INTRODUCTION:

Leaf blight of sesame caused by Alternaria spp. (Alternaria sesami Kawamura and Alternaria alternata Fr Keissler) is an important foliar disease which causes yield loss to the extent of 28.9 per cent (Prasad et al., 1997)¹. The fungus is known to cause seed rot, pre and post emergence death of seedlings and infect all the above ground parts resulting in considerable loss in yield both quantitatively and qualitatively (Ellis and Holiday, 1970 and Naik et al., 2004)^2,3. The disease symptom mainly manifested on the leaf surface as concentric minute brown spots, later enlarged with characteristic concentric rings at the centre to produce a target like pattern. Stem bending symptom was also observed (Rani and Thirupathaiah, 1983)⁴. Symptoms on the capsules started with minute water soaked lesion, later turned to black circular, such pods drop off. Several Alternaria spp. are known to produce a variety of toxins even though they are not required for their normal growth and reproduction.

In Alternaria, many non-specific toxins have been studied in detail with respect to Alternaria alternata f. sp. lycopersici (AAL toxin), A. alternata f. sp. citri tangerine (ALT toxin), A. alternata f. sp. kikuchiana (AK toxin), A. alternata infecting sunflower (AS toxin), A. brassicae (Destruction B) and A. helianthi (Deoxyradium and radicin) (Thomma, 2003)⁵. Whereas the toxin produced by A. macrospora inhibited seed germination of cotton up to 100 per cent (Padmanabhan and Narayanaswamy, 1977)⁶. This paper makes an effort to understand the preliminary studies on toxin production by A. sesami and its partial purification and Infra-red analysis. In addition, induction of systemic resistance was also attempted in sesame by challenge inoculation with A. sesami.

The non-toxic synthetic chemicals and some bioagents are known to acts as strong elicitors of plant defense reaction leading to induced resistance and enhanced broad spectrum of resistance against pathogens (Meteraux et al., 1991)⁷. Hence, an attempt was made to induce the defense system in susceptible cultivar through seed treatment with inducing agents and which might provide a reasonable solution to the non availability of resistant cultivars against specific pathogen.

MATERIAL AND METHODS:

Collection of Alternaria sesami isolate

The isolate of A. sesami was obtained from the blight infected leaves of sesame by using standard tissue isolation technique. The pure culture of the fungus was obtained by hyphal tip isolation. Further the identity of fungus was confirmed by depositing the culture at the National Bureau of Agriculturally Important Microorganisms, Indian Agricultural Research Institute, New Delhi. The pure culture of A. sesami obtained from sesame cultivar E8 was cultured on Czapeck’s agar medium. Nine days old culture is transferred to Czapecks broth in conical flasks incubated at 27 ± 1⁰ C for 11 days. Later culture medium was filtered using Whatman No. 42 filter paper. The toxin was extracted from culture filtrates using water saturated butano (Bhaskaran and Kandaswamy, 1978)⁸.

Partial purification of toxin

Culture filtrate was centrifuged at 12000 rpm for 10 minutes to remove spores. The supernatant was reduced to 1/5^th of its original volume by evaporating at 46⁰ C in hot water bath. Gradually two volumes of acetone were added to the culture filtrate with constant stirring till the precipitation was completed and allowed to stand overnight at 4⁰ C. The precipitation was removed by centrifugation at 2000 rpm for 10 minutes. The acetone was removed from supernantent liquid by hot water bath at 40⁰C, the remaining solution was adjusted to 1/5^th of original volume with water. This solution was extracted three times with two parts of aliquots of water saturated 1-butanol. The water phase was discarded and the Butanol phases were combined and kept on hot water bath at 40⁰C till complete dryness. The residue from the Butanol phase was dissolved in 100 ml water extracted three more times with 200 ml of aliquots of water saturated Butanol. The water phase was again discarded and the Butanol phases were combined and dried.

Plate 1: Bioassay of purified toxin of A. sesami on seed germination and seedling growth of sesame.

The dried residue from the combined Butanol phase was dissolved in 200 ml of water. This solution was extracted twice with 400 ml aliquots of diethyl ether and ether phases were discarded. The water phase was taken to dryness by hot water bath. Moisture was completely removed and dried product was stored in clean and air-dried bottle. The toxin was obtained as crystalline brown powder.

Infra-red analysis of toxin

The final residue obtained was weighed and it was designated as purified toxin. The purified toxin was subjected to infra-red analysis to confirm the chemical nature of compound. The infra-red spectrum was run with the sample Nujol followed by purified toxin (Fig 1). The toxin thus obtained was used for bioassay.

Bioassay of purified toxin on sesame and tomato seedlings

Different concentrations of purified toxin of A. sesami viz., 2000, 1000, 500, 250, 100 and 50 ppm were tested on sesame and tomato seedlings. The top portion of 25 days old healthy seedlings of sesame and tomato were placed in toxin of 10 ml solution and suitable control was maintained using sterilised distilled water. Effect of toxin on these plants was recorded after 24 h and 48 h exhibiting the necrosis, chlorosis, epinasty and drooping symptoms.

Bioassay of purified toxin on sesame and tomato seeds

Thirty seeds of sesame were soaked in 10 ml of 50, 100, 250, 500, 1000 and 2000 ppm concentration of purified toxin for an h. Later they were spread on moistened blotting paper and each treatment was replicated thrice containing thirty seeds. Equal number of healthy seeds were soaked in sterile distilled water, which serves as control. Observations on germination per cent of both the seeds were recorded after six days. The per cent inhibition of seed germination, shoot and root length was evaluated by the formula given by Vincent (1947)⁹.

Induced systemic resistance

Resistance inducing chemicals such as salicylic acid, sodium nitrate, mannitol, potassium nitrate, sucrose, potassium dihydrogen phosphate and magnesium sulphate were evaluated on the mycelial growth of A. sesami which is a predominant species causing leaf blight of sesame. The fungus A. sesami, was grown on PDA medium for nine days. The PDA medium was prepared and melted. The resistance inducing chemicals were added to the melted medium to obtain the desirable concentration based on the molecular weight of the chemical. Twenty ml of this medium was poured in each sterilized petri plate. Suitable check was maintained without addition of resistance inducing chemicals. Five mm mycelial disc taken from the periphery of 10 days old colony was placed in the Petriplate and incubated at 27 ± 10⁰ C for 9 days. Three replications were maintained for each treatment. The diameter of the colony was measured in two directions and average was recorded and the per cent inhibition of growth was calculated by using the formula given by Vincent (1947)⁹.

In vivo testing of induced resistance

The efficacy of different bioagents viz., Pseudomonos flourescens (I and E), Bacillus subtilis(E) and Bacillus subtilis(I), leaf extract of Occimum spp., Prosopis julifer (10%) and resistance inducing chemicals, salicylic acid and magnesium sulphate (1%) were used for induction of resistance. The seeds of sesame cultivar E8 were soaked in the above mentioned treatments for four h followed by 30 minutes shade drying and the seeds were inoculated with spore suspension (1 X 106 spores/ml) of A. sesami. The seeds treated with the fungus alone and distilled water was maintained as check for comparison. Forty seeds were sown separately in pots having sterilized soil for each treatment. The seed germination and seedling vigor were recounted after nine days of sowing and calculated by using the formula given by Abdulbaki and Anderson (1976)¹⁰.

RESULTS AND DISCUSSION:

Culture of A. sesami

The culture started appearing on the PDA slants after a week’s inoculation. Based on the type of growth, sporulation and measurements the fungus was identified as Alternaria sesami. The identity was confirmed by depositing the culture at the National Bureau of Agriculturally Important Microorganism (NBAIM), Indian Agricultural Research Institute, New Delhi with ITCC No. 170.83.

Partial purification of toxin

Many plant pathogenic fungi produce toxic metabolites in culture media and plant tissues, which take part in pathogenesis and symptom expression (Wood et al., 1972)¹¹. A. helianthi produces a toxin under in vitro and plant tissues as reported by early workers (Bhaskaran and Kandaswamy, 1978; Islam and Maric, 1980 and Amaresh and Nargund, 1999)^{8, 12, 13}. In the present study pure toxin was recovered as crystalline brown powder.

Bioassay of purified toxin on seed germination and seedling growth

The different concentrations of purified toxin were used for bioassay on sesame and tomato seed germination, root and shoot length elongation and induction of necrotic symptoms on sesame and tomato seedlings.

Table 1. Effect of purified toxin on seed germination and seedling growth of sesame.

Treatments	% inhibition of seed germination over control *	% inhibition of seedling growth over control *
Treatments	% inhibition of seed germination over control *	Shoot length	Root length
50 ppm	6.67 (14.60)	7.39 (17.20)	16.43 (23.42)
100 ppm	12.23 (20.41)	13.04 (22.42)	23.15 (28.18)
250 ppm	18.90 (25.72)	13.92 (23.39)	26.86 (31.00)
500 ppm	35.57 (36.57)	26.09 (30.84)	47.22 (43.31)
1000 ppm	56.67 (48.83)	55.22 (48.50)	56.48 (48.50)
2000 ppm	64.25 (81.10)	64.78 (51.85)	62.97 (51.87)
S. Em ± C. D. at 1%	0.586 2.625	0.453 1.83	0.463 1.86

* Mean of three replications.

The maximum inhibition of seed germination (64.25 %) of sesame, shoot length (64.78 %) and root length (62.97 %) was noticed at 2000 ppm concentration (Table 1 and Plate 1&3). In bioassay of tomato seeds the inhibition of tomato seed germination (82.21 %), shoot length (83.2 %) and root length (67.62 %) was observed which was much higher, indicating much more sensitivity of tomato to toxin than sesame (Table 2 and Plate 2&4). Least inhibition of seed germination, root and shoot length of both the crop seeds was observed at 50 ppm concentration tested. The findings are in agreement with Islam and Maric (1980)¹², Robeson and Strobel (1984)¹⁴ and Lu et al (1987)¹⁵ who also demonstrated inhibition of shoot elongation and root elongation in sesame.

Bhaskaran and Kandaswamy (1978)⁸ also reported toxicity of culture filtrate of A. helianthi on sunflower. Mahabaleshwarappa (1981)¹⁶ also made similar observation while working with A. carthami on safflower.

Table 2. Effect of purified toxin on seed germination and seedling growth of tomato.

Treatments	% inhibition of seed germination over control *	% inhibition of seedling growth over control *
Treatments	% inhibition of seed germination over control *	Shoot length	Root length
50 ppm	13.33 (21.29)	7.82 (16.03)	21.00 (27.22)
100 ppm	17.77 (24.88)	24.44 (29.49)	39.43 (38.89)
250 ppm	24.44 (29.59)	36.48 (37.13)	46.72 (42.91)
500 ppm	31.11 (33.84)	40.80 (39.67)	51.97 (46.13)
1000 ppm	51.11 (44.61)	49.72 (44.81)	63.18 (52.64)
2000 ppm	82.22 (65.32)	83.02 (65.67)	85.54 (67.65)
S. Em ± C. D. at 1%	0.329 1.472	0.259 1.162	0.276 1.236

* Mean of three replications.

In the present study, purified toxin was also subjected to Infra-red analysis, which revealed that the toxin comprising lot of hydrogen bounded OH group. The nature of the peak indicates that the compound may contain more than two OH groups. This was known by the peaks exhibited through spectrum of compound (Fig. 1). However, Amaresh and Nargund (2005)¹⁸ identified a compound which is a derivative of furone molecules with 3 propyl 4 hydroxy 20 X 0-2-3 dehydrofurones groups based on the production of infrared by nuclear magnetic resonance mass spectrum using CaCl3 + DMSO-d6 chemical.

Table 3. Efficacy of resistance inducing chemicals in inhibiting the mycelia growth of A. sesami.

Sl. No	Induced chemicals	Per cent inhibition
1	Sucrose	65.18 (53.81)
2	Mannitol	66.67 (54.70)
3	Potassium Nitrate	55.81 (48.41)
4	Sodium Nitrate	67.10 (54.92)
5	Salycilic acid	68.18 (55.59)
6	Magnesium sulphate	61.23 (51.41)
7	Potassium Orthophosphate	62.58 (52.28)
S. Em ±C. D. at 1%		0.236 (1.019)

Induced Resistance

Some of the chemicals were used to induce resistance in host plant, which defend against pathogenic infection. This systemic acquired resistance will spread to untreated parts thus providing protection against pathogens. The present investigation showed that salicylic acid and sodium nitrate at 10 mM concentration were effective in inhibiting the mycelial growth of A. sesami by 68.18 and 67.10 per cent respectively (Table3).

Vigor index is one important criteria for observing induction of resistance. The efficacy of resistance inducing chemicals, botanicals and bioagents were further studied in vivo condition also (Table 4).

Table 4. Effect of systemic resistance inducing agents on seed germination, seedling growth and vigor of sesame, challenged with A. sesami.

Sl. No	Treatments	Germination Per Cent	Mean Shoot Length	Mean Root Length	Vigor Index
1	Pseudomonas fluorescens (I)	86.95	6.50	2.67	794.84
2	Pseudomonas fluorescens (E)	97.75	6.97	3.60	1029.85
3	Bacillus subtilis (I)	82.20	7.83	2.37	839.32
4	Bacillus subtilis (E)	77.78	7.27	2.97	807.56
5	Occimum spp. (10%)	91.03	7.33	3.13	938.46
6	Prosopis julifera (10%)	93.24	7.60	3.70	1059.83
7	Salicylic acid (1.0%)	91.03	8.27	3.90	1138.28
8	Sodium Nitrate (1.0%)	93.28	6.97	3.90	1008.96
9	Inoculated Control	19.99	3.77	1.30	102.39
10	Uninoculated Control (Healthy)	97.75	8.43	3.73	1089.27
	S. Em ±	0.412	0.263	0.338	18.22
	C. D. at 1%	1.676	1.071	1.377	74.13

The study indicated that the maximum germination per cent was observed in P. fluorescens (E), Prosopis julifera, Occimum spp, Sodium nitrate and salicylic acid with 97.75 per cent, 93.28 per cent, 93.24 per cent and 91.03 per cent respectively. The maximum shoot and root length was observed in salicylic acid treatment (8.27 cm and 3.9 cm). Overall higher vigor index was noticed in salicylic acid treated sesame seeds (1138.28). The efficacy was high in salicylic acid, Prosopis julifera and P. fluorescens (E) treatments. The higher vigor index was more in these treatments. Ratnam et al. (2001)¹⁹ also noticed effective control of A. helianthi by salicylic acid treatment in sunflower. The results are also in accordance with Mosa (2002)²⁰, who reported salicylic acid and P. fluorescens strain (Pf5) seed treatment as the ideal inducers of resistance and in reducing the disease. Ramegowda and Naik (2008)²¹ also reported higher vigor index of cotton seedlings by use of salicylic acid and Bacillus subtilis bioagent against A. macrospora, the causal agent of leaf spot obtained from Bt cotton. The present study not only indicated the property of these chemicals in inhibiting the growth of mycelium which but also it can be extended to field for managing disease after working out the dosage and timing of such treatments.

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Received on 23.08.2012 Modified on 30.08.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1176-1181