Arne Tronsmo

Biological and integrated controls of Botrytis cinerea on apple with Trichoderma harzianum

Biological and integrated controls of dry eye rot on apple caused by Botrytis cinerea were possible under natural field conditions by the use of the antagonistic fungus Trichoderma harzianum. A fungicide-resistant isolate of T. harzianum, P1, controlled dry eye rot better than the fungicide-sensitive parent strain both alone and together with reduced dosages of the fungicide vinclozolin. Although integrated control was not significantly more effective than biological control or chemical control by itself, the combination of two different control methods may provide a more consistent control and reduce the risk of fungicide-resistance developing in the pathogen. Use of fungicide-resistant strains of the biocontrol agent is also necessary as long as other diseases on the crop are controlled by fungicides.

Antagonism of Nutrient-Activated Conidia of Trichoderma harzianum (atroviride) P1 Against Botrytis cinerea.

ABSTRACT The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum (atroviride) P1 to suppress Botrytis cinerea was investigated in laboratory, greenhouse, and field trials. Preliminary nutrient activation at 21 degrees C accelerated subsequent germination of the antagonist at temperatures from 9 to 21 degrees C; at >/=18 degrees C, the germination time of preactivated T. harzianum P1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated T. harzianum P1 conidia reduced in vitro germination of the pathogen by >/=87% at 12 to 25 degrees C; initially quiescent conidia achieved this level of suppression only at 25 degrees C. Application of quiescent T. harzianum P1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by >/=85% at 24 degrees C, but only by 35% at 12 degrees C. Preactivated conidia reduced infection by >/=60% at 12 degrees C. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperature of 19 degrees C, whereas only preactivated conidia were effective in the field at a mean temperature of 14 degrees C on the day of treatment application. An antagonistic mechanism based on initiation of germination in sufficiently concentrated inocula suggests that at suboptimal temperatures the efficacy of Trichoderma antagonists might be improved by conidia activation prior to application.

Effect of Germination Initiation on Competitive Capacity of Trichoderma atroviride P1 Conidia.

ABSTRACT Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1 x 10(7) P1 conidia/ml were coinoculated with 1 x 10(5) Botrytis cinerea conidia/ml, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient activated before inoculation. Pregermination respiration also affected competitive capacity of the antagonist on solid substrates, where respiratory CO(2) stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R(2) >/= 0.97, P < 0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only 2 h of incubation on a nutrient-rich substrate at 23 degrees C. These results indicate that nutrient-induced changes preceding germination in Trichoderma conidia can either enhance or decrease their biological control potential, depending on environmental conditions in the microhabitat.

Coproduction of chitinolytic enzymes and biomass for biological control by Trichoderma harzianum on media containing chitin

Abstract Production of N -acetyl-β-glucosaminidase (EC 3.2. 1.30), chitobiosidase, and biomass by Trichoderma harzianum is dependent on media composition but not on inoculum concentration. Growth of T. harzianum in simple mineral salt solutions plus single carbon sources, including chitin, gave low enzyme activity in the culture filtrate and few conidia. Addition of any of several complex materials, such as V8 juice, yeast extract, or proteose peptone, substantially increased enzyme and spore yield. If sucrose (0.5%) was added together with chitin, enzyme yields were increased after 6, but not after 3 days of culture, relative to chitin alone. The source of chitin was also important; higher yields of enzymes or conidia were obtained with a purified colloidal chitin than with an unpurified chitin from crab shells. Addition of insoluble polyvinylpyrrolidone to the culture increased yields of enzymes. All activity was present as extracellular enzymes; grinding fungal mycelium did not increase the amounts of enzymes detected. An economical medium consisting of a mineral salts solution, colloidal chitin, and sucrose that supported high yields of both conidia and chitinolytic enzymes was identified. Such coproduction may aid commercialization of T. harzianum by reducing the costs of two separate and valuable products.

Profile and Morphology of Fungal Aerosols Characterized by Field Emission Scanning Electron Microscopy (FESEM)

Fungal aerosols consist of spores and fragments with diverse array of morphologies; however, the size, shape, and origin of the constituents require further characterization. In this study, we characterize the profile of aerosols generated from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum grown for 8 weeks on gypsum boards. Fungal particles were aerosolized at 12 and 20 L min−1 using the Fungal Spore Source Strength Tester (FSSST) and the Stami particle generator (SPG). Collected particles were analyzed with field emission scanning electron microscopy (FESEM). We observed spore particle fraction consisting of single spores and spore aggregates in four size categories, and a fragment fraction that contained submicronic fragments and three size categories of larger fragments. Single spores dominated the aerosols from A. fumigatus (median: 53%), while the submicronic fragment fraction was the highest in the aerosols collected from A. versicolor (median: 34%) and P. chrysogenum (median: 31%). Morphological characteristics showed near spherical particles that were only single spores, oblong particles that comprise some spore aggregates and fragments (<3.5 μm), and fiber-like particles that regroup chained spore aggregates and fragments (>3.5 μm). Further, the near spherical particles dominated the aerosols from A. fumigatus (median: 53%), while oblong particles were dominant in the aerosols from A. versicolor (68%) and P. chrysogenum (55%). Fiber-like particles represented 21% and 24% of the aerosols from A. versicolor and P. chrysogenum, respectively. This study shows that fungal particles of various size, shape, and origin are aerosolized, and supports the need to include a broader range of particle types in fungal exposure assessment.

Submicronic Fungal Bioaerosols: High-Resolution Microscopic Characterization and Quantification

ABSTRACT Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min−1 airflow. The overall median numbers of aerosolized submicronic particles were 2 × 105 cm−2, 2.6 × 103 cm−2, and 0.9 × 103 cm−2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

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.

Genetic Manipulation for Improvement of Microbial Biocontrol Agents

During the last few years genetic modification of microorganisms to improve their biological control of plant pathogens has created some very interesting results, which indicates that molecular biology can be used not only to understand the mechanism behind biological control, but also to improve biocontrol ability. It is, however, important to remember that in most strains, the ability to control disease will be explained by more than one mechanism, and the host for genetic modification has to be ecologically fit in the environment where the biocontrol is to be performed. Modification of a biocontrol mechanism may result in an improved biocontrol ability, but may also give unexpected effects. The effect of a new or modified biocontrol agent must therefore be carefully determined empirically for each host-pathogen system. There is also in the public a resistance against introduction of genetically modified organisms into the environment, which probably will slow down the introduction of genetically modified biocontrol agents in many countries. So far only a few modified organisms are on the market, e.g. the deletion mutants A. radiobacter strain K1026 (Section 25.3.3) and the Ice- mutant of P. syringae (Section 25.3.6), plus the protoplast fusant T. harvanum strain 1295-22 (Bio-Trek 22) (Section 25.2.2), but with the great demand for alternatives to chemicals in plant protection it is expected that more modified organisms soon will become commercially available.