Figure 1.
Different stages of M. phaseolina infected jute (C. capsularis) plant.
Jute seedlings were grown under controlled conditions (A), Three approximate distinctive areas are shown in infected leaf (B): infected area (Inf), invaded area (Inv), responsive area (Res), (C) represents healthy jute plant, (D) represents dark brown lesion of infected stem, (E), (F) and (G) represent symptoms of charcoal-rot disease in infected stem of jute.
Figure 2.
Morphological characteristics of M. phaseolina.
Longitudinal sections of M. phaseolina infected Jute stem showing sclerotia (A), hyphal network (B). Light micrograph of globose pycnidia and conidia along with released conidia of M. phaseolina from the pycnidium (C). Aerial hyphae of M. phaseolina (D).
Figure 3.
Detection of NO in M. phaseolina infected Jute stem stained with DAF FM-DA by fluorescence microscopy.
Images represent cross sections (A–D) and longitudinal sections (E–H) of jute stem showing the bright green fluorescence corresponding to NO, bar = 60 µm (F). The red colour corresponds to the autofluorescence. (A) represents control stem cross section, (C) represents infected stem cross section, (B) and (D) are the corresponding bright fields respectively. Bar = 250 µm (B). Figures are representative of at least six independent experiments.
Figure 4.
Detection of NO in longitudinal sections of mid rib portion of M. phaseolina infected Jute leaf.
Leaf sections were stained with DAF FM-DA showing the presence of NO as bright green fluorescence (A, D and G). Red colour corresponded to chlorophyll auto fluorescence (B, E and H). Merge images represent both NO and autofluorescence (C, F and I).
Figure 5.
Detection of RNS and RSNO in Jute stem.
DHR 123 and Alexa fluor 488 Hg-link phenylmercury were used for detecting RNS and RSNO in control and infected jute stem cross sections respectively. Images are control (A) and infected (C) jute stem cross sections showing the bright green fluorescence corresponded to the detection of RNS. (B) and (D) are the corresponding bright fields of (A) and (C) respectively. The red colour corresponds to the autofluorescence. Detection of RSNO in control (E) and infected (G) stem cross sections showing the bright green fluorescence. (F) and (H) are corresponding bright fields respectively. Figures are representative of at least six independent experiments. Bar = 250 µm.
Figure 6.
Quantitative measurement of RSNO in control and M. phaseolina infected jute leaf extract.
RSNO contents were measured in crude leaf extracts according to the method described in experimental procedures. Results are expressed as mean ± SD, for n = 3 experiments. P≤0.01, using one-way ANOVA.
Figure 7.
Secretome analysis of M. phaseolina and xylanase induced NO production in jute leaf discs.
M. phaseolina were grown on wheat bran under Solid State Fermentation (SSF) (A). (B) represents M. phaseolina culture grown on birch wood xylan containing agar plate. (C) represents silver stained gel M. phaseolina secretome. M corresponds to molecular weight markers and lane 1 represents the fungal secretome. Lane 2 represents zymogram analysis of xylanase. Achromatic white bands represent strong xylanase activity of the fungal secretome. (D) represents Congo Red stained plate of M. phaseolina culture grown on birch wood xylan containing agar plate (B) showing light yellow coloured hallow zone for xylanase activity. Panel (E) and (G) represent control and xylanase treated leaf discs cross sections respectively. Xylanase induced NO production was detected with NO specific fluorophore DAF FM-DA showing the bright green fluorescence corresponded to the presence of NO. The orange yellow colour corresponds to the autofluorescence. (F) and (H) are the corresponding bright fields of (E) and (G) respectively. Figures are representative of at least six independent experiments. Bar = 400 µm.
Figure 8.
Detection of NO in the micro particle present in the mycelia of M. phaseolina.
Micro particle, Mp; mycelial lumen, ML; fungal mycelium, FM; cell wall of fungal mycelium, CW. Individual or separate micro particles shown in panel (A), (B) and (C). Panel (D) and (E) represent diffusion of NO within the mycelia as shown by bright green fluorescence in presence of DAF-FM. Panel (F) is the corresponding bright field of panel (E).
Table 1.
List of the 22 NOS sequences of various organisms collected from NCBI database.
Figure 9.
Motif enrichment analysis of the reductase domain of M. phaseolina MS6 with the four fungal NOS sequences by MEME.
Enriched motif (A) found in 5 NOS sequences from the species M. phaseolina, N. parvum, C. gloeosporioides, G. graminicola, A. oryzae using MEME. (B) represents positions of the enriched motif in the five fungal NOS sequences. The motif is actually the Flavodoxin domain in these 5 necrotrophic pathogens. (C) represents the actual sequence of the enriched motif in the five fungal NOS sequences. The very low p-value denotes the high stringency in occurrence in each of the sequence.
Figure 10.
Motif enrichment analysis of the putative oxygenase domain of M. phaseolina MS6 with the four fungal NOS sequences by MEME.
Enriched motif (A) found in 5 NOS sequences from the species M. phaseolina, N. parvum, C. gloeosporioides, G. graminicola, A. oryzae using MEME. (B) represents position of the enriched motif in the five fungal NOS sequences. The motif is actually the Oxygenase domain in these 5 necrotrophic pathogens. (C) represents the actual sequence of the enriched motif in the five fungal NOS sequences. The very low p-value denotes the high stringency in occurrence in each of the sequence.
Figure 11.
Flavodoxin/nitric oxide synthase and Oxygenase domain of Nitric oxide synthase of M. phaseolina MS6.
Panel A represents the predicted domain structure of Flavodoxin/nitric oxide synthase which was found to be present in the reported amino acid sequence of M. phaseolina MS6. The Flavodoxin domain corresponds to amino acids 95 to 243. Panel B represents the predicted domain structure Oxygenase domain which was found to be present in the reported amino acid sequence of M. phaseolina MS6.
Figure 12.
Putative CaM binding motif within the oxygenase domain of M. phaseolina MS6.
The stretch of amino acids with a score of 9 represents probable CaM binding region.
Figure 13.
Determination of GR (A), Catalase (B) and APx (C) in control and M. phaseolina infected leaf extract.
Enzymes were assayed in crude leaf extracts according to the method described in Materials and Methods. Results are expressed as mean ± SD, for n = 3 experiments. p≤0.01, using one-way ANOVA.