Guy R. Knudsen

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.

Isothiocyanates produced by Brassicaceae species as inhibitors of Fusarium oxysporum

Glucosinolates contained in members of the Brassicaceae release isothiocyanates potentially useful in controlling Fusarium oxysporum pathogens in conifer seedling nursery soils. Our objective was to determine the toxicity of individual isothiocyanates to different growth stages of the fungus. Bioassays with four F. oxysporum isolates were conducted using sealed containers in which 0.3 μl of 2-propenyl, ethyl, buty, phenylethyl, benzyl, or phenyl isothiocyanate was allowed to volatilize. Propenyl and ethyl isothiocyanates were the most fungistatic of those compounds tested. The same concentrations of propenyl and ethyl isothiocyanates that inhibited mycelial growth completely suppressed conidial and chlamydospore germination of all isolates. Other isothiocyanates including ethyl, benzyl, and phenethyl were also fungitoxic to F. oxysporum conidia and chlamydospores. Reduction in pathogen populations resulting from a green-manure crop are likely achievable since chlamydospores are sensitive to isothiocyanate. Pathogenic F. oxysporum isolates infesting nursery soils would likely be most suppressed by species of plants such as Brassica carinata, B. nigra, and B. juncea, which contain glucosi-nolates that release high concentrations of propenyl isothiocyanate.

Potential for biocontrol of Sclerotinia sclerotiorum through colonization of sclerotia by Trichoderma harzianum

In the laboratory, Trichoderma harzianum strain ThzID1, formulated as mycelial fragments in alginate pellets with or without wheat bran incorporated, colonized sclerotia of Sclerotinia sclerotiorum in raw or steamed soil. Incidence of colonization was higher in steamed soil than in raw soil, higher at 25 C than at 15 C, and higher at −500 kPa than at −50 kPa but was not affected by bran in pellets. Some sclerotia were colonized by indigenous Trichoderma spp. Pellets containing T. harzianum were added to soil in a pea (Pisum sativum) field or field microplots at densities of 4 × 10 2 − 2 × 10 4 pellets per square meter, along with sclerotia of S. sclerotiorum (.)

Inhibition of Aphanomyces euteiches f. sp. pisi by Volatiles Produced by Hydrolysis of Brassica napus Seed Meal

Seed meal from Brassica napus (rapeseed) produced volatile fungitoxic compounds potentially of value in the control of Aphanomyces root rot of pea. Hyphal growth, germination of encysted zoospores, and oospore survival and inoculum potential, were determined in the presence of volatiles produced from B. napus seed meal. Volatile compounds from B. napus meal completely suppressed mycelial growth and germination of encysted zoospores on agar. In growth chamber bioassays, pea (Pisum sativum) seed inoculated with zoospore suspensions and incubated 24 h in the presence of volatiles from rapeseed meal had 50% lower root rot disease severity than in the absence of meal. Volatile compounds passing through soil also significantly decreased survival and inoculum potential of oospores. Gas chromatographic analysis of rapeseed tissues and the volatile compounds evolved from tissues showed that substrate glucosinolates were hydrolyzed enzymatically to produce mainly isothiocyanates. Non-autoclaved rapeseed meal produced significantly higher levels of volatile compounds than did autoclaved meal. Also, volatile compounds produced from autoclaved meal were dominated by nitriles, whereas isothiocyanates were more common volatile products from non-autoclaved meal. Our results indicate that B. napus allelochemicals responsible for toxic effects toward A. euteiches f. sp. pisi are enzymatic hydrolysis products of glucosinolates.

Soil amendment effects of rape (Brassica napus) residues on pea rhizosphere bacteria

Abstract Rhizosphere bacteria were isolated from pea (Pisum sativum) plants grown in soil amended with a green manure of high-glucosinolate rape (Brassica napus ‘Dwarf Essex’), and from pea plants grown in soil without amendment. Total viable counts of bacteria and fluorescent Psuedomonas spp., and proportionate representation of actinomycetes and Gram-negative bacteria in the pea rhizosphere community were not significantly different for amended and non-amended pea. Carbon substrate utilization patterns obtained from Biolog GN microplates were compared. Rhizosphere isolates from the two treatments, that differed by less than 50% in utilization of specific carbon substrates, were considered to use those substrates in a similar manner. Cluster analysis was used to evaluate relationships of strains according to their abilities to utilize the different carbon substrates. Non-amended pea and amended pea isolates used 88% of the carbon substrates in a similar manner, and cluster patterns were similar for the amended and non-amended pea strains. The number of nodules formed on amended and non-amended pea roots by Rhizobium leguminosarum were not significantly different. Amendment of soil with rape residues did not appear to significantly alter the pea rhizosphere population and community attributes measured.

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.

Efficacy of Trichoderma harzianum as a biological control of Fusarium oxysporum in container-grown Douglas-fir seedlings

Inoculating a soilless medium with encapsulated Trichoderma harzianum did not affect any aspect of Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) seed germination or subsequent growth. Results of inoculating medium with a known pathogenic isolate of Fusarium oxysporum alone, or concurrently with T. harzianum, were the same: high levels of damping-off, low amounts of hypocotyl and root disease in midsummer, and significant reductions in height growth. When seedling roots grew through T. harzianum-inoculated medium before growing into a mixture of T. harzianum-F. oxysporum-inoculated medium, mortality was reduced about 50%. Although contamination by resident Fusarium occurred, subsequent root colonization was significantly reduced in T. harzianum-amended growing medium.

Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil.

ABSTRACT One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.

Colonization of Sclerotinia sclerotiorum sclerotia by a biocontrol isolate of Trichoderma harzianum, and effects on myceliogenic germination

Abstract Sclerotia of Sclerotinia sclerotiorum were incubated on cultures of Trichoderma harzianum. Myceliogenic germination decreased by 50% within 1 day and continued to decrease over time. Quantitative PCR showed a decrease in Sclerotinia DNA for older sclerotia, but not fresh sclerotia. Trichoderma DNA increased and persisted inside older sclerotia but not fresh sclerotia.

Simulation of Russian wheat aphid movement and population dynamics on preferred and non-preferred host plants☆

Abstract A computer simulation model was developed to investigate spatial and population dynamics of apterous Russian wheat aphids ( Diuraphis noxia ) on preferreed (wheat) and non-preferred (oat) hosts. The location, development, reproduction, movement, and mortality of individual aphids within a population were predicted at simulated one-day time steps. Reproductive rate and mortality were modeled as stochastic processes, which were functions of both aphid chronological age and host favorability. An index of host favorability wwas established for each plant in a 25× 25-plant grid containing wheat oats. Host favorability was dependent on host species and aphid density on that plant. Aphid movement was modeled as a stochastic process, with probability of movement dependent on insect age and host plant favorability. The distance moved was proportional to the density of aphids on the plant the aphid was leaving, with random direction of movement. The model was calibrated with data from experiments in which Stephens wheat or Border oat plants in growth chambers were infested with adult female aphids, and populations monitored after 0, 3, and 7 days. Ability of the model to predict observed populations and plant infestation after 14 and 21 days was then tested. Use of the model to generate predicted spatial patterns of aphids on differential hosts is discussed.