Isolation and fusion of protoplasts from the phytopathogenic fungus sclerotium rolfsii(sacc.)

Abstract

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5)/g mycelia and 2.8x10(5)/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Keywords: Pathogenicity; Protoplast; Sclerotium rolfsii; fusion; osmotic stabilizer.

Figure 1. . Effect of different osmotic stabilizers on protoplast yields of strains A and R of Sclerotium rolfsii , lysing enzyme 15mg/ml at pH 5.8 1g mycelia and incubation period 24 hours

Figure 2. . Effect of different concentrations of lysing enzyme on protoplast yields of strains A and R of Sclerotium rolfsii , lysing enzyme 15mg/ml at pH 5.8 1g mycelia and incubation period 24 hours

Figure 3. . A. Purified protoplasts in media. Regeneration of protoplasts of Sclerotium rolfsii. B. Onset of a regenerating protoplast forming a bud like structure. C. Germ tube formation from the bud. D. Formation of a hypha from germ tube. E. Elongation of hypha, (Magnification, 400 X)

Figure 4. . Fusion of protoplast of strains A and R of Sclerotium rolfsii , A. Close contact of two fusing protoplasts, B. rupture of membranes of fusing protoplasts, C. Complete rupture of membranes of fusing protoplasts and merging of cytoplasmic contents (Magnification, 400 X)

Figure 5. . Parent strains A and R of Sclerotium rolfsii and fusants F1, F2, F3 and F4. Strain A has thin mycelia, produces large number of small-sized sclerotia. The sclerotia are present all over the Petri dish. Strain R shows thick mycelia with large sized sclerotia few in numbers. F1 shows aerial growth pattern of mycelia and whitish in color. The sclerotia are produced in irregular manner in the Petri dish. F2 has thick mycelia with complete absence of sclerotia. F3 exhibits aggregation of small sized sclerotia, black in color. The aggregations of sclerotia are present at the periphery of the Petri dish. F4 shows small size sclerotia in chains present at the periphery of the Petri dish.

Figure 6. . Chlorophyll fluorescence in beans leaf (two months old plants) infected with parent strains A and R and fusants F1, F2, F3 and F4 and control. The vertical bars represent standard errors

Figure 7. . Chlorophyll a in beans leaf (two months old plants) infected with parent strains A and R and fusants F1, F2, F3 and F4 and control. The vertical bars represent standard errors

Figure 8. . Chlorophyll b in beans leaf (two months old plants) infected with parent strains A and R and fusants F1, F2, F3 and F4 and control. The vertical bars represent standard errors