Evaluation of Seed Transmission of Rhizoctonia solani and Seed Mycoflora of Ajwain

In vitro study was conducted Department of Plant Pathology, Rajasthan College of Agriculture, Udaipur (Rajasthan) and evaluated the seed transmission of R. solani from the seeds of eight popular cultivars of ajwain viz., Ajmer Ajwain-1, Ajmer Ajwain-2, Ajmer Ajwain-93, Pratap Ajwain, Lam selection-1, Gujarat Ajwain-1, Azad Ajwain and local cultivar. It was found that maximum recovery of the pathogens was from local cultivar and Ajmer Ajwain-93 exhibited lowest recovery of pathogens. It was also found in this study that there some differential results in recovery of R. solani from seeds of cv. Gujarat Ajwain-1 and cv. Azad Ajwain. Seed samples were collected and results revealed that in all seed mycoflora were detected in both blotter and agar plate test methods from almost all the seed samples and these fungi were Aspergillus niger, Aspergillus flavus, Aspergillus ochraceeous, Alternaria alternata, Rhizopus oryzae, Rhizoctonia solani, Dreschslera australiensis and Fusarium sporotrichioides.


INTRODUCTION
Ajwain also (Trachyspermum ammi L.) known as Bishops weed and Carom seed, is one of the most important seed spices crop its, belongs to family Apiaceae is a native of Egypt. Ajwain is erect, glabrous or minutely pubescent branched annual herb which grows up to 75-80 cm in height. In India it is widely distributed and its production is concentrated mainly in Rajasthan followed by Gujarat, Madhya Pradesh, Bihar, Utter Pradesh, Punjab, Tamil Nadu, Andhra Pradesh and West Bengal, respectively. Since ancient time the state of Rajasthan and Gujarat has emerged as seed spices bowl. Whose dried fruit of seeds are used as spices. In Rajasthan, it is cultivated in the districts of Chittorgarh, Udaipur, Jhalawar, Baran, Rajsamand, Bhilwara and Kota covering an area of 11658 hectares with the production and productivity is 4672 tonnes per annum and 401 kg/ha, respectively [1]. They are carrier of many important seed mycoflora inciting various diseases, which results in considerable losses in yield. Ajwain seeds carry a number of mycoflora. Although majority of them are saprophytes, a few are potentially pathogenic capable of ruining the crop. Several factors limiting the production of the crop in which poor health of the seed is one of the major factors which takes heavy toll of the crop at all the stages right from seedling to harvest and also during transport and storage. Seed germination and seedling growth greatly influenced by seed mycoflora [2].
One of the factors responsible for this is the use of contaminated seeds by farmers for the sowing purpose because several fungi are responsible for deterioration of ajwain grains/seeds in storage causing reduction in the germination potential and chemical constituents of seeds [3]. The aim of the present study to investigate the present status of seed mycoflora of Ajwain in Rajasthan and their incidence on seeds associated with fungi.

In vitro Evaluation of Different Seed
Mycoflora of Ajwain

Blotter method
Seed transmission of R. solani was studied on eight ajwain cultivars viz., Ajmer Ajwain-1, Ajmer Ajwain-2, Ajmer Ajwain-93, Pratap Ajwain, Lam selection-1, Gujarat Ajwain-1, Azad Ajwain and local cultivar. The sterilized plastic Petri plates of 90 cm layered with fresh and sterilized blotting paper on both the surfaces of Petri plate were used. Seeds of each cultivar were surface sterilized using 0.1 per cent mercuric chloride solution for one minutes followed by three washing with sterilized distilled water and then air dried. The blotter paper lined in the Petri plates were then moistened by using sterilized distilled water. Ten inoculated (R. solani) seeds per plate were used and maintained five replications for each cultivars, standard untreated check for each cultivar was also maintained with five replications for comparison. The plates were incubated at 28±1°C for 7 days and blotters were aseptically moistened on alternate days with sterilized distilled water to provide adequate moisture. Observation for seed germination, seedling mortality and healthy seedlings were recorded after seven days incubation.

PDA medium
Seed transmission of R. solani in all the eight cultivars was also studied using PDA medium. Twenty ml of sterilized melted PDA medium was aseptically poured in sterilized Petri plates and was allowed for 2 hrs to solidify. Seeds of all the cultivars were surface sterilized (using 0.1 per cent mercuric chloride solution for one minute followed by three washings with sterilized distilled water then dried). Ten seeds per plate were used keeping five replications for each cultivar. Standard untreated check for each cultivar was also maintained for comparison. The plates were incubated at 28±1°C for 7 days. Observation for seed germination, per cent seed germination, seedling mortality and healthy seedlings were recorded after seven days incubation.

In vitro Evaluation of Different Cultivars of Ajwain for Seed Transmission of Pathogens (R. solani)
Seed samples were collected from mandies of ajwain growing districts i.e., Udaipur, Pratapgarh, Chittorgarh and Rajsamand of the Southern Rajasthan. Further, seed samples collected from seed producing agency, which produced seeds using improved cultural practices. All the collected seed samples were kept in cloth bags and brought to the laboratory following store at room temperature for further studies. Samplings were done by methods suggested by ISTA, [4]. Mycoflora associated with seeds samples were collected at post-storage stage and isolated by using two incubation methods i.e. Blotter Method and Agar Plate Method [5,6].

Blotter method
From each sample, four hundred seeds were selected and analyzed randomly. White blotting papers were cut into circles of 9 cm of diameter and sterilized in autoclave at 121°C, 15 psi for 15 minutes. Three circles of blotter papers were placed at the bottom of sterilized Petri-dishes aseptically and moistened by sterilized distilled water. Ten seeds were placed at an equal distance in each Petri dish. These dishes were incubated at 28± 1°C with 12 hours of light alternating with 12 hours of dark period. The seeds were examined on 7 th day of incubation for emanating fungal colonies. Observed seed mycoflora developed on seeds in Petri dish and then examined the mycoflora under low and high objectives of compound light microscope.

Agar plate method
Two hundred seeds were taken from each sample for isolation of seed born mycoflora. Seeds were surface sterilized with 0.1 per cent mercuric chloride solution for one minutes followed by 3 washing with sterilized distilled water. Each sterilized Petri dishes contain 20 ml of PDA medium was used for incubation of seeds. Aseptically ten seeds per Petri dish were incubated at 28±1°C with 12 hours with alternating light and dark period. The fungal colonies emanating from seeds were examined from 3 rd and 8 th day of incubation. Isolation of mycoflora from ajwain seeds were carried out and maintained on 2 per cent PDA medium. Further, identification of the isolates was made in the laboratory by using compound microscope.

In vitro Evaluation of Different
Cultivars of Ajwain for Seed Transmission of Root-rot Pathogens R. solani

Blotter method
The effect of root rot causing pathogens R. solani was studied on eight different cultivars of ajwain.
The surface sterilized and un-sterilized ajwain seeds were aseptically kept on sterilized and wet blotter paper in Petri dishes and observed after 7 days of incubation at 28±1°C. The data revealed that the pathogens R. solani was recovered from almost all the test cultivars of ajwain.

CONCULSION
The present study shows that the seed transmission of R. solani from the seeds of eight popular cultivars of ajwain. It was found that maximum recovery of the pathogen was from local cultivar and lowest in Ajmer Ajwain-93. Seeds samples of ajwain were detected in both blotter method and agar plate test from almost all the seed samples and there were maximum fungi was found Aspergillus spp.