Linda Hjeljord

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.

Relative Contribution of Various Sources of Botrytis cinerea Inoculum in Strawberry Fields in Norway

To identify the most important sources of inoculum of Botrytis cinerea (causal agent of gray mold) in commercial strawberry (Fragaria × ananassa) fields in Norway, soil and overwintered plant material were collected from planting beds and alleys at five locations in 2000 to 2002 (13 samples altogether). Plant material was sorted by category (e.g., leaves, stems, mulch, and weeds). After subsamples of each material were incubated for 5 days at 20°C at high humidity, conidiophores of B. cinerea growing from mycelia and sclerotia were counted. Overwintered plant debris within planting beds yielded more than 96% of total conidiophores counted, the remainder originating from plant debris collected from alleys or soil. Overwintered strawberry plant debris produced 98% of the conidiophores within planting beds and 80% of the conidiophores in the alleys, while the remaining was produced by weeds. Senescing and dead leaf laminae produced 45% of the conidiophores while stem residues (i.e., petioles, stolons, inflorescences, and unidentifiable stem parts) produced 50% and mummified fruit produced 5% within planting beds. The contribution of sclerotia, compared with mycelia, to conidiophore production varied greatly between fields and years. Overall, 47% of the total number of conidiophores produced in plant material within planting beds originated from sclerotia. More than 90% of the conidiophores from sclerotia were found in dead stem residues.

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.

First Report of Colletotrichum acutatum in Strawberry in Norway

Anthracnose caused by Colletotrichum acutatum J. H. Simmonds was detected in strawberry (Fragaria × ananassa Duch.) for the first time in Norway in 1999. Symptoms were found in greenhouse grown strawberries in the cultivar Korona. Symptoms were typical of strawberry anthracnose: sunken, brown, and firm lesions appeared on maturing fruits. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on acidified potato dextrose agar. Colonies grown on potato dextrose agar (PDA) were pale to mouse gray and became dark greenish to blackish in reverse. Conidia were formed in orange to salmon pink masses in the center of the culture. Conidia in cultures were 16.5 (13.8 to 18.8) × 4.5 (3.8 to 5) μm, and were hyaline, cylindrical, with pointed ends, and aseptate. Setae were never observed in culture or on fruits. The fungus did not form an ascigerous stage in culture. Mycelial growth rate at 25 to 26°C on PDA was 8.1 to 8.4 mm per day. Morphological characters and growth rate were in accordance with previous reports on C. acutatum (1,2). The isolated fungus was confirmed to be C. acutatum by both the International Mycological Institute, Egham, England, and Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Kochs postulates were fulfilled by inoculating ripe and unripe fruits on strawberry plants with the isolated fungus. Fruits were either sprayed with a conidial suspension (106 conidia per ml) or slightly wounded with a needle that had been dipped in a conidial mass from a pure culture of C. acutatum. Symptoms appeared after 4 days at 20°C, and after 5 days, brown, sunken, circular lesions reached a size of 1 cm in diameter on wounded, ripe fruits. In unripe fruits the lesions developed more slowly, and in unwounded fruits sprayed with a conidial suspension, large, irregular spots developed. Leaves were inoculated by placing a small block of agar at the base of petioles on intact strawberry plants. The tissue underneath the agar was either unwounded or slightly wounded with a needle. After 20 days (at 20 to 25°C) some necrosis developed on both unwounded and wounded petioles. No symptoms were observed in the crown tissue where the inoculated petioles were attached. The fungus was readily reisolated from both fruits and petioles, after which typical morphological characters developed in culture as described above. References: (1) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (2) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.

ABSTRACT Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.