Trichoderma protoplast fusion: a tool for improving biocontrol agents

Abstract

Protoplasts from two auxotrophic mutants of Trichoderma harzianum Rifai (ATCC 32173), obtained from young thalli following cell wall digestion by NovoZym 234, were fused in 33% PEG suspended in 10 mM Tris-HCl and 10 mM CaCl2, pH 7.5. The frequency of fusion between lysine- and arginine-requiring auxotrophs resulting in prototrophic strains was about 5%. These prototrophic strains were classified into parental and nonparental types. Colonies developed from single conidia of the nonparental phenotype exhibited prototrophic parental or recombinant phenotypes. The ability of both prototrophic and parental strains to overgrow the soil-borne pathogenic fungi Rhizoctonia solani, Sclerotium rolfsii, and Pythium aphanidermatum in dual cultures was used to evaluate their antagonistic capability. The antagonistic abilities of the prototrophic strains were found to vary with each pathogenic fungus. The prototrophic strain A2 overgrew all the pathogenic fungi more rapidly than the parental strains. Strain A2 effectively controlled Rhizoctonia damping-off of cotton seedlings, in the greenhouse, when compared with the parental strains. Protoplast fusion appears to be a useful tool for combining desirable traits from parental strains to produce improved biocontrol strains. Key words: Trichoderma harzianum, biocontrol, protoplast fusion.

Influence of Protoplast Fusion in Trichoderma Spp. on Controlling Some Soil Borne Diseases

“…Strains of T. harzianum (sensitive to carbendazim and resistant to Thiophanatemethyl) and T. viride (sensitive to Thiophanate-methyl and resistant to carbendazim) mycelia were subjected to lytic activity with different fungal enzyme such as β-glucuronidase and driselase at different conversation for 2 hrs with intermittent agitation [18]. The enzyme…”

“…Among different concentrations of tested lysing enzymes, KCl as osmotic stabilizer was optimal for the release of protoplasts from different Trichoderma spp. However, in previous study highest protoplasts were obtained from T. harzianum using Novozym 234 at 10 mg/ml with 0.6 M KCl [18]. Other author used 10 mg/ml of Novozym 234 with 0.6 M sucrose to isolate maximum number of protoplasts from T. harzianum and Trichoderma koningii [7], and also, obtained maximum protoplasts from Trichoderma roseum using Novozym 234 in combination with chitinase and cellulase, each at 5 mg/ml [23].…”

“…The enzyme reacted with the cell wall of mycelial mass became swelled up and round hyaline globules by the from 40% to 60% of PEG is suitable for protoplasts fusion in different fungi [23,24]. However, Pe'er and Chet [18], used 33% PEG for protoplast fusion in T. harzianum. Therefore, concentration of PEG is highly critical for effective fusion of protoplasts [5,24].…”

Phylogeny and antagonistic activity of some protoplast fusants in Trichoderma and Hypocrea

“…Protoplasts were fused using a procedure similar to that described by Pe′er and Chet (1990). 1 ml of the suspension containing 10 6 protoplasts in STC buffer (0.6 M sorbitol; 10 mM CaCl2; 10 mM Tris-HCl at pH 7.5) was prepared and equal number of protoplasts from H. koningii and T. asperellum strains was mixed.…”

Pathogenicity and its mode of action in different sedentary stages of Heterodera glycines (Tylenchida: Heteroderidae) by Verticillium lecanii hybrid strains

“…Lalithakumari (2000) reported that protoplast fusion is a valuable tool for strain improvement by facilitating the recombination of whole genomes and the development of hybrid strains in fungal biological control agents. Furthermore, improvement in the antagonistic activity of hybrid strains was observed when compared with parental strains (Pe'er and Chet, 1990;Couteaudier et al, 1996;Prabavathy et al, 2006).…”