In present paper, studies related to seed -borne nature of F.oxyporum in Ridge gourd and its role on disease transmission have been described.

Review of literature has given in heading result and discussion.

108 Seeds samples of Ridge gourd were collected separately in paper bags from different districts of Rajasthan. The seed samples were stored under natural conditions and then subjected to dry seed examination for disease symptoms. They were screened by Standard Blotter Test (ISTA1999). 4 Samples from different districts carrying infection of F. oxysporum were used for location and transmission studies. Both symptomatic and asymptomatic seeds were used for histopathological study. Pathogen location was studied by component plating, cleared whole-mount preparation of seed coat, aleurone layer cotyledons and embryonal axis and microtome section of whole seeds in selected samples. For component plating 20 seeds of each category were soaked in water for 5 hours and components (seed coat, aleurone layer, cotyledons and embryonal axis) were dissected aseptically and plated on moistened blotter for a week to isolate the pathogen. In whole -mount preparation ,10 seeds of each category were boiled individually in distilled water for 10-15 minutes (because of its hard seed coat), cooled and dissected to separate seed components. Seed components were kept separately in 10% aqueous solution of KOH for 5-20 min depending of the softening of the tissue, washed thoroughly in light warm water and boiled in 5 ml of lactophenol containing 2 ml of cotton blue for 3-4 min. The stained softened seed components were kept on a slide in a drop of polyenyl alcohol and pressed gently to spread the tissue uniformly (Singh et al. 1977). For microtome sectioning 10 seeds of each category were boiled in water for 10 min,fixed in 70% ethanol for 48 hour, dehydrated through Tertiary Butyl Alcohal series and embedded in paraffin wax. One or two transverse incisions were made with a blade into each seed to ensure paper dehydration infiltration and embedding. The paraffin blocks were cut to expose the tissue and immersed in 1% aqueous solution of sodium lauryl sulphate for12 hr, washed thoroughly in distilled water and transversed to a mixture of glycerine and acetic acid (1:1 v/v) for seven days for further softening. Serial microtome sections were cut (20-25 µ thick) and stained sections were mounted in DPX (Jonhnson ,1940). Disease transmission of seed -borne inoculum was determined by using blotter method (10 seeds / plate) and water agar seedling symptom test (1 seed/tube) using two replicates of 100 seeds /category/sample for each test (Khare et al.1977). The seeds were pretreated with 2% of sodium hypochlorite solution for 2-3 min. before sowing.

Seeds of 51 samples out of 108 samples were covered with thin whitish hyphae or white crust carried infection of F. oxysporum with incidence of 2.25-19.00%. The incidence of pathogen in incubation test varied from 4-61% in untreated ,1-48 % in pretreated and 1-51% in PDA test.

In component plating the recovery of pathogen was 5-10% in seed coat of asymptomatic seeds. In weakly symptomatic seeds, the incidence was 50,45% in seed coat ;40,30% in aleurone layer;30,20% in cotyledons and 35,30% in embryonal axis of both the samples. Heavily infected seeds showed presence of thin, hyaline mycelium on all components of seed with higher incidence ,80,70% in seed coat,65,55% in aleurone layer, 50,40 % in cotyledons and 60,50% on embryonal axis. (Plate 1, fig. a, b, c, d &e).

In cleared whole- mount preparation, mycelium was confined to seed coat 0-5% only in asymptomatic seeds, while it was observed in all parts of weakly and heavily symptomatic seeds. In weakly infected seeds it was 40,30% in seed coat;32,25% in aleurone layer; 25,15%in cotyledons and 30,20 % in embryonal axis of two samples studied. In heavily infected seeds it was 70,60% in seed coat;55,45% in aleurone layer;40,30% in cotyledons (Plate 1, fig. e & f) and 50,40% in embryonal axis of both the samples studied. (Table 1)

In microtome section asymptomatic seed showed thin hyaline mycelium which was confined to seed coat only but in weakly infected seeds branched, thin, hyaline, septate mycelium was observed in all layers of spermoderm, tissue of hilar fissure, aleurone layer and cotyledons. Mycelial aggregation in epidermal, sub-epidermal palisade layers, parenchymatous cells and inner epidermis showed deformation of cells. Mycelium was both inter as well as intra-cellular in epidermal,sub-epidermal  layers( sclerotic cells) and radially elongated palisade cells. Cells were loosened because of mycelial aggregation but cellular content remained as such. Hyphae also ramified in aleurone layer and cotyledons and in trachiedal bars.

Heavily infected seeds (somewhat shriveled) showed thin, hyaline mycelium in all components of seed. Epidermal palisade cells were heavily colonized by inter and intra cellular hyphae. Microconidia and hyphae invaded in sclerenchymatous layers of hypodermis and spread inter and intra-cellularly all around in spongy parenchyma and form thick mat. Polygonal aleurone cells were completely destroyed and replaced by mycelial mat of F. oxysporum.  The mycelium invaded from seed coat in to cotyledons through aleurone layer. Heavy infection caused disintegration and lysis of cells. (Plate 2, fig. a, b, c, d, e &f) Similar effects has also been observed in seeds of guar and sesame (Bhatia, Singh and Singh, 1995; Dubey and Singh 2005).

Seed-borne infection of F. oxysporum caused failure of seed germination, seed rot, seedling infection and wilting. Disease symptoms appeared with in 2^nd and 3^rd day of incubation as browning of radicle later small pale yellow discolored patches formed on hypocotyl which became more darker after 48 hr of its initiation, then symptoms appeared on cotyledons as irregular necrotic patches.Even  cotyledonary leaves also revealed mycelial growth on its surface. Severity of symptoms caused mortality of seedling which was 1,5,7% in Rg 25 and 1,6,3% in Rg32 samples in asymptomatic, weakly symptomatic and heavily symptomatic category. Total pre and post emergence losses were 6,8% in asymptomatic; 41,44% in weakly symptomatic seeds and 95,90 % in heavily infected seeds of sample number 25 and 32 respectively. (Table 2) (Plate 3 fig. a, b, c, d, e,& f).

The study indicates that the mycelium of F.oxysporum remains extra-embryonal  in asymptomatic as well as both extra and intra -embryonal in symptomatic  category.  Extra-embryonal mycelium plays an important role in disease transmission of F.oxysporum .Seeds carrying heavy extra and intra-embryonal infection remains ungerminated and these seeds help in increase the inoculum of this fungi in soil. Mycelium present on seed coat adds the primary source of inoculum of disease in soil also.

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