This dataset contains measurements of fungal growth, lipid composition, and spore germination under different fatty acid treatments in the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Fungal cultures were established under asymbiotic conditions using a modified synthetic medium supplemented with defined fatty acid combinations, including myristate alone or in combination with palmitate and/or palmitoleate. Cultures were incubated for four months, followed by optional storage at 4 °C for one month. For each treatment, fungal biomass (dry weight), spore number, and spore diameter were quantified. Lipid composition was determined for triacylglycerol and phospholipid fractions using gas chromatography–mass spectrometry (GC–MS), including relative abundances of individual fatty acids and degrees of saturation. Spore germination rates were assessed before and after low-temperature storage.

These data can be used to investigate relationships between exogenous fatty acid supply, lipid composition, and fungal performance, and may support further analyses of lipid metabolism and optimization of asymbiotic culture systems for AM fungi.

In vitro asymbiotic culture and low temperature strage.

Sterile spores of Rhizophagus irregularis DAOM197198 were purchased from Premier Tech and used as a seed fungus. Detached hyphae in the spore suspension were removed by density-gradient centrifugation using gastrografin (Sugiura, 2020). Five mL of 0.5% gellan gum (FUJIFILM Wako Chemicals) containing 3 mM magnesium sulfate was poured into a 60 mmφ Petri dish and allowed to solidify at room temperature. A cork borer was used to make a hole 8 mm in diameter and 2 mm deep in the center of the gel, into which a suspension containing approximately 500 spores was added. After the water in the spore suspension evaporated, a small amount of 0.5% gellan gum containing 3 mM magnesium sulfate was poured in and allowed to solidify at room temperature. Next, the gel was covered with 5 mL of modified SC liquid medium (0.85 g L^-1 yeast nitrogen base [MP Biomedicals], 0.5 g L^-1 synthetic complete [Formedium], 2.5 mM ammonium sulfate, 0.5 mM glycerol, 2.5 mg L^-1 thiamine HCl, 2.5 mg L^-1 nicotinic acid, 2.5 mg L^-1 pyridoxal phosphate) (Sugiura, 2020) with one of the following supplements: (1) 0.5 mM potassium myristate, (2) 0.5 mM potassium myristate and 0.5 mM potassium palmitate, (3) 0.5 mM potassium myristate and 0.5 mM palmitoleic acid neutralized with 200 mM potassium hydroxide, and (4) 0.5 mM potassium myristate, 0.5 mM potassium palmitate, and 0.5 mM neutralized palmitoleic acid. Cultures were grown in a CO₂ incubator at 28°C, 2% CO₂, and in the dark. After 4 months of incubation, the whole petri dish was transferred to a refrigerator and stored at 4°C for 1 month.

Measurement of fungal biomass and spore production.

Fungal biomass was measured according to Sugiura and co-workers (Sugiura, 2020) with some modifications. After discarding liquid medium from Petri dishes, the gel was melted by incubating in citrate buffer (8.3 mM trisodium citrate, 1.7 mM citric acid, and 1% Triton X-100; pH 6.0) at room temperature with rotation. The melted gel was removed using a pipet. The gel removal process was repeated several times. The tangled fungal materials were recovered using forceps and transferred to an antistatic 1.5-mL tube. The sample was centrifuged at 3,500 rpm for 10 min at room temperature using a swing rotor. After the removal of the supernatant, the fungal pellet was washed with 1 mL of pure water by centrifuging at 3,500 rpm for 10 min several times. The pellet was dried at 70°C for 48 h. Total fungal dry weight was measured with a micro analytical balance (BM-252, A&D). The number of seed fungus spores was counted in advance under a dissecting microscope. The standardized growth increment of R. irregularis was calculated as follows:

Standardized growth increment (μg) = DW_t / N_p - DW_p

where DW_t is the total fungal dry weight, N_p is the number of seed fungus spores, and DW_p is the dry weight of a seed fungus spore.

              Digital images of spores were captured using a stereomicroscope equipped with AxioCam (Carl Zeiss). Spore number and diameter were measured using AxioVision (Carl Zeiss).

Germination assay.

Next-generation spores were cut out one by one under a stereomicroscope using a scalpel and tweezers. The spores were placed on 0.3% gellan gum gel and germinated in a CO₂ incubator at 28°C, 2% CO₂, and in the dark. Four weeks later, spores were observed under a stereomicroscope and the percentage of spores that showed germ tube development was determined as the germination rate.

Lipid analysis.

After 4 months of asymbiotic culture of R. irregularis, gels including spores of seed fungus were removed. The remaining gels were recovered and melted with citrate buffer as described above. Fungal materials were freeze-dried. Lipids of the fungal materials were extracted by incubating in 500 μL chloroform:methanol (2:1) containing 0.001% butylated hydroxytoluene overnight followed by sonication for 30 min twice. After filtration using a  0.45 μm PTFE syringe filter, the organic solvent was evaporated under N₂ flow. The lipid was dissolved in 600 μL chloroform. The Strata silica column (1 mL bed volume, Phenomenex) was equilibrated with 7 mL chloroform. A lipid solution was applied to the silica column. To extract non-polar lipid fraction, 2 mL chloroform was added to the column. After adding 2 mL of 2-propanol:acetone (1:1) to the same column, the phospholipid fraction was collected by adding 2 mL methanol. The organic solvent in non-polar lipid fraction was evaporated under N₂ flow, Triacylglycerol (TAG) fraction was recovered from non-polar lipid fraction by thin-layer chromatography with hexane:diethylether:acetate (80:30:1). TAG was extracted in 500 uL chloroform by sonicating for 30 min, and then filtrated using a PTFE syringe filter. The organic solvent was evaporated under N₂ streaming. Fatty acid methyl esters were prepared by incubating the purified TAG in 1,800 μL hexane and 180 μL 1 M methanolic KOH as described previously (Ichihara, 1996). The phospholipid fraction was evaporated under N₂ streaming and incubated in 400 μL hexane and 40 μL 1 M methanolic KOH. An aliquot of the upper hexane layer was applied to GC-MS. The GC-MS data were recorded with a GCMS-QP2010 Plus (Shimadzu) and an SH-Rxi-5ms column (30 m×0.25 mm, 0.25 μm film, Shimadzu). Specific conditions were used for chromatographic separation: injection 1 μL (splitless, 60 s valve time), injector temperature 200°C, carrier gas He (at 35 cm s^-1), transfer line temperature 300°C, ion source temperature 230°C, electron energy 70 eV. The temperature of the column oven was programmed in three steps: 60°C for 2 min, followed by an increase to 160°C at 25°C min^-1, and then an increase to 280°C at 4°C min^-1. The identification and quantification of fatty acid methyl esters were performed in scan mode. The composition of fatty acids was determined by peak areas.