Arbuscular Mycorrhizal Fungus Alters Alfalfa (Medicago sativa) Defense Enzyme Activities and Volatile Organic Compound Contents in Response to Pea Aphid (Acyrthosiphon pisum) Infestation

Pea aphid (Acyrthosiphon pisum) infestation leads to withering, reduced yield, and lower quality of the host plant. Arbuscular mycorrhizal (AM) fungi have been found to enhance their host plants’ nutrient uptake, growth, and resistance to biotic stresses, including pathogen infection and insect pest infestation. Therefore, we evaluated the effects of AM fungus Rhizophagus intraradices on alfalfa defense responses to pea aphid infestation. Aphid infestation did not affect the colonization of AM fungus. The inoculation of AM fungus, on average, enhanced alfalfa catalase and the contents of salicylic acid and trypsin inhibitor by 101, 9.05, and 7.89% compared with non-mycorrhizal alfalfa, respectively. In addition, polyphenol oxidase activities significantly increased by six-fold after aphid infestation in mycorrhizal alfalfa. Moreover, the fungus significantly (p < 0.05) improved alfalfa shoot N content, net photosynthetic and transpiration rates, and shoot dry weight in aphid infected treatment. The aphid infestation changed the total volatile organic compounds (VOCs) in alfalfa, while AM fungus enhanced the contents of methyl salicylate (MeSA). The co-expression network analysis of differentially expressed genes (DEGs) and differentially expressed VOCs analysis showed that three DEGs, namely MS.gene23894, MS.gene003889, and MS.gene012415, positively correlated with MeSA both in aphid and AM fungus groups. In conclusion, AM fungus increased alfalfa’s growth, defense enzyme activities, hormones, and VOCs content and up-regulated VOC-related genes to enhance the alfalfa’s resistance following aphid infestation.

The infestation of aphids also decreases photosynthesis [7] and photosynthates' transport to the roots [8]. For example, feeding by Spodoptera exigua significantly improved net photosynthetic rate and transpiration rate of mycorrhizal plants of peanut (Arachis hypogaea) and tomato (Lycopersicum esculentum) [7]; bird cherry-oat aphid (Rhopalosiphum padi) Glomeromycota in Beijing, China. The inoculum consisted of dry soil containing AM fungus spores (>100 spores per g), mycelium, and white clover root fragments, which were prepared in pot cultures of AM fungus grown on the white clover (Trifolium repens) in the greenhouse. The alfalfa growth medium was mixed with pre-sieved sand and soil using a 2 mm sieve at a 1:3 weight ratio. Sand and soil were purchased from the flower market in Lanzhou, China. All the growth medium components were sterilized by autoclaving at 121 • C for 1 h twice in three days [26].

AM Fungus Inoculation and Aphid Infestation
A two-stage experiment was established using alfalfa and aphids in a plant incubator (RXM-508C; Ningbo Jiangnan Instrument Factory, Ningbo, China) with 12 fluorescent lights (300 µmol·m −2 ·s −1 ) at 25 • C and 12 h of dark at 25 • C (watered with tap water every three days). The alfalfa seeds were surface-sterilized with 10% hydrogen peroxide (H 2 O 2 ) for 10 min and then rinsed three times with sterile distilled water before germinating on wet filter paper in the dark at 25 • C for 48 h. Before transplantation, 50 g of AM fungus were added to the growth medium at a depth of 2-3 cm in each of eight pots (AM treatment). The control pots contained the same growth medium with a 50 g sterile growth medium each (non-mycorrhizal treatment, NM). Five germinated seeds were then transplanted in a 12 × 10 × 8 cm pot and thinned to four seedlings after one week.
We captured pea aphids from an alfalfa field at the Yuzhong campus of Lanzhou University, China and then selected and reared the single adults on broad bean plants. We then identified the newly emerged aphid offspring morphologically and molecularly, as described previously [28]. At 51 days after planting (DAP), half of the alfalfa plants inoculated with or without AM fungus were infested with five similar size adult pea aphids (A+). The remaining half of the alfalfa plants were not infested with pea aphids (A−).
2.3. AM Colonization, Shoot N, P Uptake, Dry Weight, Defense Enzyme Activity, and Hormone Content At harvest (60 DAP), 0.5 g of fresh shoots from each pot were used to determine the enzyme activities, by measuring polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as described previously [29]. We measured the concentrations of jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) contents; nitric oxide (NO); total phenols; and trypsin inhibitors from 0.4 g of fresh shoots using ELISA kits (Plant JA, Plant SA, Plant ABA, Plant NO, Plant total phenols, and Plant trypsin inhibitor) following the manufacturer's instructions (mlbio; Shanghai Enzyme-linked Biotechnology, Shanghai, China) [26]. About 1.0 g of fresh shoots from each pot was used for the VOCs' content analysis using static headspace SPME-gas chromatography coupled with mass spectrometry (GC-MS) [30,31], while the remaining fresh shoot tissues were used to measure the dry weight. Thus, the total plant shoot dry weight was calculated from the fresh and dry weight ratios. In addition, 0.2 g of plant dry shoots were digested in 5 mL H 2 SO 4 to determine the total N and P by a flow injection analyzer (FIAstar 5000 Analyzer; FOSS, Munkedal, Sweden) with the Kjeldahl method [32].
Approximately 0.15 g of the root tissues from each pot were used for AM colonization tests as previously reported [33]. Roots were cut into 1 cm-long pieces and cleaned in 10% KOH for 40 min at 60 • C water bath. The samples were then treated in 1 M HCl for 30 s, washed three times with distilled water, and stained in trypan blue overnight at room temperature. They were then washed three more times with distilled water and maintained in a 1:1:1 solution of 5% lactic acid: glycerin: water (v/v/v). The root samples were then used to determine the degree of mycorrhizal colonization using a compound microscope (SOFTOP BH200M-R, Ningbo, China) [33,34].

The Transcriptome Analysis for Plant Leaves
At harvest (60 DAP), 0.1 g of fresh shoots from the four seedlings in each pot was randomly collected, flash-frozen immediately in liquid nitrogen, and stored at −80 • C before total RNA extraction. The total RNA was isolated from the leaf tissues of three biological replicates per treatment (NMA−, NMA+, AMA−, AMA+) using TRIzol Reagent following the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). The residual genomic DNA was removed from the RNA samples using DNase I (Takara Bio, Kusatsu-Shiga, Japan). RNA quality and quantity were determined using the 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA) and ND-2000 (NanoDrop Technologies, Wilmington, DE, USA). The high-quality RNA sample (OD 260/280 = 1.8-2.2, OD 260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, >2 µg) was selected to construct the sequencing library [36].
Accordingly, 1 µg of the total RNA was used to construct the RNA-seq transcriptome libraries using the TruSeq TM RNA sample preparation kit following the manufacturer's protocol (Illumina, San Diego, CA, USA). The mRNA was isolated by the polyA selection method using oligo(dT) beads and fragmented using the fragmentation buffer. The doublestranded cDNA was synthesized using random hexamer primers (Illumina, San Diego, CA, USA), following the protocol of the SuperScript double-strand cDNA synthesis kit (Invitrogen, Waltham, MA, USA). The synthesized cDNA was end-repaired and phosphorylated, and the 'A' base was enriched following the Illumina library construction protocol. After cDNA quantification, 200-300 bp target fragments were amplified by PCR using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) for 15 PCR SeqPrep cycles. The libraries were paired-end sequenced on the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length) [37].

Volatile Organic Components Analysis
For each pot, 1 g of fresh shoots was immediately frozen in liquid nitrogen after harvesting and stored at −80 • C. A total of four treatments (NMA−, AMA−, NMA+, AMA+) with four biological replicate samples were separately ground to powder in liquid nitrogen to isolate the volatiles [39]. The powder was transferred immediately to a 20 mL headspace vial containing 2 mL saturated NaCl solution (Agilent, Palo Alto, CA, USA). The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent). At the time of SPME analysis, each vial was placed at 60 • C for 10 min, and then a 65 µm divinylbenzene/carboxen/polydimethylsilioxan fiber (Supelco, Bellefonte, PA, USA) was exposed to the headspace of the sample for 20 min at 60 • C.
The desorption of the VOCs from the fiber coating was carried out in the injection port of the GC apparatus (Model 7890B; Agilent) at 250 • C for 5 min under the splitless mode. The VOCs were identified and quantified using an Agilent Model 7890B GC and a 7000D mass spectrometer (Agilent) equipped with a 30 m × 0.25 mm × 1.0 µm DB-5MS (5% phenyl-polymethylsiloxane) capillary column. Helium was used as the carrier gas at a linear velocity of 1.0 mL/min. The injector temperature was kept at 250 • C and the detector at 280 • C. The oven temperature was programmed from 40 • C (1 min), increasing at 5 • C/min to 280 • C. The mass spectra were recorded in electron impact (EI) ionization mode at 70 eV. The quadrupole mass detector, ion source, and transfer line temperatures were set at 150, 230, and 280 • C, respectively. The mass spectra were scanned in the range of m/z 30-350 amu at 1 s intervals. The volatile compounds were identified by comparing the mass spectra with the data system library (NIST) and linear retention index [40].
Principal component analysis (PCA) was used to differentiate the samples and identify marker metabolites. Afterwards, the variable influence on projection (VIP) was used to illustrate the variables that contributed to the separation. The orthogonal partial least squares discriminant analysis (OPLS-DA) extension was then used to compute the variation of X, which is predictive (correlation between X and Y), and the part of the variation which is orthogonal (unrelated) to Y [41].

Statistical Analysis
Data of AM colonization; fresh weight; dry weight; total N; total P; JA; SA; NO; ABA; phenolic and trypsin inhibitor concentrations; and the activities of CAT, POD, SOD, and PPO are presented as the means ± standard error of the mean (SEM) of four biological replicates. Data of the net photosynthetic and transpiration rates are presented as SEM of 4 biological replicates. The data were subjected to analysis of variance (ANOVA) using the R statistical program (version 4.2.0). Comparisons between the means were determined by Turkey's HSD test (p < 0.05). The percentage of AM colonization and net photosynthetic rate were SQRT transformed while the transpiration rate was log transformed to achieve normality. The RNA-seq analysis was performed using the free online platform of Majorbio Cloud Platform (www.majorbio.com, accessed on 9 October 2022). The VOCs analysis was performed using the free online platform of Metware Cloud (cloud.metware.cn, accessed on 9 October 2022).

AM Fungal Colonization, Plant Growth, Shoot N and P Uptake
The mean percentags of AM fungus colonization were 45% and 43.33% in the mycorrhizainoculated alfalfa without and with pea aphid infestation, respectively. No mycorrhizal structures were observed in the non-fungal-inoculated plant roots. The infestation of aphids did not significantly affect AM colonization ( Figure S1; p > 0.05). AM fungus increased alfalfa shoot fresh weight by 100% (Figure 1a; p = 0.0043) and shoot dry weight by 64.15% (p = 0.0151; Figure 1b). Moreover, the alfalfa shoot total N was increased 40.60% by AM fungus (p = 0.0083), and mycorrhizal alfalfa with aphid infestation had the highest shoot total N ( Figure 1c) and shoot total P (p = 0.0373; Figure 1d). AM fungus significantly increased the net photosynthetic rate of alfalfa infested by aphids (p < 0.0001; Figure 2a), and the transpiration rate of mycorrhizal alfalfa at 45.00% was higher than that of nonmycorrhizal alfalfa at 59 DAP (p = 0.03867; Figure 2b) (Table S1), which is consistent with the higher biomass of mycorrhizal plants.  intraradices (AM) and infested by A. pisum (A+) or without inoculation with R. intraradices (NM) and un-infested by A. pisum (A−) before aphid infesting and after aphid infesting. Boxes show first quartile, median, and third quartile. Whiskers extend to the most extreme points within 1.5 × box lengths, and the points are values that fall outside the whiskers. Different letters above bars or pairs of bars represent significant difference between different treatments before aphid infesting or after aphid infesting in the comparison at p < 0.05, as determined by a Tukey's HSD test.
3.2. The Activities of Plant Defense Enzymes, Jasmonic Acid, SA, Trypsin Inhibitors, Total Phenolics, ABA, and NO Concentration in Alfalfa AM fungus alone did not affect plant defense enzyme activity; however, its interaction with aphid-affected plant CAT and PPO activities indicates that AM fungus plays an important role only when the plant is exposed to aphid stress (Table S1). Aphid infestation significantly increased plant CAT and PPO, especially in mycorrhizal plants. The PPO activities were significantly increased by 184% and 600% after aphid infestation in nonmycorrhizal alfalfa and mycorrhizal alfalfa, respectively (p = 0.0054) (Figure 3a). The CAT activity in mycorrhizal alfalfa was 101% higher than in non-mycorrhizal plants after aphid infestation (p = 0.0304; Figure 3c). However, pea aphid infestation decreased the POD activity (p = 0.0046; Figure 3b) but did not affect the SOD activity in alfalfa plants ( Figure S2) (Table S1). Aphid infestation significantly increased JA concentration by 8.59% (p = 0.0335; Figure 3d), and AM fungus colonization increased SA concentration by 9.05% in alfalfa (p = 0.0337; Figure 3e). There was no interactive effect of aphid infestation and AM colonization on JA and SA. Trypsin inhibitor concentration in mycorrhizal alfalfa was 7.89% higher in non-mycorrhizal plants (p = 0.0174). Furthermore, aphid infestation significantly increased trypsin inhibitor concentration in non-mycorrhizal alfalfa, while AM fungus increased trypsin inhibitors either in aphid-infested or non-infested alfalfa ( Figure 3f; Table S1). This indicates that AM fungus colonization activated the plant's trypsin-inhibitor-related defense process. However, there were no significant differences in the contents of total phenols, ABA, and NO across the treatments ( Figure S3).

Volatile Organic Components in Alfalfa
One hundred fourteen VOCs were identified, including terpenes, alcohol, aromatics, heterocyclic compounds, aldehyde, ketone, alkanes, ester, phenol, ether, and olefin. The differential VOCs were satisfied by a VIP > 1 and a p value < 0.05 in different samples. The colonization of AM fungus increased the total contents of VOCs (p = 0.0296; Figure 4a)   Seven different VOCs, including three esters, two alcohols, one aldehyde, and one alkane, were detected in four groups (NMA− vs. AMA−, NMA− vs. NMA+, AMA− vs. AMA+ and NMA+ vs. AMA+). The amounts of butanedioic acid, methyl ester, and dimethyl ester were higher in mycorrhizal alfalfa than in non-mycorrhizal alfalfa plants. Pea aphid infestation decreased 2-Octen-1-ol, (E)− and MeSA in the non-mycorrhizal alfalfa leaves but increased the concentration of Pentadecanal, (E)-2,6-dimethylocta-3,7diene-2,6-diol; Nonane, 5-(2-methylpropyl)-; and 3-Hexen-1-ol, acetate, (E)− in the plants inoculated with mycorrhiza and infested with pea aphids. The concentrations of 2-Octen-1ol, (E)−; MeSA; (E)-2,6-Dimethylocta-3,7-diene-2,6-diol; and nonane, 5-(2-methylpropyl)were higher in the mycorrhizal alfalfa infested with pea aphids than the non-mycorrhizal alfalfa infested with aphids (Table 1). The PCA showed AM fungus colonization, and pea aphid infestation affected VOC composition and content. The VOCs were highest in the pea aphids fed on the mycorrhizal alfalfa. A part of VOCs was consistent in four different treatments ( Figure S4). The OPLS-DA showed the AMA− and AMA+ treatments were separated from the other treatments, while the NMA-and AMA+ treatments were separated from the NMA+ and AMA− treatments ( Figure S5a). Sixty compounds had a high discriminatory power (i.e., VIP > 1), and all were associated with the four treatments, the top 20 listed by decreasing order of VIP value, with numbers referring to the volatile compounds listed in Table S2: (76), (44)

Differentially Expressed Genes and VOCs Analysis
The co-expression network analysis of differentially expressed genes (DEGs) and differentially expressed VOCs was conducted to examine the relationship between DEGs and differentially expressed VOCs in the aphid-infested alfalfa plants (Pearson correlation coefficient > 0.8 or < −0.8). Sixty-six genes were found to be associated with four metabolites in NMA+ vs. AMA+, of which MS.gene071245 positively regulated (E)-2,6-Dimethylocta-3,7-diene-2,6-diol and nonane, 5-(2-methylpropyl)-in NMA+ vs. AMA+. Similarly, three DEGs were found to be associated with butanedioic acid, methyl ester, and dimethyl ester in NMA− vs. AMA−. Moreover, three and two DEGs were associated   (Tables S3 and S4). These results were consistent with the content of MeSA in different treatments.

Discussion
The present study reveals that the biochemical mechanism of AM fungus modulates plant responses to pea aphid infestation. We did not observe the negative influence of aphids on the growth of non-mycorrhizal and mycorrhizal plants. We assume this may be due to the short period of the experiment. In addition, alfalfa plants may have a compensation for adverse effects of aphid infestation. The infestation of the aphids induced alfalfa to absorb P and N in mycorrhiza-inoculated plants, which may compensate for the loss caused by aphids feeding on plant growth as revealed by higher fresh and dry weights in AMA+. Furthermore, the increased shoot total P content in plant leaves can increase the net photosynthetic rate in plants [42]. This is consistent with the higher net photosynthesis in mycorrhiza-inoculated plants infested with pea aphids. Insect feeding usually decreases plant photosynthesis, reducing the transportation of photosynthesis to the host plant's roots [8]. Subsequently, this affects the colonization of the AM fungi and nutrient acquisition from the host plants [7]. Alfalfa was exposed to pea aphid stress as the symbiotic relationship changed owing to the carbon-lipid exchange with N and P absorption by the AM fungus [43]. Our results show AM colonization was not affected by aphid infestation, which shows the symbiosis of AM fungus and alfalfa was stable, and mycorrhiza retains its function in enhancing plant P uptake as this was proved by the higher P and N content in mycorrhizal plants. Babikova et al. found that the alteration of metabolism of alfalfa by the AM fungus antagonized the infestation of pea aphids by accumulating total P in the leaves [44]. In our study, the stable AM fungal colonization in alfalfa protected plants from aphid infestation by nutrient uptake and higher photosynthetic rate.
Our results show aphid infestation causes alfalfa defense enzyme activity, especially reactive oxygen species (ROS) related enzymes, such as PPO, POD, and CAT; this agrees with previous studies [45][46][47][48]. For example, Mahanil found the ROS related enzyme was increased when tomato was exposed to the common cutworm [46]. Another study found host plant POD activity increases to resist insect infestation with identification of B6T173 (ZmPrx35) as the prevailing peroxidase in highly insect-resistant maize (Zea mays, p84C3) kernels by activity-directed purification, which include activities of antioxidant enzymes in three species of Ludwigia weeds on feeding by Altica cyanea. Though ROS are produced in the plant to resist biotic stresses, a large quantity of ROS might damage nucleic acids and proteins in the cells, polyunsaturated fatty acids on the cell membranes, and subcellular organelle membranes [48]. The present study found the POD activity in alfalfa significantly decreased after pea aphid infestation, indicating reduced ROS in the legumes, which may benefit plants' stress tolerance [49]. In addition, the activity of CAT increased in mycorrhiza-inoculated alfalfa plants infested with pea aphids compared to non-fungal-inoculated plants, which may be related to a signal for the induction of defense genes in plants [50]. Therefore, this study suggests that AM fungus could alter a plant's response to aphids by increasing plant P, N uptake, plant growth, and defense enzyme activity.
Our research shows that JA significantly increases in the alfalfa after being infested with pea aphids, while AM fungus enhances SA concentration in the legumes. However, JA and SA did not consistently change in alfalfa in response to pea aphid infestation, with JA acting as the altering hormone in the legumes' response to pea aphid infestation. The AM fungus-induced SA might serve as a growth, antioxidant, and metabolic activity regulator for alfalfa. Plant hormones such as JA and SA are essential endogenous molecules involved in plant development and direct and indirect defense systems [51]. Jasmonic acid and SA are the primary signaling pathways in plant defense [52,53]. The JA signaling pathway plays an essential role in plant growth, activation of plant resistance genes, and enhanced antioxidant enzyme activity [54,55]. The SA signaling pathway regulates growth, enhances the plant antioxidant system, and increases metabolic activity [56,57]. Alfalfa shows a stronger response to aphid infestation than to AM colonization, which indicates JA and SA pathways are closely related to plant response to biotic stress [13], while AM fungus mostly enhances a plant's JA content when a plant is exposed to stress conditions. The plant defense system shares a complex relationship with the hormonal pathway, including coordination, prioritization, and cross-talk [58,59]. Trypsin inhibitors contribute to the tolerance of host plants to heavy herbivore pressure by deterring insect herbivory [60]. The high levels of trypsin inhibitors in mycorrhiza-inoculated plants and pea aphid treatments indicate trypsin inhibitors may play important roles in plant response to AM colonization and aphid infestation [13]. AM fungus could induce trypsin-inhibitor accumulation in the same way that occurs in the presence or absence of aphids. The higher rate of trypsin inhibitors in mycorrhizal plants is perhaps present before the arrival of the aphids and therefore limits the infestation of the plants by the aphids. The number of VOCs detected in this study (114) was larger than commonly reported in similar studies. For example, 17 VOCs were reported in a study of V. faba response to aphid attack [61], 10 in a study of Z. mays response to application of herbivore beetle extract [62], and 33 in a study of site differences in VOC profiles of Calluna vulgaris [63]. The reason for the larger number of VOCs detected here is unclear. One possibility is that SPME extraction of VOCs from frozen leaf material was more efficient than vapor-phase VOC capture in live-plant headspaces used in many studies [20].
The AM fungus induced elevated levels of overall contents and resulted in apparent changes in the ratios of seven individual volatile compounds. Many previous studies have shown that insect herbivores can exploit VOC cues to avoid inferior hosts [64,65]. Thus, despite the large sizes of mycorrhiza-inoculated alfalfa plants, more quantities of VOCs in the fungal-inoculated plants may repel aphids. Subtle quantitative and qualitative differences also exist between the VOCs' contents in inoculated and non-mycorrhizainoculated alfalfa after pea aphid infestation [66]. There are ester, ketone, and terpenes in the top 20 VOCs that had a high discriminatory power in the four treatments. Among these, 3-Octanone and gamma-terpinene in alfalfa can enhance plant resistance to aphids. The 3-Octanone also elicits higher Electroantennogram (EAG) responses in the vetch aphid Megoura viciae Buckton (Homoptera, Aphididae) [67], while gamma-terpinene repels the insect through its toxic property [68,69].
The VOCs such as MeSA are the key compounds in mycorrhiza-alfalfa response to pea aphid infestation. MeSA is synthesized from SA mainly through carboxyl methyltransferase in the plant [70] as a long-distance signal associated with systemic acquired resistance (SAR) [71]. MeSA can induce plant growth and development and enhance defense responses [72]. Moreover, plants emit MeSA into the atmosphere [73] to repel aphids and attract their parasitoids [74,75]. MeSA attracts aphid predators, such as syrphid flies (Diptera: Syrphidae) and green lacewings (Neuroptera: Chrysopidae) to decrease Aphis glycines Matsumura (Hemiptera: Aphididae) abundance [76]. In the current study, pea aphid infestation reduced MeSA content, benefiting the insects' infestation but reducing the predators' discovery. AM fungus increased the total content of VOCs, which likely influenced the plants' response to pea aphid infestation and predator attraction. The key DEGs, including MS.gene23894, MS.gene003889, and MS.gene012415, which regulate MeSA content, were down-regulated in the NMA− vs. NMA+ and up-regulated in the NMA+ vs. AMA+. Generally, MeSA increases the abundance of natural enemies in numerous food crops and has been commercially used as slow-release lures for pest control [77].

Conclusions
Colonization of the AM fungus R. intraradices promotes plant growth, N uptake, net photosynthesis, and transpiration rates when alfalfa is exposed to aphid infestation, thus compensating for the negative influence caused by pea aphids. The infestation of pea aphids activates alfalfa PPO activities and JA concentration, while AM fungus colonization enhances plant-defense enzyme activities when plants are exposed to pea aphid infestation, and the content of SA in mycorrhizal alfalfa is higher than non-mycorrhizal alfalfa. AM fungus changes alfalfa's response to pea aphids, and plants have complex defense pathways in response to insect infestation. Moreover, AM fungus increases MeSA, which is decreased by pea aphid infestation. In addition, the MeSA-regulated key genes in alfalfa are upregulated by AM fungus after pea aphid infestation.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8121308/s1, Figure S1: AM fungi colonization of M. sativa inoculated with R. intraradices (AM) and infested by A. pisum (A+) or without inoculation with R. intraradices (NM), and un-infested by A. pisum (A−). All values are shown as means ± SEM of four biological replicates. Different letters above bars represent significant difference in the comparison at p < 0.05, as determined by Tukey's HSD test. SEM, standard error of the mean.; Figure S2: SOD activcity of M. sativa inoculated with R. intraradices (AM) and infested by A. pisum (A+) or without inoculation with R. intraradices (NM) and non-infested with A. pisum (A−). All values are shown as means ± SEM of four biological replicates. Different letters above pairs of bars represent significant difference in the comparison at p < 0.05, as determined by a Tukey's HSD test. SEM, standard error of the mean. SOD, superoxide dismutase.; Figure S3: (a) Total phenols concentration, (b) ABA concentration, and (c) NO concentration of M. sativa inoculated with R. intraradices (AM) and infested by A. pisum (A+) or without inoculation with R. intraradices (NM) and non-infested with A. pisum (A−). All values are shown as means ± SEM of four biological replicates. Different letters above pairs of bars represent significant difference in the comparison at p < 0.05, as determined by a Tukey's HSD test. SEM, standard error of the mean. ABA, abscisic acid.; Figure S4 Table S2.; Table S1: Main effects of AM fungus (inoculation or uninculation), pea aphid (infested or uninfested) and their two way interactions on AM colonization, Shoot fresh weight, Shoot dry weight, Shoot total N, Shoot total P, PPO, CAT, POD, SOD, SA, JA, ABA, NO, Trypsin inhibitor, Total phenols, Total VOCs. p < 0.05 are highlighted in bold.; Table S2: The compounds of 114 VOCs in Medicago sativa roots inoculated with Rhizophagus intraradices (AM) and infested with Acyrthosiphon pisum (A+) or non-inoculated with R. intraradices (NM) and non-infested with A. pisum (A−).; Table S3: Methyl salicylate related differentially expressed genes in NMA+ vs AMA+; Table S4: Methyl salicylate related differentially expressed genes in NMA− vs NMA+.