Fungal radial growth rate and asexual sporulation assays

By Kathy Price,2014-05-06 12:23
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Fungal radial growth rate and asexual sporulation assays


    2 Fungal radial growth rate and asexual sporulation assays. For radial growth assays, spore

    suspensions of the isolates ARSEF 2575, Ect A-18, KO B1-3, KO 37-2, KO 43-1, and KO 8-18 3

    64 /ml. An aliquot (3 ;l) of each were prepared in 0.05% Silwet L-77 and adjusted to 1 x 10

    5 suspension was transferred to sterile 5 mm diameter paper disks placed in the center of 90 mm

    plates of ? SDAY or CDA. Cultures were incubated at 25?C in the dark. Replicates were 6

    7 conducted in quadruplicate, and colony diameters were recorded daily for 12 d.

    68 For asexual sporulation assays, an aliquot (100 ;l) of each conidial suspension (1 x 10/ml)

    of ARSEF 2575, Ect A-18, KO B1-3, and KO 8-18 was spread on ? SDAY plates in triplicate 9

    10 and incubated under continuous fluorescent light at 25?C for 10 d. The contents of each plate 11 (medium plus fungal colony) were blended for 20 s in 50 ml water, transferred to individual 250 12 ml flasks, and combined with an additional 50 ml of water used to rinse the blender. To reduce 13 spore aggregation, Silwet L-77 was added to 0.1% final concentration, followed by incubation of 14 the flasks on an orbital shaker at 200 rpm and ambient temperature for 3 h. Nine aliquots of the 15 homogenized material were sampled from each flask and diluted 100-fold in water. Conidium 16 concentration in each aliquot was assessed using a hemacytometer. The number of spores 17 produced by each replicate was estimated as the average of the nine aliquots multiplied by the 18 dilution factor (100x) and the volume of the blended culture (122 ml).

    19 Colony hydrophobicity assays and radial growth of ?manps1 mutants in response to

    20 hydrogen peroxide. For assaying hydrophobicity of sporulating cultures, two-wk-old cultures of 21 control and ?manps1 mutants were treated with 100 ;l droplets of sterile water on the surface of

    22 the culture for several hours to evaluate whether there were detectable changes in the ability of 23 the water to be absorbed by the culture.


1 O) assays, experiments were conducted using CDA alone or For hydrogen peroxide (H22

    2 amended with 10 or 16 mM HO, a reactive oxygen species generator. Approx 5,000 conidia of 22

    ARSEF 2575, Ect A-18, KO B1-3, KO 8-18, KO 37-2, or KO 43-1, prepared in 0.05 % Silwet L-3

    4 77, were transferred to a 5 mm paper disk placed in the center of each test plate (three replicates 5 per treatment). The diameter of each colony was recorded after 11 or 12 days in two independent 6 experiments.

    7 Conidial germination assays with ?manps1 mutants. A 5 ml aliquot of ? SDAY with

    8 0.05% Silwet L-77 (added after autoclaving) was transferred to each 9 cm diameter plate and 9 spread equally across the plate to produce a thin layer from which spores could be visualized at 10 multiple time points by microscopy. Plates were prepared fresh for each assay to avoid 11 premature dessication. Dry spores of ARSEF 2575, Ect A-18, KO 37-2, KO 43-1, or KO 8-18 12 were transferred from ? SSAY to the surface of ? SDAY plus Silwet L-77 assay medium by 13 moistening a sterile Q-tip on a clean agar surface, loading the Q-tip with spores from ? SSAY 14 plates, and swabbing the spores across the entire surface of the plate in both directions. Dry 15 spores were used rather than calibrated spore suspensions in water to avoid possible leaching of 16 polar metabolites from spores into the suspending medium (4), which could affect germination.

    o17 All inoculated plates were incubated at 25C in the dark, without sealing, in a closed plastic

    18 container with a moistened paper towel at the bottom.

    19 Conidia were photographed at two or three time points within a 36 h period after plating to 20 capture events before and during germination. The specific timing of data recording for each 21 experiment varied depending on whether a treatment altered spore germination relative to the 22 control. Photos were taken so that treated and untreated germlings from at least one time point in 23 each experiment could be easily viewed, scored, and compared before mycelial growth became


    21 so extensive to obscure visibility of individual spores and germination tubes. An approx 1.5-cm2 piece of agar was cut from the center of the plate, transferred to a microscope slide, and a cover

    slip was mounted with a drop of water. Digital photographs were taken at 40X magnification 3

    4 with a Macrofire digital camera linked to an Olympus BH-2 epifluorescence microscope, 5 documenting a minimum of four randomly chosen and well-separated areas. The status of at least 6 100 conidia was recorded for each time point within each treatment, recording the data for all 7 spores within a single field before moving on to the next field. A spore was considered 8 germinated if any evidence of a germ tube was present, no matter the length. 9 One- and 3-wk-old conidia of ?manps1 mutants and control strains were assayed to

    10 determine whether aging of conidia under standard growth conditions affected viability of 11 mutant strains. Germination rate (germ tube length at a particular time point), spore length, and 12 area of hydrated spores of ?manps1 mutants were compared with control strains, using a pen on

    13 a graphic pad to measure pixels with the image processing software ImageJ 1.37v (1). Pixels 14 were converted to microns at 8.1 pixels/micron. Spore germination of each strain was also 15 assayed on medium containing HO (5, 7.5, and 10 mM) or 0.8 M KCl in comparison with 22

    16 spores of each strain germinated under standard conditions without a stress treatment. 17 To assess germination of conidia on cockroach wings, wings of German cockroach (Blattella

    18 germanica) were collected from adults that had been treated with CO then frozen before 2

    o19 removal of wings, which were stored at -20C. Wings were surfaced sterilized following the

    20 methods of Wang and St. Leger (6), except using 0.6% sodium hypochlorite rather than 5%. 21 Each surface-sterilized wing (5 per fungal isolate) was transferred to water agar (1.5%) and 22 inoculated with spores of ARSEF 2575, Ect A-18, KO 8-18, KO 37-2, or KO 43-1 by swabbing 23 the wing surface with a conidia-laden Q-tip as described above for conidial germination assays.


    o1 C for 25 h, viewed, and photographed as described above. Inoculated wings were incubated at 25

    2 Conidial adhesion assays. Attachment to hydrophobic polystyrene. Test plates (60 mm

    33 polystyrene Petri dishes) were filled with 5 ml sterile water containing 75 ;l of a 4 x 10/ml

    4 spore suspension of ARSEF 2575, Ect A-18, KO B1-3, KO 8-18, KO 37-2, or KO 43-1. Each 5 plate was swirled gently ten times, and then left to sit for 1 h at ambient temperature to promote

    attachment of spores. Water was removed with a disposable transfer pipette. Plates were rinsed 6

    7 once with 5 ml sterile water by swirling the liquid in the plate for 20 rotations then excess 8 supernatant was removed. Plates were overlaid with 8 ml of cooled (45?C), molten ? SDAY 9 with 0.075% Igepal CA-630 (ICN Biomedicals Inc., Costa Mesa, CA), a colony growth restrictor. 10 For reference plates, 75 ;l of each spore suspension was mixed in 8 ml medium and poured into 11 60 mm plates to solidify. Both adhesion assay and reference test plates were prepared in 12 triplicate and incubated at 25?C in the dark for 7 d. Attachment rate of conidia for each test plate 13 was calculated as follows:

    14 CFU/Cfu x 100 ipi

    15 where CFU is the CFU counted for the fungal isolate i on the test plate p; Cfu= average CFUs ipi

    16 counted on the reference plates for strain i.

    17 Attachment to hydrocarbon. Bacterial adhesion to hydrocarbon (BATH) assays were

    18 conducted using the method of Singh et al. (5), which they adapted for fungal conidia. Conidia of 19 ARSEF 2575, Ect A-18, KO 8-18, and KO 43-1 were assayed. Both 1-, 2-, and 3-wk-old conidia 20 produced on barley agar or 10-, 20-, and 30-d-old conidia produced on ? SSAY were harvested 21 in 0.1 M phosphate-buffered saline, pH 7.2, filtered through cheesecloth to remove mycelium, 22 and the concentration was adjusted to 0.4 OD. Conidial suspensions were treated with benzene 620


    1 as described previously for the BATH assay, and cell surface hydrophobicity (CSH) was 2 calculated (5). Triplicate samples were assayed for each isolate.

    3 Attachment to surfaces with varying levels of polarity. Qualitative assessment of adhesion to

    4 hydrophobic, weakly polar, and hydrophilic surfaces was conducted using modified approaches 5 of Holder and Keyhani (2). Permanox Lab-Tek tissue culture treated plastic slides (no. 177437; 6 Lab-Tek Chamber Slide System, Nalge Nunc International, Rochester, NY) were used as a 7 weakly polar surface. A charged hydrophilic surface was created by coating Permanox Lab-Tek 8 plastic slides with 1% poly-D-lysine hydrobromide (no. P-7280; Sigma-Aldrich Co.) followed by 9 drying. Siliconized glass slides with a highly hydrophobic surface were prepared using 10 Sigmacote (Sigma-Aldrich Co.) as in Holder and Keyhani (2). Conidia of ARSEF 2575, Ect A-11 -old ? SSAY plates in sterile distilled water, 18, KO 8-18, and KO 43-1 were collected from 10-d

    12 filtered through sterile cheesecloth, pelleted by centrifugation, and diluted to a final

    613 spores/ml in 0.1 M Tris-HCl, pH 8. An aliquot of 100 ;l (untreated concentration of 1 x 10

    14 Permanox plastic and Sigmacote-treated glass) or 500 ;l (poly-D-lysine-treated Permanox

    15 plastic) was transferred to each slide chamber, with 10 replicates per isolate. After 30 min 16 incubation at ambient temperature, each chamber was washed with 50 mM Tris-HCl, pH 8.0, and 17 numbers of conidia bound to each surface were counted along a diagonal of the chamber. Then a

    nd18 2 rinse was applied, followed by a second set of counts.

    19 To evaluate binding of conidia to charged surfaces, protocols similar to those described for 20 hydrophobic interaction chromatography were followed (5) but resins were used as described in 21 Leland et al. (3). CM-Sephadex C-50 (Pharmacia, Uppsala, Sweden) was equilibrated with 10 22 mM MES, pH 6.0, and DEAE-Sephadex A-50 (Pharmacia) was equilibrated with 10 mM Tris, 23 pH 7.5. Conidia of ARSEF 2575, Ect A-18, KO 8-18, KO 37-2, and KO 43-1 were collected as


    61 /ml. A 1-ml aliquot was loaded onto a 1-ml above, but then diluted in distilled water to 1 x 10

    2 column of each resin, and then rinsed with 5 ml of the appropriate buffer for each column. The

    rinsate containing unbound conidia was collected for counting via hemacytometer. 3

    4 Scanning electron microscopy (SEM). Dried conidia prepared for BAW assays and stored

    5 at -20?C for 2 mo were assayed for viability before SEM analyses. Conidia were exposed in 6 culture dishes for 15 min to a warm vapor of 25% glutaraldehyde solution soaked into Whatman 7 filter paper. After standing for approx. 30 min, samples were lightly touched with a double-sided 8 carbon adhesive tab on an aluminum specimen stub, then sputter-coated with a thin layer of gold. 9 The coated samples were examined with a Quanta 200 FEG scanning electron microscope (FEI 10 Co., Inc., Hillsboro, OR) operated in the high vacuum, secondary electron imaging mode, and 11 several digital images were recorded of each isolate (ARSEF 2575, Ect A-18, KO 8-18, KO 37-2, 12 and KO 43-1) at magnifications ranging from 2,500X to 50,000X, with most images viewed at 13 25,000X in order to resolve nanometer-scale features.



    16 1. Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. 2004. Image processing with ImageJ.

    17 Biophotonics Internat. 11:36-42.

    18 2. Holder, D. J., and N. O. Keyhani. 2005. Adhesion of the entomopathogenic fungus

    19 Beauveria (Cordyceps) bassiana to substrata. Appl. Environ. Microbiol. 71:52605266.

    20 3. Leland, J. E., D. E. Mullins, L. J. Vaughan, and H. L. Warren. 2005. Effects of media

    21 composition on submerged culture spores of the entomopathogenic fungus, Metarhizium

    22 anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying

    23 stability among three spore types. Biocontrol Sci. Technol. 15:379-392.


1 4. Qazi, S. S., and G. G. Khachatourians. 2007. Hydrated conidia of Metarhizium

    2 anisopliae release a family of metalloproteases. J. Invert. Pathol. 95:48-59.

    3 5. Singh, T., R. Saikia, T. Jana, and D. K. Arora. 2004. Hydrophobicity and surface 4 electrostatic charge of conidia of the mycoparasitic Trichoderma species. Mycol. Prog. 5 228. 3:219

    6 6. Wang, C., and R. J. St. Leger. 2005. Developmental and transcriptional responses to

    7 host and nonhost cuticles by the specific locust pathogen Metarhizium anisopliae var.

    8 acridum. Eukaryot. Cell 4:937-947.




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