Trypan blue staining was performed as previously described (Bowling et al., 1997 (link)). Leaves were immersed in lactophenol blue solution (Sigma-Aldrich, St. Louis, United States) and reacted for overnight. Stained leaves were placed in a 60% chloral hydrate solution and finally equilibrated with 50% glycerol.
Lactophenol blue solution
Manufactured by Merck Group
Sourced in United States, Germany
Lactophenol blue solution is a laboratory reagent used as a staining agent. It is primarily used for the microscopic examination and identification of fungi, such as yeasts and molds. The solution contains lactophenol, a mixture of phenol, lactic acid, and glycerol, which acts as a preservative and clearing agent, allowing for the visualization of fungal structures.
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8 protocols using lactophenol blue solution
1
Trypan Blue Leaf Staining Protocol
2
Fungal Conidial Germination Assay
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Germination was assessed from each culture medium, using the recovered dry conidia (conidial powder). The microbiological parameter percentage of germination at 36 and 48 h was assessed. For each treatment, 1 g samples picked up aseptically were diluted in 0.1% v/v Tween® 80 solution, and decimal dilutions were done until 10–2. An aliquot of 100 µL of 10–2 dilution was plated on water agar plus 2% w/v yeast extract (Difco®, Thermo Fisher Scientific, U.S.A.), 2% v/v V8 juice (V8® Vegetable Juice, Campell’s, U.S.A.), and 0.00008% w/v benomyl [Benlate 50% w/w (WP), DuPont, Spain]. The Petri plates were incubated at 25 ± 2 °C for 36 and 48 h, and the germ tube growth was stopped with a lactophenol blue solution at each time (Sigma-Aldrich®, Merck KgaA, Germany). Germinated and non-germinated conidia were read in an optical microscope (400X magnification; CH30, Olympus®, Japan), and at least 100 conidia were scored per replicate. Conidia was considered germinated when its germ tube was at least twice its diameter (Ekesi et al. 1999 (link); Milner et al. 1991 (link)). The results were reported as a percentage of germination at 36 and 48 h.
3
Fungal Microscopy Protocol for Identification
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The description of the macroscopic characters and the maintenance of the monosporic cultures was carried out in PDA medium. The description of the microscopic structures was made from monosporic cultures incubated at 25 °C with a photoperiod of 12 h for 14 days in Spezieller Nahrstoffarmer agar (SNA)47 and carnation agar medium48 (link). Each isolate was mounted in a lacto-phenol blue solution (Sigma-Aldrich, Munich, Germany), and examined under a light microscope (Olympus Cover 015, Life Science Solutions). The description of the colonies was made taking into account colony color in the front and back direction, descriptions of aerial mycelium, conidial size, shape, and conidia color of each species. Morphological characteristics of the isolate were then compared with identification guides such as Introduction to food and airborne fungi, The Fusarium laboratory manual and Fungi and food spoilage19 ,20 ,49 .
4
Characterization of Fungal Vegetative Growth
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For comparing vegetative growth, mycelial colony diameter was measured after growing the wild type and transformants on V8, oatmeal, and CM agar medium at 25°C without light for 6 days. The hydrophobicity of vegetative hyphae was tested by dropping sodium dodecyl sulfate (SDS) and water on 7-day-old mycelia. Conidiation was evaluated by counting conidia harvested in 5 ml distilled water from 7-day-old OMA medium. Lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) was used to distinguish conidiophores from mycelia (Kim et al., 2009 (link)). Conidial germination was performed by placing the conidial suspension (5 × 104 /ml) on hydrophobic coverslips and counting germinated conidia 2 h later. Appressorium formation was conducted by incubating a conidial suspension on hydrophobic coverslips and hydrophilic slide glasses with or without the addition of 5 mM exogenous cAMP (Fu et al., 2018 (link)). Surface hydrophobicity was tested by dropping water or SDS solution onto 14-day-old mycelia bearing conidia and evaluated by comparing mycelial wettability. Experiments were repeated three times with three replicates for each repeat. All data were analyzed according to Duncan’s test at P < 0.05.
5
Broiler Litter Temperature, Moisture, and Fungal Contamination
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Litter temperature (°C) was measured with a Testo 925 thermocouple thermometer (Testo SE & Co. KGaA, Lenzkirch, Germany) at 3 cm below litter surface at five points of the broiler house (four corners and one in the centre). At these points we also took one litter sample per point into sterile bags (Aptaca Spa, Canelli, Italy) and transported them to the laboratory for analysis on the same day. Litter moisture (%) was determined using the gravimetric method by calculating weight loss after drying, whereas pH was determined electrometrically using a WTW inoLab pH 720 pH meter (Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany). More detailed descriptions are available elsewhere (8 (link), 34 (link)).
Litter fungal contamination was determined as follows: 10 g of each sample was first diluted 10-fold with sterile distilled water and then 100 μL of the dilution plated on Sabouraud dextrose agar (Biolife, Milan, Italy) and incubated at 25 °C for 5–7 days. The fungi were identified based on macroscopic appearance of grown colonies and on microscopic examination of spores stained with lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) (35 , 36 ). The concentration was expressed as colony forming units per gram of litter (CFU/g). Analytical procedures were performed in triplicate.
Litter fungal contamination was determined as follows: 10 g of each sample was first diluted 10-fold with sterile distilled water and then 100 μL of the dilution plated on Sabouraud dextrose agar (Biolife, Milan, Italy) and incubated at 25 °C for 5–7 days. The fungi were identified based on macroscopic appearance of grown colonies and on microscopic examination of spores stained with lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) (35 , 36 ). The concentration was expressed as colony forming units per gram of litter (CFU/g). Analytical procedures were performed in triplicate.
6
Microscopic Fungal Examination Protocol
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According to the manufacturer's instructions, the ECLIPSE Ni-E light microscope augmented with an F-mount camera with a digital sight of 50M (NIKON Corp., Tokyo, Japan) was employed here. A sterile spatula was used to take a disc of 6mm diameter from each mycelial growth and insert it in the center of a sterile slide. Lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the slides, which were then covered with sterile coverslips and inspected at 40 × power51 (link).
7
Fungal Isolate Phenotypic Characterization
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The phenotypic variant of the fungal isolate was determined by visual inspection. The fungus was grown in PDA broth at 28 °C for 7 days, then the Petri plate was photographed. The hyphae and conidia from the fungal cultures were stained with lactophenol blue solution (LPCB), Sigma-Aldrich, Saint Louis, MO, USA) and observed under a phase-contrast light microscope using an Olympus BX51 with a 60× objective UPlanF1.
8
Fungal Isolate Characterization Protocol
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Fungal isolates were grown on potato dextrose agar (PDA) and malt extract agar (MEA) at 25 °C in the dark to facilitate the colony morphology, texture, color, and conidiation for microscopic examination. Conidiation was induced by culturing the colony (after scratched-off mycelium) at 12h–12h light–dark condition. Temporary slides were prepared for each isolate, mounted in lactophenol blue solution (Sigma-Aldrich, Munich, Germany), and examined under a light microscope (Zeiss, Oberkochen, Germany). In order to describe colony color, descriptions of aerial mycelium, conidial size, shape, and conidia color of the strains were observed using a BX50 microscope (Olympus, Tokyo, Japan) with Artcam 300 MI digital camera (Artray, Tokyo, Japan). Colors were named using “A Mycological Colour Chart” [42 ]. Morphological characteristics of the isolate were then compared with previous descriptions.
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