AMF inocula [Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schubler] consisting of spores, soil, hyphae and infected clove (Trifolium repens L.) root fragment from a stock culture of F. mosseae.The inoculated dosage was 10 g of inocula per pot containing approximately 720 spores calculated by microscopy before experiment. There was 2100 infective propagules/g in the inoculum as determined by MPN assay . Seeds were surface sterilized by immersion in 70% ethanol for 5 min, rinsed four times with distilled water, and placed on wet filter paper in Petri dishes at 28 ℃ for germination. After 3 days, the germinate seeds were transplanted into 13 cm × 13 cm plastic pots containing 0.88 kg organized soil substrate (organic manure, soil and decomposed straw = 1:2:1). Half of the pots (AM plants) were inoculated with 10 g of F. mosseae per pot. Non-AM plants received the same weight of autoclaved inocula. The inocula were placed adjacent to each seeding root. The organic substrate was collected from the greenhouse of Institute of Vegetables and Flowers, CAAS and sterilized for 4 h at 160 ℃ , with the chemical properties as follows: pH 7.26, 11.1% organic matter, 150 mg/kg available phosphorus, 451 mg/kg available nitrogen and 518 mg/kg available potassium. The experimental pots were placed in solar greenhouse at an average temperature of 28 ℃ /20 ℃ (day/night) with photon flux density of 600 µmol m^-2 s^-1 and 85% relative humidity.The seedlings uniformed in size were transferred to a growth chamber subjected to different temperature conditions at 20 days after inoculation. The experimental design consisted of four treatments crossing two mycorrhizal inoculations levels (non-AMF and F. mosseae) with two temperature levels with photon flux density of 100 µmol m^-2 s^-1 (25 ℃ /15 ℃ , 15 ℃ /10 ℃ , day/night). (1) Normal temperature (NT): 10 g of sterilized inoculua, 25 ℃ /15 ℃ (day/night); (2) AMF-inoculation (AMF): 10 g of inocula, 25 ℃ /15 ℃ (day/night); (3) low temperature (LT): 10 g of sterilized inocula, 15 ℃ /10 ℃ (day/night); (4) AMF-inoculation under low temperature (AMF + LT): 10 g of inocula, 15 ℃ /10 ℃ (day/night).The experimental design was a completely randomized block design and thirty plants were arranged in each replication. On 45 days after inoculation, phenolic compounds contents, enzymes and gene transcription were determined.

AMF-inoculated cucumber seedlings had significant higher fresh weight and dry weight than non-AMF inoculated control plants under both normal (25/15 ℃ ) and low temperature (15/10 ℃ ) treatment. Under chilling stress, AMF inoculation significantly improved the content of related secondary metabolites including phenols, flavonoids, lignin, DPPH activity and phenolic compounds compared with the non-AMF control. Furthermore, large increments were observed in a number of enzymatic activities related to secondary metabolism and antioxidant system in AMF-inoculated seedlings under low temperature, such as glucose-6-phosphate dehydrogenase (G6PDH), shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO), guaiacol peroxidase (G-POD), caffeic acid peroxidase (CA-POD) and chlorogenic acid peroxidase (CGA-POD). As well, the expression of stress-related marker genes was enhanced in AMF-inoculated seedlings in comparison with the non-AMF control. Furthermore, AMF symbiosis decreased hydrogen peroxide (H₂O₂) content under low temperature.

Consistent with the changes of enzyme activities, the relative transcriptional level of genes related to secondary metabolism increased significantly in response to AM fungi inoculation in cucumber roots . Moreover, under chilling stress, the expression levels of WRKY30, PR-1, C4H, CCOMT, CAD, G6PDH, PAL, LPO, and POD genes in the AMF-inoculated seedling were 2.46, 2.90, 1.84, 2.47, 1.96, 2.52, 1.89 and 1.93 folds respectively compared with those under low temperature alone.