Characterisation of potential antigen(s) of Tilletia indica teliospore walls to develop a specific immunoassay for Karnal bunt detection

Figures & data

Figure 1.  Morphological examination of different spores/teliospores of wheat fungal pathogens at 40× magnification. (A) Tilletia indica; (B) Tilletia foetida; (C) Puccinia recondita; (D) Puccinia striiformis; (E) H. sativum; (F) Fusarium spp.; (G) Aspergillus niger; (H) Ustilago nudo tritici.

Figure 2.  Determination of optimum number of teliospores by indirect ELISA using anti-teliospore antibodies.

Figure 3.  Determination of optimum antibody dilution by indirect ELISA using anti-teliospore antibodies.

Table 1. Cross-reactivity of anti-teliospore antibodies of Karnal bunt with other fungal spores.

	ELISA reading (OD 405 nm)
	Reading in triplicate
Fungal pathogen	R1	R2	R3	Mean – blank (0.185)	Percent cross-reactivity (%)
Fusarium sp.	0.299	0.205	0.319	0.089	8.8
H. sativum	0.256	0.203	0.265	0.056	5.6
T. indica	1.354	1.117	1.098	1.005	100
T. foetida	0.794	0.885	0.813	0.645	64.17
P. recondita	0.800	0.739	0.678	0.554	55.12
P. striiformis	0.768	0.793	0.815	0.607	60.3
A. niger	0.359	0.208	0.221	0.077	7.7
U. nudotritici	0.351	0.207	0.443	0.148	14.7

Table 2. Effect of various treatments on immunoreactivity of teliospores and the spores of different fungal pathogens using anti-teliospore antibodies of T. indica.

	ELISA readings (OD 405) using T. indica antiserum
Name of the treatment	T. indica	Loss of reactivity (%)	P. recondita	Loss of reactivity (%)	T. foetida	Loss of reactivity (%)	P. striiformis	Loss of reactivity (%)
Control	1.008	0	0.626	0	0.717	0	0.679	0
Heat treatment	0.888	12	0.585	6.6	0.696	3.0	0.608	10.5
Trypsin treatment	0.957	5.1	0.394	37.1	0.500	30.3	0.392	42.3
Periodate treatment	0.430	57.4	0.252	59.8	0.271	62.3	0.211	69.0
Methanol treatment	1.002	0.6	0.570	9.0	0.678	5.5	0.643	5.4
Periodate + Trypsin treatment	0.112	88.9	0.031	95.1	0.046	93.6	0.047	93.1

Figure 4.  (A). Protein profile analysis of spores/teliospores walls of different fungal pathogens of wheat by SDS-PAGE (12%). (B). Identification of shared immunoreactive epitopes in spores/teliospores walls of common fungal pathogens of wheat by western blotting. M – Marker, Lane 1 – Fusarium spp.; Lane 2 – Helminthosporium sativum; Lane 3 – Tilletia indica; Lane 4 – Tilletia foetida; Lane 5 – Puccinia recondita; Lane 6 – Puccinia striiformis; Lane 7 – Aspergillus niger; Lane 8 – Ustilago nuda tritici.

Table 3. Comparison of common bands identified by SDS-PAGE and identification of immuno-reactive proteins in spores/teliospores walls of different fungal pathogens using western blotting.

	Common band
Fungal pathogen	Low (25–35 kDa)	Medium (38–45 kDa)	High (60–80 kDa)	Total immuno reactive bands	Number of unique bands	Molecular weight of unique bands (kDa)
T. indica	27	42	67	3	2	34.0
	33		80			15.0
T. foetida	30	42	67	4	−	−
	27
P. recondita	−	39	67	3	−	−
			60
			80
P. striiformis	34	42	60	3	1	63.0
	30