The isolates were grown in unfiltered Oatmeal Agar (OA) (60 g of oatmeal flakes, 12.5 g of agar, 1 L water) for twenty-one days and microscopical analysis was performed directly or using the slide culture technique, with the slides being stained with lactophenol cotton blue (Sigma-Aldrich, St. Louis, USA). Microscopical observations were conducted with a light microscope and photographed (Leica DM 4000B + Leica DFC 490 digital camera (Leica, Wetzlar, Germany)). A holotype and ex-type living cultures were deposited in Micoteca da Universidade do Minho (MUM), Braga, Portugal.
Lactophenol cotton blue
Manufactured by Merck Group
Sourced in United States, Germany
Lactophenol cotton blue is a staining solution used in microbiology and mycology to visualize and identify fungal structures. It contains lactophenol, a preservative, and cotton blue, a dye that stains fungal cell walls. The solution is used to prepare wet mounts for microscopic examination of fungal samples.
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Lab products found in correlation
Zoe fluorescent cell imager, by Bio-Rad (2 mentions)
Optiphot 2 microscope, by Nikon (2 mentions)
Dfc490, by Leica (1 mentions)
Dm4000b, by Leica (1 mentions)
Nile red, by Merck Group (1 mentions)
Jem 1400 series 120 kv transmission electron microscope, by JEOL (1 mentions)
Potato dextrose agar (pda), by Merck Group (1 mentions)
Ef s 10 18 mmf 4.5 5 6 is stm, by Canon (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Mm 4 64, by Enzo Life Sciences (1 mentions)
Ds fi2 camera, by Nikon (1 mentions)
Dc300f, by Leica (1 mentions)
Dmrb e light microscope, by Leica (1 mentions)
Tryphan blue, by Merck Group (1 mentions)
Ds ri1, by Nikon (1 mentions)
Eclipse e200, by Nikon (1 mentions)
12 protocols using lactophenol cotton blue
1
Oatmeal Agar Fungal Cultivation and Microscopy
2
Evaluating Fungal Virulence and Appressoria
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To evaluate the virulence, mycelia‐colonized plugs (0.5 mm diameter) from wild type, ∆SsFoxE3, SsFoxE3‐C, ∆SsAtg8, and SsAtg8‐C were harvested from colony margins of 48 h‐old PDA cultures and then inoculated on tomato, pepper, and soybean leaves. The diameter of the lesions were measured after 48 h and each experiment was repeated three times. Compound appressoria of strains were observed on hydrophobic (glass slide) and onion surfaces, stained with lactophenol cotton blue (Sigma). The formation of compound appressoria was observed at 6 and 12 hpi under a microscope. The experiments were conducted three times.
3
Microscopic Examination of Fungal Cells
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Germination of conidia was examined with a Nikon Optiphot 2 Microscope (Tokyo, Japan). Lipid bodies within fungal cells were stained with 2.5 μg/mL Nile red (Sigma-Aldrich) solution for 20 min at room temperature and examined by a ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, USA) microscope equipped with the 530-nm excitation and 635-nm emission wavelength filters. Transmission electron microscopy (TEM) was performed using a JEOL JEM-1400 series 120 kV Transmission Electron Microscope (Jeol, Tokyo, Japan). Samples were treated with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and 1% osmium tetraoxide, and immersed in LR White Resin after being dehydrated with an ethanol series. Samples were cut to thin slices, stained with uranyl acetate and lead citrate, and examined. Photographs were taken with an Gatan Orius SC 1000B bottom mounted CCD-camera (Gatan Inc., Pleasanton, CA, USA). For penetration studies, leaf samples inoculated with fungal strains were sectioned to thin slices by hand using a sharp razor blade, stained with lactophenol cotton blue (Sigma-Aldrich), and examined by light microscopy.
4
Fungal Spore Micromorphology Analysis
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Spores were harvested when 5-day-old fungal colonies were drenched in sterile water and dislodged from the PDA (Potato dextrose agar, Sigma-Aldrich) medium using a sterile spatula. The mycelial suspension was then strained through cheesecloth to obtain a spore suspension. The micromorphological characteristics, such as the presence or absence of septation, spores and conidiophores, were visualised using a modified version of the slide culture technique by Johnson (1946) [35 (link)]. A sterile microscope slide was placed in a petri dish containing moist filter paper. Thereafter, a 1 cm2 section of PDA was transferred to the microscope slide, and 10 µL of a spore suspension was added to the centre of the PDA square. A cover slip was placed on top of the PDA, and the petri dish was sealed with parafilm. The plates were incubated on a benchtop at room temperature for 2 weeks until mycelial growth was visible. Following the incubation, the coverslip was removed and placed on a clean microscope slide containing lactophenol blue staining solution (Lactophenol cotton blue, Sigma-Aldrich) and viewed under the microscope (Zeiss PrimoStar, 40×) (Carl Zeiss (Pty) Ltd., Cape Town, South Africa). All images were captured using a Canon 80D digital camera (lens: Canon EF-S 10–18 mmf/4.5–5.6 IS STM).
5
Microscopic Characterization of Fungal Organelles
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Leaves were inoculated, hyphae and conidia were stained with lactophenol cotton blue (Sigma‐Aldrich), and samples were examined with an Optiphot 2 microscope (Nikon). Conidia were photographed, and sizes were measured using ImageJ. TEM was carried out as previously described (Wu et al., 2020 ) to examine autophagy and organelles in hyphae and conidia. Hyphae were stained with 1 μg/ml MM 4‐64 (Enzo) to track endocytosis (Fischer‐Parton et al., 2000 (link)) or with 100 μM MDC (Sigma‐Aldrich) to detect autophagic vacuoles such as autophagosomes, amphisomes, and autolysosomes (Biederbick et al., 1995 (link)). Hyphae were stained for 30 min and observed by a ZOE fluorescent cell imager (Bio‐Rad) microscope with excitation at 588 nm and emission at 734 nm for MM 4‐64 and with excitation at 335 nm and emission at 518 nm for MDC. Red fluorescence from mCherry was visualized with excitation at 543 nm and emission at 560–615 nm. Hyphae were examined after staining with 40 μM DCFHDA (Sigma‐Aldrich) using excitation at 504 nm and emission at 529 nm. GFP fluorescence was visualized with excitation at 450–490 nm and emission at 505–550 nm. Vacuoles were stained with CMAC (final concentration, 100 μM; Thermo Fisher Scientific) and visualized with excitation at 353 nm and emission at 466 nm.
6
Compound Appressoria and Penetration Efficiency Analysis
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Compound appressoria of WT, ΔSsStuA and ΔSsStuA-C were observed on glass slides by inoculating agar disks (d = 0.7 cm) and cultivated in a humidifier box at 25 °C incubator. After 48 h incubation, taken pictures and calculated the area of compound appressoria using Image J version 1.50i [29 (link)]. The morphology of compound appressoria of WT, ΔSsStuA and ΔSsStuA-C strains on glass slides was observed by microscopy. Onion epidermis was used to observe the penetration efficiency difference between the compound appressoria of wild-type, ΔSsStuA and ΔSsStuA-C strains. The onion epidermis was inoculated with agar disks (d = 0.5 cm) of strains and cultivated in a humidifier box, after 12 h and 24 h incubation, the invasion hypha was stained with lactophenol cotton blue (Sigma, St. Louis, MA, USA) for 2 min, then rinsed with distilled water and observed by DIC microscopy. The morphology, number, and weight of sclerotia were photographed and calculated after 2 weeks incubation.
7
Fungal Spore Attachment and Larval Tissue Analysis
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Dead larvae treated with fungal spores were stained with lactophenol cotton blue (Sigma-Aldrich, Density: 1.16 g/mL at 20 °C) on grease free slides to detect the fungal spore attachment and the presence of germinating hyphae. Fungal ME treated dead larvae were stained with alizarin (0.02%) (Sigma-Aldrich, 97%), to stain the alimentary system of the dead anopheline larvae for the detection of the effect of fungal ME on the larval internal tissue system; cover slips were added to the grease free slides over the sample and gently pressed flat with fingertip and the slides were observed under a compound microscope (Olympus CX31)67 at different magnifications (4x, 10x and 40x).
8
Macromorphological and Microscopic Characterization of Fusarium Isolates
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Macromorphological examinations were conducted on isolates grown on potato dextrose agar (PDA; Merck, Kenilworth, NJ, USA) plates incubated at 28 °C for fourteen days. The diameter and the colour of the colony surface and substrate mycelia (reverse side/bottom of agar plate) were recorded. For microscopic examination, each isolate was sub-cultured on approximately 1 cm2 blocks of agar containing a piece (0.5 cm2) of sterile carnation leaf agar (CLA). The agar blocks were placed on a sterile microscope slide, covered with a sterile cover slip, and incubated inside a petri dish for 12 h periods of light (day) and 12 h periods in the dark (night) by using a 103 V fluorescent light bulb (TL-D Standard Colours; Philips & Co., Eindhoven, The Netherlands) as a light source at room temperature (28 °C) for four to seven days until sufficient hyphal growth was observed on the cover slip. For microscopic observation, a drop of lactophenol cotton blue (Sigma-Aldrich, St. Louis, MO, USA) was applied to the fungal mycelia on the cover slip and the cover slip was placed face-down onto a microscope slide. The length and width of 30 randomly selected macroconidia were measured to determine the mean length and width for each isolate. Descriptions of the morphological characteristics were adopted from the Fusarium Laboratory Manual [57 ].
9
Microscopic Analysis of Fungal Structures
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Fully expanded 21-day-old leaflets from ILWL180, and 14-day-old leaflets from ILL7537 and ILL6002 were detached separately for each isolate and time point. Immediately after excision, for each time point, one leaflet from each of four replicates of each host/isolate interaction was sterilized, inoculated and incubated as described by Sambasivam et al. (2016) . After 6, 12, 20 or 30 hpi leaflets were fixed and cleared to remove chlorophyll by immersing them in ethanol:glacial acetic acid (1:2 v/v) for 36 h with at least one change of clearing solution at 24 h (Sambasivam et al., 2016) . Cleared leaflets were stained with lactophenol cotton blue (Sigma Aldrich) for 5 min and visualized for fungal structures using a BHC light microscope (Olympus). Images were captured using a digital sight DS-Fi2 camera (Nikon).
10
Visualizing Fungal Structures in Leaf Tissue
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Fungal structures were visualised by staining the cleared leaf tissue with lacto-phenol cotton blue (Sigma Aldrich) for 5 min or tryphan blue (0.05%; Sigma Aldrich) in a phosphate buffer for 10 min. The stained leaf tissue was washed in 50% ethanol or distilled water for 2 to 3 times in order to remove the excess dye.
The leaf tissues were then mounted in water on a glass microscope slide and covered with a cover slip. The samples were examined with a Leica DMRBE light microscope fitted with a Leica DC300F digital camera.
Images were captured using Leica IM50 v.4 software.
The leaf tissues were then mounted in water on a glass microscope slide and covered with a cover slip. The samples were examined with a Leica DMRBE light microscope fitted with a Leica DC300F digital camera.
Images were captured using Leica IM50 v.4 software.
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