Evaluating Arbuscular Mycorrhizal Fungi Colonization and Gene Expression in Walnut

After three months of treatments, the plants were harvested. Plant height, stem diameter, and total biomass were measured directly before harvesting. At the same time, a portable Li-6400 photosynthetic apparatus (Li-Cor Inc., Lincoln, USA) was used to determine leaf gas exchange starting at 9:00 am on a sunny day before harvest. The soil attached to the roots was gently shaken off for hyphal length analysis, based on the method outlined by Bethlenfalvay and Ames (1987) (link). A portion of root segments were cut, and root mycorrhizal staining was performed using trypan blue method described by Phillips and Hayman (1970) (link). After microscopic observation, the root mycorrhizal colonization rate (%) was estimated as the percentage of the length of root segments colonized by AMF to the total length of root segments examined.
Eight plants from each treatment were divided equally into two parts, one of which was immediately frozen in liquid nitrogen and then stored at -80 °C for analysis of gene expressions. The other part was killed at 105 °C for 3 min after chlorophyll determination, then dried at 75 °C to constant weight, ground to powder, and passed through a 2 mm sieve for P concentration determination. The ICP Spectrometer (IRIS Advantage, Thermo, Waltham, USA) was used to analyze leaf P concentration. The concentration of glucose, fructose and sucrose in leaves was determined according to the colorimetric method described in detail by Wu et al. (2015b) (link). The concentration of chlorophyll components was extracted with 80% acetone and determined using the method described by He et al. (2022) (link).
The sequences of PAP genes (PAP10 and PAP12) and PT genes (PT3;1 and PT3;3) in Arabidopsis were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov) and then compared with genome-wide of walnut (http://aegilops.wheat.ucdavis.edu/Walnut/data.php). The primer sequences (Supplementary Table S1) of JrPAP10, JrPAP12, JrPT3;1, and JrPT3;3 genes were designed using Primer5 premier 5.0 software and synthesized by Shanghai Bioengineering Co., Ltd. (Shanghai, China). Total RNA of leaf samples was extracted using an EASY spin Plus plant RNA kit (Aidlab). The reverse transcription of RNA was performed using the PrimeScript™ RT reagent kit with gDNA eraser kit (Takara). The 18S rRNA of walnut was used as the reference gene for qRT-PCR amplification. qRT-PCR was performed using the fluorescent dye method (2×AceQ^® qPCR SYBR^® Green Master Mix) with three biological replicates per treatment. The 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative gene expression, in which the relative expression was normalized by the treatment with non-inoculation of D. spurca at P₁₀₀ levels.