Groundnut is an important oilseed crop, and it severely infected with S. rolfsii. Trichoderma sp. is widely distributed antagonistic fungi that used for the control of Sclerotium rolfsii. Six different Trichoderma spp. in vitro efficacy and it producing microbial Volatile Organic Compounds (mVOCs) assessed against the S. rolfsii through dual culture assay and paired plate assay respectively. The Trichoderma sp. T(SP)-20 exhibited the maximum percentage inhibition (84.44%) followed by T(AR)-10 (75.44%) and TNAU TA (75.33%) in dual culture assay. Among them T. longibrachiatum T(SP)-20 and T. asperellum T(AR)-10 effectively reduced the mycelial growth of S. rolfsii by 61.11 and 54.44 per cent respectively in paired plate assay. Trichoderma spp. co cultured with S. rolfsii produced antimicrobial properties having mVOCs such as Isolongifolan-7-ol,Trans-sesquisabinene hydrate, Bis(2-ethlhexyl)phthalate, Methyl 2-methoxypropenote, Butane,1-propoxy, Pentane,2,dimethyl, 1,3-Dioxolane-4-ethanol,2,2,4-trimethyl, Heptadecane,8-methyl, (1R,5R)-4-methylene-1-(R)-6 methylhept-5 which could induce the plant growth promotion and inhibit the S. rolfsii compared to Trichoderma sp. cultured alone. Trichoderma longibrachiatum effectively reduced the mycelial growth of S. rolfsii. Which exhibited several mode of actions, among them mVOCs effectively reduce the mycelial growth of Sclerotium rolfsii.
Research Article
Unraveling of the mVOCs produced by Trichoderma spp. against Sclerotium rolfsii using Gas Chromatography-Mass Spectrometry
https://doi.org/10.21203/rs.3.rs-1562350/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted 20 Apr, 2022
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Sclerotium rolfsii
Trichoderma spp.
mVOCs
antimicrobial
GC-MS
Sclerotium rolfsii is a notorious soil borne pathogen that affects a wide range of hosts particularly groundnut and produce the destructive symptoms. It causes various symptoms like seedling damping-off, stem rot, stem canker, crown blight, tuber rot, and bulb blight that might lead to yield loss. In India, S. rolfsii is a big issue, which yield loss is up to 11% at normal condition and 10%-25% in heavily infected fields (Prasad et al., 2012) and the losses can escalate up to 80 per cent. It produces melanin containing resting structure (sclerotia) that survive long period of time and cause the infection. Chemical based management of S. rolfsii is makes a residuel effect in the soil and development of the cross resistance (Backman, 1997). Trichoderma has been substantially used to manage the many soil and foliar borne pathogens. It produces the different inhibitory mechanism viz., competition, parasitism, antibiosis, lysis, and induced systemic resistance, among them, mVOCs (microbial Volatile Organic Compounds) play a crucial role in plant disease reduction and growth promotion. The mVOCs such as pyrazines, acids, ester, aldehydes, benzenoids, terpenes, ketones and alcohol that act against the pathogen (Effmert et al., 2012). Sridharan et al. (2020) profiled mVOCs during the interaction of Trichoderma sp.with S. rolfsii and Macrophomina phaseolina such as longifolan, caryophyllene, 1-Butanol 2-methyl reduced the mycelial growth, sclerotia production and induce the plant growth promotion. The current research focused on antagonistic activity of Trichoderma spp. against S. rolfsii and which produced mVOCs against the S. rolfsii. Beyond that during bipartite interaction between the pathogen and antagonist, pathogen and antagonist alone which mVOCs unregulated and down regulated by analyzed through GC-MS.
Test pathogen and antagonist
The soil borne and resting structure producing pathogen, Sclerotium rolfsii IS (BDI)-8 (Athelia rolfsii Acc. No MZ277282) isolated from stem rot infected groundnut plants and the antagonist T(AR)-10 (Trichoderma asperellum Acc. No MZ277326), T(SP)-20 (Trichoderma longibrachiatum Acc. No MZ277378), T(VT)-3 (T. hamatum Acc. No MZ675442), T(BI)-16 (T. longibrachiatum Acc. No MZ802988), T(TK)-23 (T. citrinoviride) isolated from the rhizosphere region of the groundnut and Trichoderma asperellum (TNAU -TA) obtained from TNAU used for this study.
Dual culture assay
Six different isolates of Trichoderma spp. viz., T(AR)-10,T(SP)-20, T(VT)-3, T(BI)-16, T(TK)-23 and TNAU TA were tested in vitro against S. rolfsii using a dual culture approach (Dennis and Webster, 1971). The investigation used seven-day-old cultures of Trichoderma spp. and S. rolfsii IS (BDI)-8. The test pathogen and Trichoderma sp. culture discs (9 mm diameter.) were cut out and inserted aseptically at equidistance and in opposite directions over solidified PDA media on Petri plates, then plates were incubated at 28± 2 °C. There were three replications carried out. The inhibition percentage of radial growth of the pathogen by Trichoderma sp. was compared with control (Vincent, 1947)
Percent inhibition (I) = C – T/C ×100
C = average diameter of fungal growth (cm) in control
T = average diameter of fungal growth (cm) in treatment
Paired plate assay
Inverted or paired plate assay was conducted to analyze the efficacy of volatile production efficacy of six Trichoderma spp. viz., T(AR)-10,T(SP)-20, T(VT)-3, T(BI)-16, T(TK)-23 and TNAU TA against the stem rot pathogen. A 9mm plug of Pathogen and Trichoderma sp. was placed at the center of two different Petri plates containing pda medium. The pathogen alone containing Petri plate was kept above the Trichoderma sp. containing Petri plate and both plates were sealed tightly using parafilm to avoid the leakage of the volatiles. In control only S. rolfsii was placed at the top of paired plate. Those Petri plates were incubated at 28±2℃ for five days after that, the per cent mycelia growth reduction of S. rolfsii was calculated.
Dc = average diameter mycelial growth of S. rolfsii (cm) in control
Dt = average diameter mycelial growth of S. rolfsii (cm) in treatment
Collection of mVOCs produced by Trichoderma spp.
Six iodine flasks were filled with 100 ml of PDA broth and autoclaved at 121.6 °C at 15 PSI for 20 minutes and then each flask was inoculated with T. longibrachiatum T(SP)-20, T. asperellum T(AR)-10, S. rolfsii (Sr), T(SP)-20+Sr and T(AR)-10 + Sr individually. The uninoculated served as a control. After inoculation, the flasks were secured with a stopper and parafilm was sealed to avoid the leakage of volatiles. The activated charcoal tied in the tissue paper was inserted into the flasks without touching the broth with culture inside. In order to collect volatile compounds, such flasks were incubated at 28± 2℃ at room temperature for seven days (Fig.3). The axenic culture of T .longibrachiatum T(SP)-20,T.asperellumT(AR)-10 and co-culture of Trichoderma sp with S. rolfsii (T(SP)-20+Sr and T(AR)-10 + Sr ) and axenic culture of S. rolfsii producing microbial volatile organic compounds (mVOCs) trapped in activated charcoal and that secondary metabolites was analyzed by GCMS.
GCMS analysis of mVOCs
Trichoderma longibrachiatum T(SP)-20,Trichodera asperellum T(AR)-10, Sclerotium rolfsii (Sr) ,T(SP)-20 + Sr and T(AR)-10 + Sr producing mVOCs were analyzed with a Shimadzu Gas Chromatography equipped with a mass detector Turbo mass gold containing an Elite-1 (100% Dimethyl Poly Siloxane), 30 m × 0.25 mm ID × one mM df. The conditions employed were the following: Carrier gas, Helium (1 ml/min), Oven temperature program 110 °C (2 min) to 280 °C (9 min), Injector temperature (250 °C), Total GC time (45 min).The hexane extract was injected into the chromatograph in 1.0 ml aliquots. The major constituents were identified with the aid of a computer driven algorithm and then matched the mass spectrum of the analysis with that of a National Institute of Standards and Technology (NIST) library (Version. 2.0, year-2005). Software used for Gas Chromatography-Mass Spectroscopy (GC-MS) was Turbo mass 5.1. This work was carried out in the Center of Innovation for Excellence, Agricultural College & Research Institute, Tamil Nadu Agricultural University, Madurai.
Statistical analysis
Experimental data were analyzed using analysis of variance (ANOVA) and the SPSS version 17.0. The treatment means were separated at 5 per cent significance level using Duncan’s Multiple Range Test (DMRT) and each treatments three times replicated. R package was used for the heat map construction and clustering.
Dual culture assay
The Trichoderma sp. T(SP)-20 exhibited the maximum percentage inhibition (84.44%) followed by T(AR)-10 (75.44%) and TNAU TA (75.33 %). (Table 1; Fig.1)
Paired plate assay
Paired plate assay showed the effectiveness of volatiles production by the Trichoderma spp. and it directly inhibited the mycelial growth of the stem rot pathogen. Isolate T(SP)-20 exhibited the mycelial growth reduction of 61per cent followed by T(AR)-10 (54 %), TNAU-TA (52%), as compared to the control (Table-2;Fig.2).
GCMS analysis of mVOCs
The mVOCs collected from the all the six treatments by activated charcoal, were analyzed through GC-MS (Fig.3).
GC-MS analysis of mVOCs of Trichoderma longibrachiatum T(SP)-20 showed presence of compounds such as 4-Butoxy-2-butanone, Cycloheptasiloxane, tetradecamethyl, (1R,4R)-1-methyl-4-(6-Methylhept-5en-2-yl) Tetracontane, Bicylo (9,3, pentadeca-3,7-dien-12 ol,4,8,1, Cyclohexane, 3-(1,5-dimethyl-4-hexenyl)-6. These compounds were also detected during the interaction of T. longibrachiatum and S. rolfsii. But interestingly Bis(2-ethlhexyl)phthalate, (2,3-Diphenylcyclopropyl) methyl phenyl sulfox, 3-(1,5 Dimethyl-hex-4-enyl)-2,2-dimethyl-cycl, Isolongifolan-7-ol, Trans-sesquisabinene hydrate, Methyl2-methoxypropenote9, Butane,1-propoxy, Pentane,2,dimethyl, 1,3-Dioxolane-4-ethanol,2,2,4-trimethyl of the mVOCs appeared only in the interaction of T. longibrachiatum and S. rolfsii, but not present in the T(SP)-20 GCMS result (Fig. 6a).
GC-MS analysis of the mVOCs fraction of Trichoderma asperellum T(AR)-10 and interaction between T(AR)-10 and S. rolfsii showed presence of compounds such as Tetracontane, Transsesquisabinene hydrate, (1R,4R)-1methyl 4-(6-Methylhept-5-en-2-ylHeptane,8-methyl, (1s,5s)-2-Methyl-5-((R)-6-methylhept-5-en-2Bicylo(9,3,1), pentadeca-3,7-dien 12ol,4,8,1, Cyclohexane,3-(1,5-dimethyl-4-hexenyl)-6. But interestingly 3-(1,5-Dimethyl-hex-4-enyl)-2,2-dimethyl-cyc Isolongifolan-7-ol, Heptadecane,8-methyl-, (1R,5R)-4-methylene-1-((R)-6 methylhept-5, Benzene,1-(1,5-dimethyl1-4-hexenyl)-4-methyl, 2-hexane,3,3-dimethyl, 1-Pentanol,2,2-dimethyl were present in the interaction of T. asperellum and S. rolfsii but did not occur in T. asperellum alone (Fig.6b).
The mVOCs Dotriacontane, Tetrapentacontane, Hexacosane,1-iodo, Phthalic acid, di(2propylpentyl), 2Methylhexacosane, 2-Aminoethanol,N,O-diacetyl and Heptadecane were present in S. rolfsii but absent during the interaction of Sclerotium rolfsii and Trichoderma sp. (Fig.6c). In the case of control any specific compounds not detected
The selected compounds from each interaction and their RT value, Peak Area(%), molecular weight, chemical formulae and specific role were showed in Table 3. The presence and absence of compounds; up regulation and down regulation compounds and interaction of compounds were clearly distinguished in heat map (Fig.4) and Venn diagram (Fig.5).
Heatmap construction
The presence and absence of mVOCs from axenic culture of Trichoderma spp. and co-culture with S .rolfsii was showed in heat map and it was constructed through R studio package.The upregulation and down regulation of mVOCs also easily distinquished by heat map.From the heat map, Z - score from 0 -25 based characterized, here 0 means absence of compounds that indicate bright blue (Fig.4). The volatile compounds upregulated mean the deviated from bright blue to dark red color vice versa. The mVOCs interaction of all the six culture was showed in Ven diagram (Fig.5).
Dual culture assay
Babu and Kumar (2008) reported nine Trichoderma spp. (Th-1 to Th-9) were recovered from the microflora of the groundnut rhizosphere. In the dual culture approach, the isolate Th-3 reduced S. rolfsii mycelial growth by up to 83 per cent. Similarly, Hirpara et al. (2016) discovered that Trichoderma virens NBAII Tvs12 inhibited S. rolfsii mycelial growth by coiling the surround and producing a hook-like structure inside the test pathogen's mycelium, resulting in a per cent growth reduction of 76.37 per cent and 87.91 per cent at 6 DAI and 12 DAI, respectively, and also arrested sclerotia production when compared to the control.
Paired plate assay
Yamunarani and Tjprc, (2019) reported that four Trichoderm sp. (GNTV1, GNTV1, GNTV1, GNTH1) produced the volatiles against the stem rot pathogen. Among them, GNTV1 showed higher percent reduction of S. rolfsii at 53.41 per cent over control. Sridharan et al., (2020) reported that from the inverted plate assay Trichoderma longibrachiatum EF5 produced the volatiles, which inhibited the mycelial growth of S.rolfsii (57%) and Macrophomina phaseolina (35%) and also tripartite assay of T. longibrachiatum EF5 produced mVOCs, which inhibit the per cent mycelia growth of S. rolfsii at 68 per cent and M. pahseolina at 33 per cent.
GCMS analysis of mVOCs
In this study, mVOCs were trapped by activated charcoal from without any direct contact of the S. rolfsiiand Trichoderma sp. Volatiles produced from the Trichoderma sp. inhibit the growth of S. rolfsii, where the volatiles of Trichoderma sp. escaped stem rot pathogen which played a dominant role in paired plate assay. Similarly, Pacheco et al. (2016) reported the T. harzianum, T .ressi,T. longibrachiatum inhibited the sclerotial germination of S. rolfsii. The antagonistic activity against the phytopathogens by Longifolan, Caryophyllene, and Cedrene were reported by Zhang et al. (2014). Inoue et al. (2004) reported that dead of fungi due to loss of osmotic pressure of the cell membrane of fungi induced by the sesquiterpenes. Dubey et al. (2011) reported that the antifungal activity of volatile compounds produced by Trichoderma sp. are 3 methyl heptadecanoyl, methyl cyclohexane,6-nonylene alcohol, methyl- cycloheptane,2 methyleheptadecanol, N-methyle pyrolidine, dermadin, ketorial, palmiticacid, 3-(2’-hydroxypropyl)-4-(hexa-2’-4-dineyl)-2-(5H)-furanone. Similarly Guo et al. (2019) reported the new compounds like α-selinine, limonene, and cyclohexane,1,2,4-Tris(methylene)-1 during co-cultivation of T. harzianum and Laccaria bicolar. Mukai et al. (2017) reported that the longifolan stimulated by presence of pathogen and this have antifungal property. Siddiquee observed the volatile compounds like monoterpenes, sesquiterpenes from the GCMS study of Trichoderma harzianum (Siddiquee et al., 2012). In this study, newly produced mVOCs are Isolongifolan-7-ol, Trans-sesquisabinene hydrate, Methyl 2-methoxypropenote, Butane,1-propoxy, Pentane, 2,4-dimethyl,Heptadecane,8-methyl, (1R,5R)-4-methylene-1-((R)-6 methylhept 5, Benzene, 1-(1,5-dimethyl1-4-hexenyl)-4-methyl, 2-hexane, 3,3-dimethyl, 1-Pentanol, 2,2-dimethyl played the role of antimicrobial activity.
The antagonistic fungi Trichoderma longibrachiatum T(SP)-20 and T. asperellum T(AR)-10 both are effective to reduce the mycelial growth of Sclerotium rolfsii in paired plate assay. From the GC-MS analysis, the new inhibitory compounds or mVOCs such as Isolongifolan-7-ol, Trans-sesquisabinene hydrate developed in Trichoderma spp. co-culture with S. rolfsii but not in axenic culture. Among them some mVOCs induce growth promoting activity in groundnut, that will be studied further. Trichodema longibrachiatum T(SP)-20 is the most proficient antagonist produce the mVOCs against Sclerotium rolfsii cause the stem rot diseases of groundnut and induce the growth promotion in plants.
mVOCs-microbial Volatile Organic Compounds
GC-MS- Gas Chromatography Mass Spectrometry
Acc.No- Accession Number
Viz .- Namely
% - Per cent
◦C - Degree Celsius
CD - Critical Difference
cm - Centi meter
et al., - Co-workers
etc., - et cetera
Fig. - Figure
g - Gram
ml - Milli liter
µl - Micro litre
mg - Milli gram
No.- Number
PDA- Potato Dextrose Agar
NIST- National Institute of Standards and Technology
TNAU-Tamil Nadu Agricultural University
Funding
“The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”
Competing interests
The authors declare that they have no competing interests
Authors' contributions
|
Ayyandurai M |
: |
I did all the experiments under the guidance of co authors |
|
Akila R |
: |
Chief advisor of conducting all the experiments and technical guidance |
|
Manonmani K |
: |
Analysis the data and was a major contributor in writing the manuscript |
|
Mareeswari P |
: |
Major contributor for manuscript correction |
|
Vellaikumar S |
: |
GC-MS analysis of the all the samples and standardization of the procedure and gave technical guidance |
|
Mini M.L |
: |
Interpretation of the data and role of specific chemical were analyzed |
Data Availability
The pathogen and antagonist like, Sclerotium rolfsii (Acc. No MZ277282) , T(AR)-10 (Trichoderma asperellum Acc. No MZ277326), T(SP)-20 (Trichoderma longibrachiatum Acc. No MZ277378), T(VT)-3 (T. hamatum Acc. No MZ675442), T(BI)-16 (T. longibrachiatum Acc. No MZ802988), T(TK)-23 (T. citrinoviride) and Trichoderma asperellum (TNAU -TA) were obtained from Agricultural College and Research Institute, Madurai
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Table 1. In-vitro screening of Trichoderma spp. against S. rolfsii
|
S. No |
Name of Isolates |
Radial growth of myceliaum(cm)* |
PIOC * |
|
|
T (VT)-3 |
2.42c |
73.11 (58.76) |
|
|
T (AR)-10 |
2.21b |
75.44 (60.29) |
|
|
T (BI)-16 |
6.11e |
32.11 (34.51) |
|
|
T (SP)-20 |
1.41a |
84.44 (66.76) |
|
|
T (TK)-23 |
3.12d |
65.33 (53.92) |
|
|
TNAU-TA |
2.22b |
75.33 (60.21) |
|
7. |
Control |
9.00f |
0.00 (0.00) |
|
CD(P=0.05) |
0.170 |
- |
|
PIOC – Per cent Inhibition Over Control,
*Values are the means of three replicates; means in a column followed by the same letters are not significantly different according to Duncan's Multiple Range Test at P= 0.05; Values in parentheses are arcsine transformed values
Table 2. Efficacy Volatile compounds of Trichoderma spp. against Sclerotium rolfsii
|
S. No. |
Trichoderma Isolates |
Diameter of Mycelial growth (cm)* |
Per cent reduction over control |
|
1 |
T(VT)-3 |
4.88d |
46.66 |
|
2 |
T(BI)-16 |
6.24b |
33.33 |
|
3 |
T(SP)-20 |
3.52f |
61.11 |
|
4 |
T(AR)-10 |
4.15e |
54.44 |
|
5 |
T(TK)-23 |
5.74c |
36.66 |
|
6 |
TNAU-TA |
4.35e |
52.22 |
|
7 |
CONTROL |
9.00a |
- |
|
CD (P=0.05) |
0.203 |
2.19 |
|
Table 3. mVOCs produced by Trichoderma spp. axenic culture and co-culture with Sclerotium rolfsii
|
S.NO |
Secondary metabolites |
RT |
Peak area % |
Molecular weight |
Chemical Formula |
Specific role |
Reference |
|
Axenic culture of Trichoderma longibrachiatum |
|||||||
|
1 |
Cyclohexane, 3-(1,5-dimethyl-4-hexenyl)-6 |
21.253 |
6.41 |
204.3511 |
C15H24 |
Antimicrobial |
(Al-Marzoqi et al., 2016) |
|
2 |
Cycloheptasiloxane, tetradecamethyl |
19.605 |
0.81 |
519.0776 |
C14H42O7Si7 |
Antimicrobial |
(Moustafa et al., 2013) |
|
3 |
4-Butoxy-2-butanone |
5.340 |
19.13 |
144.21 |
C8H16O2 |
Antifungal |
(Anifalaje and Ibok, 2020) |
|
Axenic culture of Trichoderma asperellum |
|||||||
|
5 |
Tetracontane |
36.619 |
7.23 |
563.0791 |
C40H82 |
Antifungal |
(Ali et al., 2020) |
|
6 |
Bicylo(9,3,1)pentadeca-3,7-dien 12ol,4,8,1 |
32.031 |
5.61 |
290.4834 |
C20H34O |
Antibacterial |
(Almulaiky and AL-Farga, 2020) |
|
7 |
(1R,4R)-1methyl 4-(6-Methylhept-5-en-2-ylHeptane,8-methyl |
23.533 |
1.26 |
204.3511 |
C15H24 |
- |
- |
|
Co-culture of T. longibrachiatum and S.rolfsii |
|||||||
|
8 |
Isolongifolan-7-ol |
24.248 |
1.85 |
222.3663 |
C15H26O |
Antimicrobial |
(Chetia and Saikia, 2020) |
|
9 |
Trans-sesquisabinene hydrate |
23.939 |
7.10 |
222.3663 |
C15H26O |
Antimicrobial Antifungal |
(Hussein et al., 2016) |
|
Co-culture of T. asperellum and S. rolfsii |
|||||||
|
10 |
Isolongifolan-7-ol |
24.250 |
1.90 |
222.3663 |
C15H26O |
Antimicrobial |
Chetia and Saikia, 2020) |
|
11 |
1-Pentanol,2,2-dimethyl |
5.343 |
25.25 |
116.2013 |
C7H16O |
Antimicrobial |
(NA, 2017) |
|
Axenic culture of S.rolfsii |
|||||||
|
12 |
2-Methylhexacosane |
31.815 |
1.79 |
380.7335 |
C27H56 |
- |
- |
|
13 |
Hexacosane,1-iodo |
30.106 |
6.20 |
492.6035 |
C26H53I |
- |
- |
|
14 |
2-Aminoethanol,N,O- |
5.348 |
11.98 |
209.265 |
C10H11NO2S |
- |
- |
No competing interests reported.