No 5 filter paper

Anthocyanin concentration was determined by the pH differential method [56 (link)]. Approximately 2.5 g of unpolished rice was extracted for anthocyanin with 24 mL of 70% acidified methanol pH 1.0 and shaking for 60 min, then filtered with Whatman No. 5 filter paper and adjusted to a total volume of 25 mL with 70% acidified methanol pH 1.0 Each 2 mL of extracted solution was prepared with KCl 0.024 M pH 1.0 and CH₃COONa 0.400 M pH 4.5. The absorbance was measured by spectrophotometer (Biochrom, Libra S22, Cambridge, England) at 520 and 700 nm wavelength.

Each fungal strain was inoculated into 200 mL potato dextrose broth (PDB) medium at 200 rpm on a rotary shaker at 28°C for 7–10 days. The fungal broth was filtered using Whatman No. 5 filter paper, and then the protein in filtrate was removed by the sevage method (Kusaikin et al., 2010 (link)). The mycelia was dried and then extracted twice with 95% ethyl alcohol (1:2/V:V) by circumfluence extraction (Cui et al., 2015 (link)). The bacteria was inoculated in beef extract-peptone medium at 200 rpm on a rotary shaker at 37°C for 48–72 h. All the extracting solution was concentrated in a rotary evaporator at 60°C and further was removed by evaporation under 40°C.

Dissected HP (150 g) from Bruckless mussels was freeze-dried to yield 40 g of dry tissue, which was extracted with EtOH (3 × 100 mL) in a Waring blender. The extract was filtered (Whatman no. 5 filter paper) and evaporated under vacuum at 35 °C. The residue was partitioned between EtOAc (150 mL) and 1.0 M NaCl (100 mL). The fractions from each step of the procedure were analyzed by LC-MS to verify the presence of AZAs and AZA-esters. The EtOAc fraction was evaporated under vacuum and the residue partitioned between hexane (50 mL) and 90% MeOH (50 mL). The methanolic fraction was evaporated under vacuum. The residue was applied to an open silica gel column (10 cm × 5.0 cm i.d.) packed with 10–40 μm silica gel (Millipore-Sigma) and sequentially eluted with hexane–EtOAc (9:1), EtOAc–MeOH (7:3 and 1:1), and MeOH (250 mL each). The 7:3 and 1:1 EtOAc–MeOH fractions were evaporated under vacuum separately, and dissolved in MeOH (10 mL) for analysis.

After harvesting at full maturity, the plant samples were dried, crushed, and sieved with a 2 mm sieve to collect fine powder to determine plant nutrient content. Plant macronutrients such as N, P & K were determined following the standard protocol of wet digestion. In brief, plant samples were digested in a 2:1 solution of sulfuric acid (H₂SO₄) and hydrogen peroxide (H₂O₂). Different apparatus were used to determine these macronutrients according to previously standard procedures. For example, the Kjeldahl apparatus was used to determine plant nitrogen (N) content [37 ], a colorimeter was used to test phosphorus (P) in plants [38 (link)], and a flame photometer was used for potassium (K) content in plant samples (Hitachi Z-2000, Tokyo). The dry ashing method was used to evaluate plant micronutrients (e.g., Ca, Zn, Fe, Mn, and Mg). This process involved the ashing of plant materials for six hours at 550 °C, adding 5 mL of hydrochloric acid, and then gradually topping with distilled water to make a 25 mL solution. Plastic vials were used to retain the slurry after it had been filtered through the Whatman No. 5 filter paper. The standard curve of the atomic absorption spectrophotometer was used to determine the micronutrient content values (Hitachi Z-2000, Tokyo).

Diplodia corticola strains were isolated from holm and cork oak trees exhibiting dieback symptoms and cankers in Algeria. The strains were identified using multi-gene phylogenetic analysis, based on DNA sequence data of the internal transcribed spacer region (ITS) and translation elongation factor (tef1-α) [15 (link)]. Pure cultures of hyphal tipped isolates were maintained in the culture collection of the Mendeleum-Institute of Genetics and Plant Breeding, Faculty of Horticulture, Mendel University in Brno, Lednice, Czech Republic. Stock cultures of selected strains were maintained on potato dextrose agar (Oxoid, Ottawa, ON, Canada) and mycelial plugs were used to inoculate 250 mL of Czapek Dox broth (Oxoid), added with 2% yeast extract in 500 mL Erlenmeyer flasks. The cultures were incubated in the stationary phase in the dark at 25 °C. After 30 days, the liquid phase was separated by using filtration on Whatman No. 5 filter paper, and the culture filtrates and respective mycelia were stored at −20 °C. The experiments were carried out in triplicate.