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).
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).