15 mixture of dichloromethane and methanol.
Transferred to a silica gel column. Column was washed to discard the
acetone by using MeOH–H₂O (85:15).
Effluent was collected and evaporated to dryness under vacuum.
Residue was dissolved in 2 ml of MeOH–H₂O (85:15), and evaporated to
dryness under vacuum for 10 min.
Final residue of peptides was quantified and dissolved in 5 ml of MeOH–
H₂O (85:15).
Cont….

38. UPLC-QTOF-MS/MS Analysis of Trichoderma
Aliquots of peptaibols(1μg/2ml of MeOH).
Analyzed using UPLC-QTOF-MS/MS(UMS, ACQUITYTM UPLC & Q-TOF MS
Premier).
MS analysis was performed using a turbo data-dependent scan.
Total current ion mass spectra were measured in between 200 and 2000 m/z.
Automatic mass calibration was by 200 ng mL-1 of leucine enkephalin (556.3 m/z).
SIMCA-P software 11.0 (Umetrics, Umea, Sweden) were used.

39. Extraction and Gas Chromatography-Mass Spectrometry (GC–MS) Analysis
of Secondary Metabolites
Trichoderma (spore suspension 4.7 × 10³ CFU/ml) was cultured in 1 liter of a mineral medium.
Incubate for 31 days at 28°C (180 rpm).
250 ml dicholoromethane was added and incubate overnight and impurities were removed
(whatman’s No. 4 ).
Stored at 2° C for 24 hours.
Solvent and water were separated (separating funnel).
Dichloromethane phase washed with distilled water (2 times) by rotary evaporator.

40. 10 μl residue was dissolved in 100 μl of methanol, passed through disposable PTFE filter.
GC-MS (AutoSystem XL GC/TurboMass MS) analysis was done.

41. Table 1. Peptaibols identified in different Trichoderma strains by UPLC-QTOF-
MS/MS analysis.
No. Strains Peptaibols Molecular Formula Molecular weight
1. T. harzianum
SH2303
Harzianin_HC_XIII Ac Aib Gln Lxx Aib Pro Ser LxxAib
Pro Vxx Lxx Aib Pro Lxx OH
1449.3743
2. T. asperellum
ZJSX5003
TCT-A_Via Ac Aib Ala Aib Aib Aib Pro Lxx
Vxx Aib Pro Lxx OH
1077.2886
Trichopolyn_I Fa Pro AHMO Ala Aib Aib LxxAla
Aib Aib AMAE
1092.6450
Tv29-14A- II a Fa Pro AHMO Ala Aib Aib LxxAla
Aib Aib AMAE
1400.8999
Trichotoxin_A-40_I Ac Aib Gly Aib Lxx Aib Gln Aib
Aib Ala Ala Aib Aib Pro Lxx Aib
Aib Glu Vxx OH
1691.0334
Trichovirin-Ib Ac Aib Gly Ala Lxx Aib Gln Ala
Vxx Aib Gly Vxx Aib Pro Lxx Aib
Aib Gln Lxx OH
1704.0444
Trichotoxin_A-40_V
Ac Aib Gly Aib Lxx Aib Gln Aib Aib
Aib Ala Aib Aib Pro Lxx Aib Vxx
Glu Vxx OH
1719.0664
Trichorzin TVB II
Ac Aib Gly Ala Lxx Aib Gln Aib Ala
Aib Ser Lxx Aib Pro Lxx Aib Aib
Gln Vxx OH
1720.0422

42. 3. T. asperellum
GDFS1009
TBV Ac Aib Ser Vxx Vxx Aib
Pro Lxx Lxx Aib Pro Aib
OH
1092.6450
Trichotoxin_A-50_F
Ac Aib Gly Aib Lxx Aib
Gln Aib Aib Ala Ala Ala
Aib Pro Lxx Aib Vxx Gln Vxx OH
1690.0410
Hypomurocin_B_IIIa Ac Aib Ala Ala Lxx Aib Gln Aib
Vxx Aib Gly Aib Aib Pro Lxx Aib
Aib Gln Vxx OH
1704.0449
Trichotoxin_A-50_I Ac Aib Gly Aib Lxx Aib Gln Aib Aib
Aib Ala Aib Aib Pro Lxx Aib Vxx
Gln Vxx OH
1718.0642
Hypomurocin_B_I Ac Aib Ser Ala Lxx Aib Gln Aib
Vxx Aib Gly Aib Aib Pro Lxx Aib
Aib Gln Vxx OH
1720.0493
Hypomurocin_B_II Ac Aib Ser Ala Lxx Aib Gln Aib
Vxx Aib Gly Aib Aib Pro Lxx Aib
Aib Gln Lxx OH
1734.0618
4. T. asperellum
ZJSX5002
Trichotoxin_A-40_II Ac Aib Gly Aib Lxx Aib Gln Aib
Aib Aib Ala Ala Aib Pro Lxx Aib
Aib Glu Vxx OH
1690.0452

43. Trichotoxin_A-50_G Ac Aib Gly Aib Lxx Aib Gln
Aib Aib Aib Ala Ala Aib Pro
Lxx Aib Vxx Gln Vxx OH
1704.0457
Trichotoxin_A-40_Va Ac Aib Ala Aib Lxx Aib Gln
Aib Aib Aib Ala Aib Aib Pro
Lxx Aib Aib Glu Vxx OH
1719.0789
Hypomurocin_B_I Ac Aib Ser Ala Lxx Aib Gln
Aib Vxx Aib Gly Aib Aib Pro
Lxx Aib Aib Gln Vxx OH
1720.0515
5. T. asperellum
HNLY1002
Trichovirin-Iib Ac Aib Gly Ala Lxx Aib Gln
Aib Vxx Aib Gly Aib Aib Pro
Lxx Aib Aib Gln Lxx OH
1704.0437
Trichotoxin_A-40_V Ac Aib Gly Aib Lxx Aib Gln
Aib Aib Aib Ala Aib Aib Pro
Lxx Aib Vxx Glu Vxx OH
1719.0745
Trichotoxin_A-40 Ac Aib Gly Aib Lxx Aib Glu
Aib Aib Aib Ala Aib Aib Pro
Lxx Aib Lxx Gln Vxx OH
1720.0466
6. T. asperellum
HNCS4002
Trichotoxin_A-50_G Ac Aib Gly Aib Lxx Aib Gln
Aib Aib Aib Ala Ala Aib Pro
Lxx Aib Vxx Gln Vxx OH
1704.0457
7. T. asperellum
GDZQ1008
Trichopolyn_I Fa Pro AHMO Ala Aib Aib
LxxAla Aib Aib AMAE
1093.6309
Trichopolyn_I Ac Lxx Aib Lxx Vxx Aib Lxx
Lxx Lxx Aib Lxx OH
1093.6372

44. Trichotoxin_A-50_E
Ac Aib Gly Aib Lxx Aib Gln Aib
Aib Aib Ala Ala Aib Pro Lxx Aib
Aib Gln Vxx OH
1690.0237
Trichovirin-Iia Ac Aib Gly Ala Lxx Ala Gln Aib
Vxx Aib Gly Vxx Aib Pro Lxx Aib
Aib Gln Lxx OH
1704.0408
TrichotoxinA-40III Ac Aib Gly Aib Lxx Aib Gln Aib Aib
Ala Ala Aib Aib Pro Lxx Aib Vxx
Glu Vxx OH
1705.0391
Trichorzin_HA_II Ac Aib Gly Ala Aib Aib Gln Aib
Vxx Aib Gly Lxx Aib Pro Lxx Aib
Vxx Gln Lxx OH
1719.0520
Trichorzin_HA_III Ac Aib Gly Ala Aib Vxx Gln Aib
Vxx Aib Gly Lxx Aib Pro Lxx Aib
Aib Gln Lxx OH
1719.0618
Trichotoxin_A-40 Ac Aib Gly Aib Lxx Aib Glu Aib Aib
Aib Ala Aib Aib Pro Lxx Aib Lxx
Gln Vxx OH
1720.0464
Hypomurocin_B_IV Ac Aib Ser Ala Lxx Aib Gln Aib
Vxx Aib Gly Vxx Aib Pro Lxx Aib
Aib Gln Vxx OH
1734.0645
8. T. asperellum
GDFS5001
TBV-B_ IVc Ac Aib Ser Lxx Lxx Aib Pro Lxx
Lxx Aib Pro Lxx OH
1148.3362

45. Trichoderma
strains
Content of antibiosis secondary metabolites (%)
Polyketide
s
Terpenes CAD NHC Alkanes Ethanols Aldehydes
SH2303 3.42 ± 0.2
d
11.82 ± 1.5
d
15.04 ± 1.2
a
1.99 ± 0.5
a
34.96 ± 2.3
d
2.59 ± 1.2
c
0.00 ± 0.0
a
ZJSX5003 3.84 ± 0.8
d
22.18 ± 3.2
d
18.42 ± 5.6
a
2.97 ± 0.8
b
21.18 ± 1.5
c
0.05 ± 0.0
a
0.18 ± 0.1
a
GDFS1009 1.15 ± 0.5
b
0.00 ± 0.0
a
45.09 ± 8.9
c
14.40 ± 1.4 15.30 ± 3.2
b
0.21 ± 0.1
a
0.23 ± 0.1
a
ZJSX5002 2.20 ± 0.9
c
2.22 ± 0.8
a
18.55 ± 4.5
a
2.41 ± 0.5
b
16.38 ± 1.5
b
2.42 ± 0.5
c
14.49 ± 2.5
c
HNLY1002 0.40 ± 0.1
a
1.43 ± 0.6
a
37.18 ± 6.5
c
6.41 ± 2.5
c
22.46 ± 2.3
c
0.22 ± 0.1
a
1.81 ± 0.5
b
HNCS4002 6.01 ± 1.5
e
4.05 ± 1.2
b
13.09 ± 2.5
a
7.93 ± 1.5
d
21.05 ± 6.2
c
3.44 ± 1.2
d
1.86 ± 0.6
b
GDZQ1008 3.75 ± 0.9
d
9.87 ± 2.5
c
28.79 ± 8.5
b
5.36 ± 1.2
c
25.86 ± 1.4
c
1.30 ± 0.9
b
0.69 ± 0.2
a
GDFS5001 5.28 ± 0.5
e
1.94 ± 0.8
a
60.14 ± 7.2
d
3.98 ± 1.2
bc
5.98 ± 3.2
a
0.16 ± 0.1
a
1.59 ± 0.8
b
Table 2. Secondary metabolites and their concentration produced by Trichoderma
strains.
The results showed mean value ± standard error (n = 3), one way ANOVA followed by multiple comparison of Duncan
test CAD - carboxylic acids and derivatives, NHC -nitrogen heterocyclic compounds; The different alphabets in the
superscript differ significantly (p < 0.05) between the Trichoderma strains.

46. Discriminant Analysis
Linear regression model equation:
Y = 0.3810y1 + 0.19082y2 + 0.13039y3 + 0.11192y4 + 0.07241y5
where Y is the integrated evaluation value of the synthesized five principal components.
The principal component score after standardization.
y1 = chitinase activity
y2 = β-1,3- glucanase activity
y3 = protease activity
y4 = peptaibols quantity
y5 = polyketide quantity

47. Trichoderma y1 y2 y3 y4 y5 Y
SH2303 1.379 −0.396 1.550 −0.981 0.446 0.572
GDFS1009 1.349 −0.505 −2.639 1.147 0.208 0.213
ZJSX5003 1.193 3.583 0.769 0.197 −0.279 1.266
ZJSX5002 0.687 −2.707 0.212 −1.628 −0.706 −0.480
HNLY1002 0.040 0.279 −1.670 −0.093 −0.527 −0.196
HNCS4002 −0.294 −1.487 1.357 2.333 1.257 0.123
GDZQ1008 1.063 0.862 −0.405 −1.469 1.536 0.470
GDFS5001 −6.186 0.406 −0.092 −0.363 −0.024 −2.331
Table 3. Principal components analysis and comprehensive scores

48. Fig. 3 A partial least-
squares discriminant
analysis (PLS-DA)
model of the UPLC-
QTOF-MS spectral
data of peptaibols
present in different
Trichoderma strains,
(a) the scores plot of
differential
Trichoderma strains
based on analysis
differential peptaibols
spots, the horizontal
and vertical axis
indicate differences
between groups and
within groups,
(b) the loading plot
show the correlation
analysis of which
peptaibols are the
major components that
determine the
difference in

49.  For peptaibols screening and discriminant analysis, SPSS version 20.0 statistical software
package (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0.
Armonk, NY: IBM Corp.) was used for principal component analysis (PCA).
 ANOVA test (one way classifications) with Duncan Post hoc multiple comparison was used
on antagonistic and enzyme activity experiments.
Statistical Analysis

50. Table 4. STATISTICAL EVALUATION STUDY OF ANTAGONISTIC TEST
Factors Code
Trichoderma
SH2303 GDFS1009 ZJSX5003 ZJSX5002 HNLY1002 HNCS4002 GDZQ1008 GDFS5001
Chitinase
activity (U) x1 4.14 ± 1.2
d
3.61 ± 0.2
c
4.07 ± 0.5
d
3.72 ± 1.2
c
2.44 ± 1.5
b
2.84 ± 1.2
b
2.89 ± 1.2
b
1.20 ± 0.2
a
β-1,3-glucanase
activity (U) x2 0.59 ± 0.2
b
0.6 ± 0.3
c
0.80 ± 0.2
d
0.50 ± 0.5
b
0.66 ± 0.2
c
0.72 ± 0.5
d
0.45 ± 0.3
a
0.62 ± 0.3
c
Protease activity
(U) x3 2.48 ± 0.9
b
2.48 ± 0.8
b
4.85 ± 0.8
d
1.72 ± 0.5
a
3.13 ± 1.2
c
2.6485 ± 0.2
b
3.8107 ± 0.5
c
2.38 ± 08
b
Peptaibol
number x4 2 ± 0.2
a
6 ± 1.5
c
7 ± 1.4
cd
4 ± 1.5
b
3 ± 0.8
b
1 ± 0.6
a
9 ± 2.4
d
1 ± 0.1
a
Polyketides
(relative %) x5 2.40 ± 0.5
b
1.15 ± 0.6
a
3.84 ± 1.5
c
2.20 ± 1.2
b
0.40 ± 0.1
a
6.01 ± 1.5
d
3.75 ± 1.6
c
5.28 ± 1.2
cd

51. Terpenes (relative
%) x6 14.53 ± 0.6
cd
0.00 ± 0.0
a
22.18 ± 4.6
d
2.22 ± 0.5
a
1.43 ± 0.6
a
4.05 ± 2.4
b
9.87 ± 1.4
c
1.94 ± 0.7
a
Alkane-including
hydrocarbon (%) x7 0.00 ± 0.0
a
15.30 ± 1.2
c
21.18 ± 2.5
d
16.38 ± 2.6
cd
22.46 ± 2.5
d
0.00 ± 0.0
a
25.86 ± 3.5
d
5.98 ± 0.9
b
Carboxylic acids
and derivatives x8 17.34 ± 1.5
b
18.42 ± 1.5
b
45.09 ± 5.6
d
18.55 ± 1.5
b
37.18 ± 4.5
c
13.09 ± 1.5
a
28.79 ± 1.8
cd
60.14 ± 4.2
e
Aldehyde-
including
hydrocarbon (%)
x9 0.22 ± 0.1
a
0.23 ± 0.1
a
0.18 ± 0.1
a
14.49 ± 2.5
c
1.81 ± 0.6b
b
1.86 ± 0.6
b
0.69 ± 0.5
a
1.59 ± 0.6
b
Nitrogen
heterocyclic
compounds
x10 2.02 ± 0.2
a
14.40 ± 1.2
d
2.97 ± 1.5
a
2.41 ± 0.8
a
6.41 ± 1.5 7.93 ± 2.4
cd
5.36 ± 1.4
c
3.98 ± 1.2
b
Alcohols (%)
x11 0.00 ± 0.0
a
0.21 ± 0.1
b
0.05 ± 0.0
a
2.42 ± 0.9
cd
0.22 ± 0.1
b
3.44 ± 1.6
d
1.30 ± 0.8
c
0.16 ± 0.05
b
Inhibitory rate of
pathogen in vitro
(%)
x12 74.12 ± 5.6
d
68.24 ± 6.5
b
74.48 ± 3.5
d
66.10 ± 5.8
b
71.3 ± 42.6
c
65.85 ± 5.6
b
65.09 ± 5.2
b
43.53 ± 5.4
a
Antagonistic
effect of pathogen
(%)
x13 64.28 ± 2.6
d
66.67 ± 7.5
d
70.67 ± 2.5
e
48 ± 4.5
b
55.11 ± 4.5
c
55.11 ± 4.5
c
63.11 ± 4.9
d
16.18 ± 1.9
a

52. In vivo Antagonistic Activity
Maize seeds were surface sterilized.
Germinate on sterile wet filter paper at 25 °C for 48 h.
Pots containing 4 kg sterilized loamy and clay soil were taken.
Conidia and mycelia fragments of freshly prepared T. asperellum strain ZJSX5003 were
harvested.
Concentrated to approximately 1 × 106 conidia/ml by centrifugation.
Two treatments were maintained:(1) CK (soil inoculated with FG alone).
2) T1 (Soil inoculated with T. asperellum strain ZJSX5003 and FG).

53. Soil was inoculated with FG at a rate of 5 g of FG biomass/kg of soil.
20 ml spore suspension of T. asperellum strain ZJSX5003 was added per pot.
Disease incidence was recorded 7 days after T. asperellum strain ZJSX5003 treatment.

54. Disease reduction = Disease index of CK—Disease index after treatment of T. asperellum
strain ZJSX5003.
DISEASE FORMULA

55. Fig. 5. Effect of T. asperellum ZJSX5003 Treatment on biological control of CSR caused by FG. CK(soil
inoculated with FG alone). T1 Soil inoculated with T. asperellum strain ZJSX5003 and FG.
T1 CK

57. ⱷ Trichovirin-Ib in T. asperellum ZJSX5003, Trichotoxin_A-50_F in T. asperellum GDFS1009,
and Trichorzin_HA_III, Trichotoxin_A-40, and Hypomurocin_B_IV in T. asperellum
GDZQ1008 were showed more antagonistic activity.
ⱷ ZJSX5003 (26 %), SG3403 (17 %), SG2303 (15 %), and GDZQ1008 (14 %) showed broad
and high antagonizing activity with polyketides and terpenes .
ⱷ Carboxylic acids and their derivatives were abundant in strain ZJSX5003 (18 %).
ⱷ The strain ZJSX5003 as potent to control FG a casual agent of CSR in maize with disease
reduction of 71 %.

58. ⱷ T. asperullum ZJSX5003 were showed higher chitinase activity(4U/L), and protease(26%)
and β-1,3-glucanase activity (47%).
ⱷ Trichoderma spp., also secrete a chemically diverse range of secondary metabolites,
including peptaibols, polyketides, terpenes, and polypeptides.
ⱷ Trichoderma produced a diverse group of metabolites that were species-specific.

59. ⱷ The antagonistic effects of T. asperellum isolate ZJSX5003 were positively correlated with
their production of peptaibols and polyketides.
ⱷ Trichoderma sp., are able to repress pathogen through synchronization of mycoparasitism
and antibiotic production.
ⱷ T. asperellum isolates produced more secondary metabolites than other strains tested
including alkanes (21 %), terpenes (22 %), carboxylic acids and derivatives (18 %), and
others.
ⱷ Trichoderma spp. strains also promote plant growth and increase immunity.

61.  T. asperellum ZJSX5003 had the best antagonistic activity.
 Seed coating with chemical fungicides were not effective against CSR due to its non-lasting
preventative effects after the seedling stage.
 Hydrolytic enzymes and secondary metabolites significantly contributed to antagonistic
activity of Trichoderma spp. against FG.
 Trichoderma spp., have an active metabolism and produce large amounts of enzymes.
 In vivo greenhouse test, confirmed that T. asperellum ZJSX5003 is an effective potential
strain against FG.

62. o T. asperellum ZJSX5003 strain is used for understanding the molecular basis of fungal
antagonism and their exploitation in improving crop production.

63. Li Y., Sun R., Yu J., Saravanakumar K. and Chen J. 2016. Antagonistic and biocontrol potential of Trichoderma asperellum
ZJSX5003 against the maize stalk rot pathogen Fusarium graminearum. Indian J Microbiol. Springer. 56(3):318–327. DOI
10.1007/s12088-016-0581-9.
Harman GE, Herrera-Estrella AH, Horwitz BA, and Lorito M.2012. Special issue: Trichoderma—from basic biology to
biotechnology. Microbiology 158:1–2. doi:10.1038/nrmicro797.
Schmoll M, Schuster A. 2010 Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol. 87:787–799.
Shoresh M, Mastouri F, and Harman G. 2010. Induced systemic resistance and plant responses to fungal biocontrol agents.
Annu Rev Phytopathol. 48:21–43.
Lorito M, Woo SL, Harman GE, and Monte E. 2010. Translational research on Trichoderma: from ‘omics to the field. Annu Rev
Phytopathol. 48:395–417.
Vinale F, Marra R, Scala F, Ghisalberti EL, Lorito M, and Sivasithamparam K. 2006. Major secondary metabolites produced
by two commercial Trichoderma strains active against different phytopathogens. Lett Appl Microbiol 43:143–148.