Yeoung-Seuk Bae

Cotransformation of Trichoderma harzianum with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils

ABSTRACT Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), β-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introducedTrichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.

Influence of a fungus-feeding nematode on growth and biocontrol efficacy of Trichoderma harzianum

ABSTRACT A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80 degrees C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.

Bacillus spp. as Biocontrol Agents of Root Rot and Phytophthora Blight on Ginseng

Ginseng (Panax ginseng) is one of the most widely cultivated medicinal herbs in Korea. However, yield losses reached up to 30-60％ due to various diseases during 3 or 5 years of ginseng cultivation in the country. Therefore, successful production of ginseng roots depends primarily on the control of diseases. The objective of this study was to select potential biocontrol agents from rhizobacteria isolated from various plant internal root tissues for the control of multiple ginseng diseases as an alternative to fungicides. Among 106 Bacillus strains, two promising biocontrol agents, Bacillus pumilus strain B1141 and Paenibacillus lentimobus strain B1146, were selected by screening against root rot of ginseng caused by Cylindrocarpon destructans in a greenhouse. Pre-inoculation of selected isolates to seed or l-year-old root of ginseng resulted in stimulation of shoot and/or root growth of seedlings, and successfully controlled root rot caused by C. destructans (P

Rhizobacteria-mediated Induced Systemic Resistance in Cucumber Plants against Anthracnose Disease Caused by Colletotrichum orbiculare

Bacterial isolates TRL2-3 and TRK2-2 showing anti-fungal activity in vitro test against some plant pathogens were identified as Pseudomonas putida and Micrococcus luteus, respectively. Pre-treatment with both bacterial isolates at the concentration 1.0 and cfu/ml in the rhizosphere could trigger induced systemic resistance in the aerial part of cucumber plants against anthracnose caused by Colletotrichum orbiculare. However, the pre-treatment with the higher concentration at 1.0 cfu/ml of both isolates could not induce resistance after challenge inoculation with C. orbiculare. As a positive control, the treatment with DL-3 amino butyric acid caused a remarkable reduction of disease severity whereas the lesions on the leaves of untreated plants developed apparently after the fungal inoculation. From these results, it was recomended that disease control using both bacterial isolates inducing systemic resistance in the field where chemical application is forbid.

A Simple and Rapid Method for Functional Analysis of Plant Growth-promoting Rhizobacteria Using the Development of Cucumber Adventitious Root System

Many plant growth-promoting rhizobacteria (PGPRs) have been known for beneficial effects on plants including biological control of soilborne pathogens, induced systemic resistance to plant pathogens, phytohormone production, and improvement of nutrient and water uptake of plants. We developed a simple and rapid method for screening potential PGPR, especially phytohormone producing rhizobacteria, or for analyzing their functions in plant growth using cucumber seedling cuttings. Surface-sterilized cucumber seeds were grown in a plastic pot containing steamed vermiculite. After 7 days of cultivation, the upper part 2 cm in length of cucumber seedling, was cut and used as cucumber cuttings. The base of cutting stem was then dipped in a microcentrifuge tube containing 1.5ml of a bacterial suspension and incubated at with a fluorescent light for 10 days. Number and length of developed adventitious roots from cucumber cuttings were examined. The seedling cuttings showed various responses to the isolates tested. Some isolates resulted in withering at the day of examination or in reduced number of roots developed. Several isolates stimulated initial development of adventitious roots showing more adventitious root hair number than that of untreated cuttings, while some isolate had more adventitious root hair number and longer adventitious roots than that of untreated control. Similar results were obtained from the trial with rose cuttings. Our results suggest that this bioassay method may provide a useful way for differentiating PGPR`s functions involved in the development of root system.

Influence of Soil Microbial Biomass on Growth and Biocontrol Efficac of Trichoderma harzianum

The hyphal growth and biocontrol efficacy of Trichodemo harzianum in soil may depend on its interactions with biotic components of the soil environment. The effect of soil microbial biomass on growth and biocontrol efficacy of T. hanianum isolate ThzIDl-M3 (green fluorescent protein transformant) was investigated using artificially prepared different levels of soil microbial biomass (153,328, or 517ug biomass carbon per g of dry soil; BC). The hyphal growth of T. harzanum was significantly inhibited in the soil with 328 or 517 g BC compared with 153 ug BC. When ThzIDl-M3 was added to the soils as an alginate pellet formulation, the recoverable population of ThzIDl-M3 varied, but the highest population occurred in 517ug BC. Addition of alginate pellets of ThzIDl-M3 to the soils (10 per 50 g) resulted in increased indigenous microbial populations (total fungi, bacterial fluorescent Pseudomonas app., and actinomycetes). Furthermore, colonizing ability of ThzIDl-M3 on sclerotia of Sclerotinia sclerotiorum was significantly reduced in the soil with high revel of BC. These results suggest that increased soil microbial biomass contributes to increased interactions between introduced T. harzianum and soil microorganisms, consequently reducing the biocontrol efficacy of 1T. harzianum.

Effect of Sclerotial Distribution Pattern of Sclerotinia sclerotiorum on Colonizing Ability of Trichoderma harzianum

Field studies were conducted over two seasons during the summers of 1997 and 1998 to investigate the effects of different spatial arrangements (random or highly aggregated) of sclerotia of Sclerotinia sclerotiorum and alginate pellet types (bran or polyethylene glycol) on colonization of sclerotia by Trichoderma spp. Treatment with alginate pellets increased the mean percentages of sclerotia colonized by Trichoderma spp. in both years. Distribution patterns of sclerotia affected the mean percentage of sclerotia colonized by Trichoderma spp. in both years, indicating that a highly aggregated distribution of sclerotia was more favorable to colonization by Trichoderma spp. The effects of the different pellet types (bran or PEG) were not significant in both years (P > 0.05). The application of higher densities (200 pellets per 1 m2) of alginate pellets resulted in higher mean percentages of sclerotia colonized by Trichoderma spp. in 1998 (P < 0.05), but did not in 1997.