Christopher K. Hayes

Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor.

ABSTRACT The goal of this research was to improve scab resistance of apple by transformation with genes encoding chitinolytic enzymes from the bio-control organism Trichoderma harzianum. The endochitinase gene, as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh by Agrobacterium-transformation. A total of 15 lines were identified as transgenic by NPTII enzyme-linked immunosorbent assay and polymerase chain reaction and confirmed by Southern analysis. Substantial differences in endochitinase activity were detected among different lines by enzymatic assay and western analysis. Eight lines propagated as grafted and own-rooted plants were inoculated with Venturia inaequalis. Six of these transgenic lines expressing endochitinase were more resistant than nontransformed cv. Marshall McIntosh. Disease severity compared with cv. Marshall McIntosh was reduced by 0 to 99.7% (number of lesions), 0 to 90% (percentage of leaf area infected), and 1 to 56% (conidia recovered) in the transgenic lines tested. Endochitinase also had negative effects on the growth of both inoculated and uninoculated plants. There was a significant negative correlation between the level of endochitinase production and both the amount of disease and plant growth.

Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination

Different classes of cell wall degrading enzymes produced by the biocontrol fungi Trichoderma harzianum and Gliocladium virens inhibited spore germination of Botrytis cinerea in a bioassay in vitro. The addition of any chitinolytic or glucanolytic enzyme to the reaction mixture synergistically enhanced the antifungal properties of five different fungitoxic compounds against B. cinerea. The chemicals tested were gliotoxin, flusilazole, miconazole, captan and benomyl. Dose response curves were determined for each combination of toxin and enzyme, and in all cases the ED50 values of the mixtures were substantially lower than ED50 values of the two compounds used alone. For instance, the addition of endochitinase from T. harzianum at a concentration of 10 micrograms ml-1 reduced the ED50 values of toxins up to 86-fold. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the highest levels of synergism with synthetic fungicides were detected for the endochitinase from T. harzianum strain P1, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The use of hydrolytic enzymes to synergistically enhance the antifungal ability of fungitoxic compounds may reduce the impact of some chemical pesticides on plants and animals.

Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds

A number of cell wall degrading enzymes (CWDEs) and cell membrane affecting compounds (MACs) that alter cell membrane structure or permeability have been assayed in vitro against phytopathogenic fungi and bacteria. Osmotin, gramicidin, valinomycin, phospholipase B, trichorzianine A1, trichorzianine B1, gliotoxin, flusilazole, and miconazole were tested in combination with three endochitinases, four exochitinases, and one glucan 1,3-beta-glucosidase from fungi, bacteria, or plants. Every combination of MAC + CWDE showed a high level of inhibition against Botrytis cinerea and Fusarium oxysporum and the interaction between the two kinds of compounds was of a synergistic nature. Different levels of synergism were obtained among the compound combinations depending upon the antifungal activity of the enzyme. When the enzyme treatment was applied subsequent to the MAC, the level of synergism was lower, indicating that degradation of the cell wall is needed to establish the synergistic interaction. The synergism with MACs was also present when the fungal cell wall was altered in a non-enzymatic manner by including L-sorbose in the growth media. The sensitivity of bacterial strains to the two trichorzianines depended upon the nature of their cell wall and could be synergistically enhanced by partial digestion of the wall. Some of the combinations showed a high level of synergism, suggesting that the interaction between MACs and CWDEs could be involved in biocontrol processes and plant self-defense mechanisms.

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.

Molecular cloning and expression of the nag1 gene (N-acetyl-β-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1

Abstract A 72-kDa N-acetyl-β-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a λgt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-β-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-β-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.

Principles in the Development of Biological Control Systems Employing Trichoderma Species Against Soil-Borne Plant Pathogenic Fungi

Control of plant pathogens has been accomplished in large part by the use of chemical pesticides. Biological control of these plant pathogens was originally thought to be futile (Garrett, 1956); however, many researchers have shown that certain filamentous fungi are capable of controlling several plant pathogenic fungi. These “biofungicides” have been under investigation for several years. Fungi in the genus Trichoderma have been shown to suppress Pythium spp. (Chet et al., 1981; Harman and Hadar, 1983), Sclerotium rolfsii and Sclerotinia sclerotiorum (Lee and Wu, 1984), Rhizoctonia solani (Elad et al., 1980; Harman et al., 1981; Lewis and Papavizas, 1987), Botrytis cinerea (Tronsmo, 1989), and Fusarium spp. (Sivan and Chet, 1986; 1989) and other pathogens (Cook and Baker, 1983) on various agronomically and horticulturally important plants. Diseases caused by these plant pathogens (e.g., seed rots, damping-off, wilts, fruit rots, and root rots) have been shown to be effectively reduced by Trichoderma. This ability is of major importance because of new restrictions on the applications of chemical pesticides (Harman and Lumsden, 1990). Also, the use of chemical fungicides may eliminate a large range of organisms, some of which may be beneficial to the environment. Biocontrol organisms should be less disruptive than chemical pesticides.

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.