SEM Analysis during in vitro fungal-fungal interaction
The SEM analysis for the interaction between T. coccinea and L. lactinea as well as T. coccinea and T. versicolor on the late stages of interaction, i.e., on the 9th, 12th and 15th days of interaction showed formation of pores on the hyphae (Fig. 2). The number of pores increased with the increase in number of days of interaction, which ultimately led to the death of the hyphae in the interaction zone.
Metabolites of the mono and dual cultures by LC-MS
The metabolic profiling of the different metabolites during the interaction of T. coccinea with T. versicolor along with that of the monocultures, i.e., T. coccinea and T. versicolor (Table 2) resulted in identification of 39 compounds. 18 of these compounds were produced exclusively during the interaction, 9 compounds were produced exclusively by T. coccinea monoculture and 13 were produced exclusively by T. versicolor monoculture. Similarly, during the interaction of T. coccinea with L. lactinea and their growth as monocultures produced 41 compounds (Table 3) of which 20 were produced exclusively during the interaction, 9 compounds were produced by T. coccinea and 12 compounds were produced by L. lactinea. The result showed that more numbers of compounds were obtained from the barrage zone as compared to the monocultures indicating the induction of metabolite production during the interaction. One of the significant results obtained from the LC-MS analysis is the detection of compounds like tyrosine, L-DOPA and melanin in the barrage zone (Fig. 3). Tyrosine and L-DOPA are involved in melanin synthesis pathway (Blagoeva 1984; Rzepka et al. 2016).
Table 2
Comparative analysis of metabolite profiles during interspecific interactions between T. coccinea - T. versicolor. ‘+’ indicates presence and ‘-’ indicates absence of a particular compound
Retention Time
|
Predicted Molecular mass
|
Adduct
|
Trametes coccinea
|
Trametes
versicolor
|
Trametes coccinea – Trametes versicolor
|
Compound
|
1.09
|
157.1
|
158.1 (M + H)
|
+
|
-
|
-
|
N-acetyl-D-proline
|
1.13
|
117.1
|
118.1 (M + H), 136.1 (M + NH4)
|
-
|
+
|
-
|
L-aspartic 4-semialdehyde
|
1.44
|
286.2
|
287.2 (M + H)
|
+
|
-
|
-
|
Cinnabarin
|
1.59
|
300.1
|
301.1 (M + H); M + ACN + H)
|
+
|
-
|
+
|
Cinnabarinic acid
|
2.29
|
147.9
|
148.9 (M + H), 171.1 (M + Na)
|
-
|
-
|
+
|
3,5-Dibromosalicylaldehyde
|
5.67
|
147.9
|
148.9 (M + H), 211.2 (M + ACN + Na)
|
-
|
+
|
-
|
Unknown
|
7.72(PDA)
|
|
|
-
|
-
|
+
|
|
7.56
|
278.1
|
279.1 (M + H), 301.0 (M + Na)
|
-
|
-
|
+
|
Unknown
|
7.74
|
240.2
|
241.2 (M + H), 481.4 (2M + H)
|
-
|
+
|
-
|
Anserine
|
8.74
|
285.3
|
286.3 (M + H), 318.3 (M + CH3OH + H)
|
-
|
+
|
-
|
Unknown
|
9.92
|
421.5
|
422.5 (M + H)
|
-
|
-
|
+
|
Unknown
|
10.31
|
313.3
|
314.3 (M + H)
|
-
|
+
|
-
|
Unknown
|
11.19
|
278.1
|
279.1 (M + H), 301.3 (M + Na)
|
-
|
-
|
+
|
Unknown
|
11.21
|
278.1
|
279.1 (M + H), 342.4 (M + ACN + Na)
|
-
|
+
|
-
|
Unknown
|
11.37(PDA)
|
|
|
-
|
-
|
+
|
|
11.77
|
229.3
|
230.3 (M + H)
|
-
|
+
|
-
|
Unknown
|
12.14
|
273.3
|
274.4 (M + H)
|
-
|
+
|
-
|
Unknown
|
12.18
|
|
|
-
|
-
|
+
|
Unknown
|
12.84
|
299.3
|
300.3 (M + H)
|
-
|
+
|
-
|
4-amino-2-methyl-5-diphosphopyrimidine
|
13.96
|
285.3
|
286.3 (M + H)
|
-
|
+
|
-
|
β-Alanine, N-isobutyryl-, decyl ester
|
14.19
|
583.2
|
584.2 (M + H)
|
-
|
-
|
+
|
Unknown
|
14.71
|
197.2
|
436.1 (2M + ACN + H)
|
-
|
-
|
+
|
L-DOPA
|
15.60
|
183.2
|
184.2 (M + H)
|
+
|
-
|
-
|
Phenoxazine
|
15.73
|
313.3
|
314.3 (M + H)
|
-
|
+
|
-
|
Unknown
|
16.39
|
240.3
|
241.3 (M + H), 279.2 (M + K)
|
-
|
-
|
+
|
D-Cystine
|
16.56
|
170.1
|
171.1 (M + H), 203.1 (M + Na)
|
-
|
-
|
+
|
D-Glyceraldehyde 3-phosphate
|
17.05
|
181.2
|
401.3 (2M + K)
|
-
|
-
|
+
|
Tyrosine
|
18.48
|
300.9
|
377.9 (M + 2K + H)
|
-
|
-
|
+
|
Unknown
|
18.73
|
633.3
|
634.3 (M + H), 678.5 (M + 2Na-H)
|
-
|
-
|
+
|
Unknown
|
19.44
|
338.3
|
339.3 (M + H), 356.4 (M + NH4)
|
+
|
-
|
-
|
Oleic acid, butyl ester
|
19.87
|
355.4
|
356.4 (M + H)
|
+
|
-
|
-
|
S-Adenosylmethioninamine
|
20.00
|
506.2
|
507.2 (M + H)
|
-
|
-
|
+
|
Fumaric acid, 3,4-dimethoxyphenyl undecyl ester
|
20.81
|
318.3
|
357.3 (M + K)
|
-
|
-
|
+
|
Melanin
|
21.76
|
427.4
|
428.4 (M + H)
|
+
|
-
|
-
|
Unknown
|
21.97
|
474.2
|
475.2 (M + H), 507.2 (M + Na)
|
-
|
-
|
+
|
Unknown
|
24.25
|
337.3
|
338.3 (M + H)
|
-
|
+
|
-
|
S-(Hydroxymethyl)glutathione
|
24.52
|
351.3
|
352.3 (M + H), 384.4 (M + Na)
|
-
|
-
|
+
|
Unknown
|
24.70
|
355.4
|
356.4 (M + H), 733.7 (2M + Na)
|
+
|
-
|
-
|
S-Adenosylmethioninamine
|
24.87
|
385.3
|
386.3 (M + H)
|
-
|
+
|
-
|
Unknown
|
25.89
|
240.1
|
|
-
|
-
|
+
|
Unknown
|
26.22
|
455.4
|
456.4 (M + H)
|
+
|
-
|
-
|
Unknown
|
Table 3
Comparative analysis of metabolite profiles during interspecific interactions between T. coccinea - L. lactinea. ‘+’ indicates presence and ‘-’ indicates absence of a particular compound
Retention time
|
Predicted Molecular mass
|
Adduct
|
Trametes coccinea
|
Leiotrametes lactinea
|
Trametes coccinea – Leiotrametes lactinea
|
Compound
|
1.09
|
157.1
|
158.1 (M + H)
|
+
|
-
|
-
|
Unknown
|
1.13
|
181.2
|
365.1 (M + H)
|
-
|
-
|
+
|
Tyrosine
|
1.44
|
286.2
|
287.2 (M + H)
|
+
|
-
|
-
|
Cinnabarin
|
1.48
|
143.0
|
144.0 (M + H)
|
-
|
-
|
+
|
Unknown
|
1.59
|
300.1
|
301.1 (M + H); M + ACN + H)
|
+
|
-
|
-
|
Cinnabarinic acid
|
2.12
|
292.1
|
293.1 (M + H)
|
-
|
-
|
+
|
Unknown
|
7.70
|
240.2
|
241.3 (M + H), 481.5 (2M + H)
|
+
|
-
|
+
|
Anserine
|
7.79
|
286.1
|
287.1 (M + H)
|
-
|
-
|
+
|
N(1)-(5-phospho-D-ribosyl)glycinamide
|
8.95
|
326.2
|
327.2 (M + H)
|
-
|
-
|
+
|
Fertaric acid
|
10.77
|
148.1
|
171.1 (M + Na)
|
-
|
+
|
-
|
2-hydroxyglutaric acid
|
11.16 (PDA)
|
|
|
-
|
+
|
-
|
|
11.19
|
278.1
|
279.1 (M + H)
|
-
|
-
|
+
|
Unknown
|
11.35
|
278.1
|
279.1 (M + H)
|
-
|
+
|
-
|
Unknown
|
11.64
|
229.2
|
230.2 (M + H)
|
-
|
-
|
+
|
Unknown
|
13.32
|
414.2
|
415.2 (M + H), 437.2 (M + Na)
|
-
|
-
|
+
|
Unknown
|
14.17 (PDA)
|
|
|
-
|
+
|
-
|
|
14.69 (PDA)
|
|
|
-
|
+
|
-
|
|
14.71
|
476.1
|
477.1 (M + H)
|
-
|
-
|
+
|
Unknown
|
14.88
|
197.2
|
395.4 (2M + H)
|
-
|
-
|
+
|
L-DOPA
|
15.58
|
226.1
|
227.1 (M + H)
|
-
|
+
|
-
|
Chorismate
|
15.60
|
183.2
|
184.2 (M + H)
|
+
|
-
|
-
|
Phenoxazine
|
16.58
|
575.3
|
576.3 (M + H)
|
-
|
-
|
+
|
Unknown
|
17.01
|
399.1
|
401.1 (M + H)
|
-
|
+
|
-
|
Unknown
|
18.44
|
382.2
|
446.0 (M + ACN + Na), 767.6 (2M + 1)
|
-
|
+
|
-
|
Unknown
|
18.48
|
383.0
|
766.5 (2M + 1)
|
-
|
-
|
+
|
Unknown
|
18.73
|
339.0
|
678.3 (M + H)
|
-
|
-
|
+
|
5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate
|
18.71
|
476.0
|
477.0 (M + H)
|
-
|
+
|
-
|
Unknown
|
19.44
|
338.3
|
339.3 (M + H), 356.4 (M + NH4)
|
+
|
-
|
-
|
Unknown
|
19.87
|
355.4
|
356.4 (M + H)
|
+
|
-
|
-
|
S-Adenosylmethioninamine
|
19.98
|
324.3
|
325.3 (M + H)
|
-
|
+
|
-
|
Uridine 5'-monophosphate
|
20.74
|
318.3
|
357.3 (M + K)
|
-
|
-
|
+
|
Melanin
|
21.76
|
427.4
|
428.4 (M + H)
|
+
|
-
|
-
|
Adenosine 3',5'-diphosphate
|
21.97
|
484.3
|
485.3 (M + H), 502.3 (M + NH4)
|
-
|
-
|
+
|
Unknown
|
22.51
|
397.4
|
398.4 (M + H), 420.3 (M + Na)
|
-
|
-
|
+
|
Phytosphingosine 1-phosphate
|
22.86
|
383.4
|
384.4 (M + H), 406.3 (M + Na)
|
-
|
-
|
+
|
Unknown
|
24.70
|
355.4
|
356.4 (M + H), 733.7 (2M + Na)
|
+
|
-
|
-
|
S-Adenosylmethioninamine
|
24.75
|
382.5
|
788.6 (2M + Na)
|
-
|
+
|
-
|
Unknown
|
24.48
|
281.3
|
282.3 (M + H)
|
-
|
-
|
+
|
Unknown
|
25.45
|
390.3
|
391.3 (M + H), 413.2 (M + Na)
|
-
|
+
|
-
|
Unknown
|
25.70
|
390.3
|
391.3 (M + H), 413.3 (M + Na)
|
-
|
-
|
+
|
Unknown
|
26.22
|
455.4
|
456.4 (M + H)
|
+
|
-
|
-
|
Unknown
|
26.28
|
535.3
|
536.3 (M + H), 599.1 (M + ACN + Na
|
-
|
+
|
-
|
Unknown
|
Table 4
Biochemical Characterization of melanin
Sl. No
|
Assay
|
Result
|
1
|
Solubility in Water
|
Insoluble
|
2
|
Solubility in Ethanol
|
Insoluble
|
3
|
Solubility in Hexane
|
Insoluble
|
4
|
Solubility in Acetone
|
Insoluble
|
5
|
Solubility in 1M KOH
|
Soluble
|
6
|
Solubility in 1M NaOH
|
Soluble
|
7
|
Reaction with Hydrogen peroxide
|
Decolorization of the pigment
|
8
|
Reaction with Hydrochloric acid
|
Precipitated readily
|
Extraction of extracellular melanin pigment
The SB broth where the two different fungi were dual cultured (i.e., T. coccinea and T. versicolor (Fig. 4A); T. coccinea and L. lactinea (Fig. 4B) showed dark brown coloration. On the other hand, no dark brown coloration was observed in the control and the monocultures of T. coccinea, T. versicolor and L. lactinea. As the color of the control did not change, it means that the difference in coloration is due to the compound secreted by the fungi and not due to the result of the oxidation of the media. An amount of 37 mg/L and 31 mg/L of extracellular pigment were obtained as a product from the culture broth, where T. coccinea – T. versicolor and T. coccinea and L. lactinea were allowed to interact respectively.
Biochemical analysis of the melanin pigment
The solubility test of the pigment showed that it was insoluble in water and organic solvents like ethanol, hexane, and acetone but soluble in 1M KOH and 1M NaOH. Also, the brown coloration of the pigment faded away when it was treated with an oxidizing agent like hydrogen peroxide. Treatment of melanin with hydrochloric acid resulted in precipitate formation (Table 3).
Characterization of the melanin pigment from FTIR spectrum
In FTIR spectrum, peaks were obtained at 3399 cm− 1, 2,926 cm− 1, 2,851 cm− 1, 1,586 cm− 1, 1385 cm− 1, 1030 cm− 1 and 618 cm− 1. Absorption at 3399 cm− 1 attributes to the polymeric OH groups. The stretching vibrations for aliphatic CH bonding appear at 2,926 cm− 1 and 2,851 cm− 1. At 1,586 cm− 1, the symmetric carboxylate stretching vibrations (COO) are detectable. The indole ring vibration / CNC stretching was observed at 1385 cm− 1. CH in-plane / CH out-of plane deformation are attributed at 1030 cm− 1. 618 cm− 1 indicates to the out-of-plane bending of the aromatic carbon-hydrogen bond (Fig. 5A). The presence of these functional groups indicates the presence of melanin.
Characterization of the melanin pigment from NMR spectrum
In NMR spectrum, peaks were obtained at 0.8 ppm, 1 ppm, 1.2 ppm, 1.3 ppm, 1.043 ppm, 2.341 ppm, 2.5 ppm, 3 ppm, 7.2 ppm. 0.8 to 1 ppm can be attributed to the CH3 groups of alkyl fragments. Peaks from 1.2 to 1.3 ppm can be attributed to the long chain methylenes. Peaks at 1.043 and 2.341 ppm are associated with the CH at aliphatic region. The signals at 2.5 ppm are associated with DMSO, which comes from the solvent deuterated DMSO or sulfonate groups bound to the pyrrole nitrogen which is relative to the occurrence of N-sulfonation. Peak at 7.22 ppm is related to the pyrrole –CH group of a carboxyl substituted indole. A peak ranging from 3 to 4 ppm is also observed tentatively near the signal from residual water in the DMSO. Peaks from 2.2 to 2.3 ppm indicates CH3 group and 2.5 to 3 ppm indicates NH groups which are connected to the indole groups. Also peaks from 7 to 7.5 ppm indicate the presence of aromatic heterocyclic ring of the pigment (Fig. 5B). The presence of these functional groups infers the compound to be melanin.
Assessment of Laccase Activity during in vitro fungal-fungal interaction
Qualitative analysis of laccase activity through plate assay indicated T. coccinea, T. versicolor and L. lactinea to be laccase positive (Fig. 6). Quantification of laccase activity revealed that the interactions increased laccase activities as compared to the enzyme activity in pure cultures. The laccase activity increased from 2nd day post inoculation up to the 8th day, and then slightly decreased on the succeeding days until the 14th day post inoculation for both the interactions (Fig. 7A, Fig. 7B). The laccase activity in case of the pure cultures also increased till the 8th days post inoculation but was less (significantly low), compared to the interaction ones. The laccase activity for T. coccinea with T. versicolor (F3-F1) and T. coccinea with L. lactinea (F3-F9) in the interaction zone was found to be 253.86 U/L and 272.25 U/L respectively. This was more as compared to the laccase activity of the monocultures of T. coccinea, T. versicolor and L. lactinea was found to be 70.64 U/L, 115.03 U/L and 134.42 U/L (p ≤ 0.05).
Assessment of Superoxide Dismutase (SOD) activity during in vitro fungal-fungal interaction
The superoxide dismutase assay showed that during the initial days of interaction (4th, 6th and 8th days) the superoxide dismutase activity was higher for the interacting fungi. But their activity gradually decreased from the 10th day post inoculation. Superoxide dismutase activity was very negligible as observed for the monocultures i.e., 1.5 U/mgFW in T. coccinea, 0.57 U/mgFW in T. versicolor and 0.79 U/mgFW in L. lactinea (Fig. 7C, D). The SOD activity for T. coccinea with T. versicolor (F3-F1) and T. coccinea with L. lactinea (F3-F9) in the interaction zone was found to be highest, i.e., 207.81 U/mgFW and 246.74 U/mgFW respectively at p ≤ 0.05 (Fig. 7C and 7D).
Gene expression profile during interaction of T. coccinea and T. versicolor
Quantitative Real time PCR (qRT-PCR) analysis of the 12 genes during interaction of T. coccinea and T. versicolor was performed and compared to the expression profile of T. versicolor control culture to understand how the interaction of the two fungal isolates affect the expression of these genes. The results of qRT-PCR analysis are shown in Fig. 8. Among the genes that showed upregulation during interaction, the lcc1 gene that encodes laccase showed 11.1-fold increased expression during interaction followed by tps (6.2-fold), cro (2.8 folds), amnO (1.7-fold) and alcDH (1.6-fold). Among the genes tested, fdo (gene encoding FAD-linked oxidoreductase), gdc (gene encoding glutamate decarboxylase), cmt (gene encoding CDF metal transporter), gst (gene encoding glutathione S transferase), alcO gene encoding alcohol oxidase) and kct (gene encoding 3-ketoacyl-CoA thiolase) showed significantly decrease in expression during interaction. The rest of the genes viz., abcT (gene encoding an ABC transporter), acdo (gene encoding aromatic compound dioxygenase), and mpx (gene encoding manganese peroxidase) did not reveal any significant difference in their expression during interaction of T. coccinea and T. versicolor.