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Introduction the soil salinity increases, the water potential of the soil The world’s population has grown rapidly over the past solution becomes lower than the water potential of the two decades, leading to a sharp increase in the global plant root cells, resulting in osmotic stress. As a result, demand for crop yields (Savary et al. 2020). How to the roots cannot absorb water and results to plants improve the yield and quality of crops has become an experiencing a physiological shortage of water (Raz- urgent global agricultural problem. Rice (Oryza sativa L.) zaq et al. 2020). Osmotic stress causes stomatal clo- is among the leading food crops worldwide, and its plant- sure, which inhibits plant uptake of C O and leads to ing area is extensive. Rice yield is of great significance a decrease in photosynthesis (Qin and Huang 2020). to the development of the world population (Zeng et al. Plants must perform osmoregulation and produce many 2001). Salt stress is an important abiotic stress factor that osmotic adjustment substances, such as proline (Pro) in threatens plant growth and development; in addition, the cells to maintain the normal expansion, growth, and salt stress is a severe problem that restricts agricultural water absorption of cells (Liu et al. 2021a). Water defi - and forestry production and ecological environment ciency in plants alters the intercellular osmotic pressure construction (Hazell and Wood 2008). It is estimated and reduces the efficiency of nutrient transport, lead - that approximately 20% of irrigated soils worldwide are ing to nutrient deficiency (Chen et al. 2020). On the affected by different degrees of salinization, and the soils other hand, ionic stress is mainly caused by the massive are continually deteriorating due to inappropriate crop accumulation of Na in the soil in plant cells (Yang and irrigation measures, excessive fertilization, unreasonable Guo 2018). Excessive Na inhibits the activity of vari- farming methods, rising sea levels leading to salt intru- ous enzymes in plants and interfere with the uptake of + 2+ 2+ sion into coastal areas, etc. (Kamran et al. 2019). Rice K, Ca, Zn , and other elements by plants (Wu et al. is generally sensitive to salt stress, which can slow rice 2018; Iqbal et al. 2018). Salt stress results to the produc- growth and reduced yields (Ahmed et al. 2021). Thus, tion of large amounts of reactive oxygen species (ROS) improving the adaptability of rice to increasingly salin- in plants, which can impact plant growth and develop- ized soil is clearly significant for effectively increasing ment and even lead to plant death (Parihar et al. 2020). rice yield to meet the growing food demand. Plants usually eliminate ROS by producing many antioxi- Salt stress mainly causes damage to plants through dant enzymes such as superoxide dismutase (SOD) and osmotic stress and ion toxicity (Quan et al. 2007). When peroxidase (POD), resisting salt stress and alleviating the Zhang et al. Rice (2023) 16:18 Page 3 of 18 damage caused by salt stress (Shehab et al. 2022). Salt differential expression of some relevant salt tolerance- stress not only affects the growth and yield of rice but related and photosynthesis-related genes was also quan- also affects the quality of rice. At present, much attention tified through qRT-PCR. The aim of the study was to is being placed on the development of new biotechnology explore some of the AMF-mediated plant responses that methods to improve the stress resistance of rice and alle- improve the salt tolerance of rice seedlings which sup- viate the damage caused by salt stress. ports the practical application of AMF compound inocu- Arbuscular mycorrhizal fungi (AMF) is an endophytic lants for rice cultivation in salinized soils. fungus that is widely distributed in nature and can form symbioses with 80% of terrestrial plants; in addi- Results tion, AMF are the class of fungus most closely related to Eec ff t of Salt Stress on AMF Inoculation and Spores agricultural production in the soil microflora (Hashem in the Rhizosphere Soil of Rice et al. 2018). AMF improve the nutritional status of the AMF compound inoculants established symbiotic rela- host plant by establishing a symbiotic relationship with tionship with rice. Figure 1 bcd shows that the roots of the host and helping the host plant absorb water and rice possess clear vesicles, hyphae, and Pi spore struc- nutrients from the soil (Abdelhameed et al. 2018). Fun- tures. However, fungal infection was not detected in neliformis mosseae (Fm) can greatly improve the salt tol- the nm treatment group, which indicated the absence erance of host plants by increasing host plant biomass, of indigenous AMF and Pi in the soil matrix (Fig. 1 a). enhancing photosynthetic intensity, antioxidant enzyme By mycorrhizal infection rate determination, we found activity, and osmoregulatory capacity (Qin and Huang that the mycorrhizal infection rate of the am 80 treat- 2020; Shahvali et al. 2020; Santander et al. 2021; Wang ment was up to 80% (Fig. 1 e); and was significantly dif - et al. 2020a, b). AMF applications show great potential ferent (P < 0.05) from the am 0 treatment. The growth for improving rice yield and enhancing rice salt toler- rate was 9.1%, however, as the concentration of salt stress ance. Piriformospora indica (Pi) can improve the toler- continued to increase, the root infection rate decreased ance of crops to adverse stress and induce plant systemic significantly (P < 0.05). Salt stress treatment significantly resistance (Singh et al. 2000). When used in combina- reduced the number of spores in rice rhizosphere soil tion with AMF, the specific functions of both fungi can (P < 0.05). Compared with the am treated rice roots at be further exploited. Moreover, there is no competition 0 mM, the spore number in the roots of rice grown under between the combined microorganisms. (Moreira et al. 80 mM, 120 mM, and 160 mM salt stress decreased by 2015). Agrobacterium rhizogenes is a kind of soil bac- 21.9%, 56.3% and 71.9%, respectively. teria that can infect a wide variety of organisms; it can infect plants and induce plants to promote plant rooting Eec ff ts of Salt Stress on Photosynthetic Gas Exchange and hairy root differentiation. In addition, Agrobacterium Parameters and SPAD of Rice rhizogenes is also a kind of mycorrhizal helper bacteria, Salt stress decreased the net photosynthetic rate of rice that contributes to the colonization of AMF in rice roots (Fig. 2 a, Table 1), and compared with the no salt treat- (Frey et al. 2007). We used two fungi and one bacterium ment, the difference was significant (P < 0.05). The AMF to create AMF compound inoculants. Through rice field compound inoculants can alleviate the effect of salt stress planting experiments, we found that AMF compound on net photosynthetic rate (A). At 80 mM and 120 mM inoculants promoted the colonization of two endophytic salt concentrations, there was a significant difference fungi in rice roots, and exhibited a stronger effect on the between the am and nm treatment groups (P < 0.05), growth and development of rice (Zhang et al. 2022). In with the most significant difference at 80 mM salt this regard, we hypothesized that AMF compound inocu- concentration. lants could increase the expression of relevant genes in Salt stress caused negative effects on the transpiration rice seedlings, resulting in increased biomass, antioxidant rate (E), stomatal conductance (GH O), intercellular C O 2 2 enzyme activity, and osmoregulatory substance content concentration (Ci), and SPAD values of rice (Fig. 2 bcde). of rice seedlings. The inoculants can regulate the cation With increasing salt concentration these parameters concentration in rice seedlings and improve salt toler- all gradually decreased compared to those of non-salt ance in rice by the above means. stress. AMF compound inoculants can alleviate the dam- In this study, we investigated the effects of AMF com - age of salt stress to rice photosynthesis, showing differ - pound inoculants on the biomass, photosynthetic gas ent results in different indicators. Under salt stress, there exchange parameters, antioxidant enzyme activities, as was a significant difference (P < 0.05) in E between am 0 + + 2+ well as the dynamic balance of Na, K , and Ca ions and nm 0, am 80 and nm 80, and am 120 and nm 120. in shoots and roots of rice seedlings grown under 0, However, the difference in E between am 160 and nm 160 80, 120 and 160 mM salt stress conditions. The relative was not significant (P > 0.05). The effect on rice GH O 2 Zhang et al. Rice (2023) 16:18 Page 4 of 18 Fig. 1 The rice root colonization of AMF under the microscope (400x) and their effective colonization in terms of colonization rate and spore number. Note: a nm treatment group; b AMF spores in rhizosphere soil; c Fm + Pi + Ar treatment group; d Fm + Pi + Ar treatment group; (e) Rice mycorrhizal colonization rate and the number of spores in the rhizosphere soil. The results are presented as the mean ± standard deviation of three replicates. Different letters indicate differences between different treatments (P < 0.05) Fig. 2 Photosynthetic gas exchange parameters and SPAD value of rice leaves between different treatment groups. Note: a A; b E; c Ci; d GH O; e SPAD. The results are presented as the mean ± standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P < 0.05) Zhang et al. Rice (2023) 16:18 Page 5 of 18 Table 1 Results of bidirectional variance analysis of effects of inoculum and salt concentration on rice seedling parameters Parameter measured Significance of sources of variation Salt AMF + PI + AR Salt × AMF + PI + AR Plant height *** *** NS Root length *** *** * Shoot Fresh weight *** *** ** Root Fresh weight *** *** ** Mycorrhizal *** *** *** Spore number *** *** *** Shoot K concentration *** *** *** Root K concentration *** *** * Shoot Na concentration *** *** NS Root Na concentration *** *** NS 2+ Shoot Ca concentration *** *** NS 2+ Root Ca concentration *** *** NS + + Shoot K /Na ratio *** *** *** + + Root K /Na ratio *** *** NS 2+ + Shoot Ca /Na ratio *** *** *** 2+ + Root Ca /Na ratio *** *** NS Shoot/root Na ratio * NS ** MDA *** *** NS SOD *** *** * POD *** *** *** Gr *** *** *** PAL *** NS NS Pro *** *** *** SPAD *** *** ** A *** *** NS E *** *** * Ci *** *** *** GH O *** *** *** ns not significant *P < 0.05 **P < 0.01 ***P < 0.001 was only significantly different between the am and nm with AMF compound inoculants significantly improved groups at 0 mM concentration (P < 0.05). In terms of Ci, plant growth parameters (P < 0.05). However, the growth the am treatment group showed a significant increase parameters of both plants inoculated with the complex (P < 0.05) at 0 mM, 120 mM, and 160 mM concentrations agent and plants not inoculated with the complex agent compared to that of the nm group. Salt stress significantly were lower under stress conditions than those of inocu- reduced the SPAD value of chlorophyll parameters in rice lated and non-inoculated plants under stress conditions. (P < 0.05), and the AMF compound inoculants relieved AMF compound inoculants significantly reduced the the damage caused by the stress. There were significant toxic effects of salt stress. The most significant difference differences from nm at 0 mM, 80 mM, and 160 mM salt was between the am 80 and nm 80 treatment groups. concentrations (P < 0.05). The plant height and root length of rice increased by 53.98% and 111.58%, respectively. Damage to rice due to Eec ff ts of AMF Compound Inoculants on Rice Biomass salt stress can be reduced by applying AMF compound Under Salt Stress inoculants. However, there is a limit to this reduction Measurement of rice growth indicators under different capacity, which tends to increase and then decrease with salt concentration treatments showed that co-inoculation increasing salt concentration (Fig. 3, Table 1). Zhang et al. Rice (2023) 16:18 Page 6 of 18 Fig. 3 Growth parameters of Oryza sativa L. in different treatment groups. Note: a plant height; b root length; c shoot fresh weight; d root fresh weight. The results are presented as the mean ± standard deviation of three replicates. Different letters indicate the difference between different treatment groups (P < 0.05) Low-salt stress (80 mM) increased the fresh weight of increase in MDA content, which significantly increased shoots in the am treatment group and the fresh weight of as the salt concentration increased (P < 0.05). The AMF roots in the nm treatment group, but the difference was compound inoculants can inhibit the increase in MDA not significant (P > 0.05). However, with the increase in content. The MDA content of the am0, am80, am120 and salt concentration, the fresh weight of shoots and roots of am160 treated groups was reduced by 12.91%, 15.79%, the am group and nm group decreased gradually. Com- 23.07% and 15.87% in each group compared to that of pared with the nm treatment group, AMF compound the nm 0, nm 80, nm 120 and nm 160 treated groups, inoculants could significantly increase the fresh weight of respectively. There was a significant effect of salt stress shoots and roots of rice under 80 mM and 120 mM salt and AMF compound inoculants on the Pro content of stress (P < 0.05), but the fresh weight of roots was signifi - rice leaves (Fig. 4 f, Table 1). Salt stress can induce a sig- cantly lower at 120 mM salt concentration. In addition, nificant increase in Pro content in rice (P < 0.05), which there was no significant difference between the am and increases with salt concentration. The AMF compound nm treatment groups as the salt concentration continued inoculants could further significantly increase the Pro to increase, which indicated that the mitigation of rice content in rice and were significantly different from growth and development under salt stress by AMF com- the nm treatment group at different salt concentrations pound inoculants is limited. (P < 0.05); the most significant difference in Pro occurred at 80 mM concentration, which was 2.12 times higher Eec ff ts of Salt Stress on the Contents of MDA, than that of the nm treatment group. Osmoregulatory Substances, and Antioxidative Enzymes Salt stress significantly affected the content of SOD in Rice in rice leaves (Fig. 4 a, Table 1). Salt stress induced an The malondialdehyde (MDA) content of rice leaves increase in SOD in rice leaves and showed a trend of treated with different salt concentrations was measured increasing first and then decreasing in the nm group. (Fig. 4 e), and the results showed that salt stress led to an Compared with the nm treatment group, the am Zhang et al. Rice (2023) 16:18 Page 7 of 18 Fig. 4 MDA contents, osmotic regulatory substances, and antioxidant enzyme activities in rice leaves. Note: a SOD; b POD; c Gr; d PAL; e MDA; f Pro. The results were the mean ± standard deviation of three replicates. Different letters indicate the difference between different treatment groups. (P < 0.05) treatment group showed a significant increase in SOD Ion Concentration and ion Homeostasis content of rice leaves (P < 0.05), which were 1.98 times, The contents of cations in the shoots and roots of rice 1.53 times, 1.61 times, and 1.93 times that of the nm seedlings were determined (Fig. 5, Table 1). It was treatment group, respectively. However, at concentra- found that more K accumulated in the roots than in tions of 80 mM, 120 mM, and 160 mM, there was no the shoots under salt stress. Salt stress decreased the significant difference among the am group (P > 0.05). content of K in rice and decreased gradually with Salt stress induced an increase in the POD content of increasing salt concentration, which was significantly rice leaves (Fig. 4 b, Table 1), and the AMF compound different from that of 0 mM (P < 0.05). However, the inoculants significantly increased the POD content content of K increased slightly under the concentra- of rice leaves in other groups except for the 160 mM tion of 80 mM salt, which was consistent in the shoots group, which was 1.87, 2.46 and 2.66 times higher and roots of rice. The addition of AMF compound inoc - than that of the nm group, respectively. Low-salt stress ulants could increase the content of K in the shoots increased the content of glutathione reductase (Gr) in and roots of rice. There were significant differences in rice leaves and inhibited the production of Gr in rice the K content in rice roots except for the 0 mM group leaves as the stress level increased (Fig. 4 c), and the dif- (P < 0.05). ference was significant (P < 0.05). The AMF compound Salt stress increased the content of N a in the shoots inoculants increased the content of Gr in rice leaves, and roots of rice. The content of Na gradually increased and there were significant differences at the levels of with increasing salt concentration. In the am and nm 80 mM, 120 mM, and 160 mM (P < 0.05). Among them, groups of rice, more Na was accumulated in the roots the am treatment group showed the highest increase at than shoots (P < 0.05) (Fig. 5 b). However, the Na con- 160 mM, which was 10.10 times that of the nm group. tent of both nonstressed plants inoculated with the com- Salt stress significantly reduced the content of pheny - pound inoculants and plants not inoculated with the lalanine ammonia-lyase (PAL) in rice leaves (P < 0.05). compound inoculants was lower than that of inoculated AMF compound inoculants increase the PAL content in plants and non-inoculated plants under stress condi- rice leaves under salt stress, but this increase is limited. tions. Except for 80 mM, other concentrations in the PAL content was significantly different between the am aboveground parts of rice were significantly different and nm treatment groups at 80 mM salt concentration from those of at 0 mM. AMF compound inoculants sig- (P < 0.05) and not significantly different at 120 mM and nificantly reduced the content of Na in the shoots and 160 mM salt concentrations (P > 0.05) (Fig . 4 d). roots of rice (P < 0.05). However, the difference in root Zhang et al. Rice (2023) 16:18 Page 8 of 18 + + 2+ Fig. 5 Aboveground and root ion contents of rice at different salt concentrations. Note: a K content; b Na content; c Ca. content; the result is the mean ± standard deviation of three repeats. Different letters represent the differences between different treatment groups. (P < 0.05) + 2+ Na content under salt-free conditions was not signifi - compound inoculants could increase the content of C a cant (P > 0.05). in the shoots and roots of rice. Compared to the roots, the shoots of rice accumulated Salt stress negatively affected the shoot and root K / 2+ + more Ca (Fig. 5 C). Salt stress decreased the content Na ratios of rice with or without AMF compound 2+ of Ca in rice, and decreased gradually with increasing inoculants (Tables 1, 2). However, the shoot ratio of rice salt concentration, which was significantly different from increased slightly at the 80 mM concentration, and the 2+ that of at 0 mM (P < 0.05). However, the content of Ca difference was not significant (P > 0.05). Compared with increased slightly under the concentration of 80 mM the nonsalt condition, the shoot of rice under salt stress salt, which was consistent in the shoots and roots of rice. showed a significant difference above 120 mM (P < 0.05), 2+ The effect of salt stress on the Ca content in the shoot while there was a significant difference in the root from of rice was not significant (P > 0.05). However, AMF 80 mM (P < 0.05). Inoculation with AMF compound + + + + 2+ + 2+ + Table 2 Eec ff ts of salt and AMF compound inoculants on shoot K /Na ratio, root K /Na ratio, shoot C a /Na ratio, root C a /Na ratio in Oryza sativa L + + + + 2+ + 2+ + NaCl(mM) TreatmentShoot K /NaRoot K /NaShoot Ca /NaRoot Ca /Na 0 nm 12.279 ± 0.671c 16.551 ± 2.217ab 1.110 ± 0.119c 0.423 ± 0.034b am 28.643 ± 2.807a 19.543 ± 0.952a 2.506 ± 0.425a 0.564 ± 0.074a 80 nm 12.831 ± 0.483bc 11.853 ± 2.699 cd 1.198 ± 0.067c 0.341 ± 0.053c am 27.253 ± 4.069a 16.977 ± 1.398ab 1.851 ± 0.205b 0.561 ± 0.037a 120 nm 3.954 ± 0.370de 7.683 ± 1.127e 0.728 ± 0.067de 0.197 ± 0.041d am 15.605 ± 0.828b 14.678 ± 2.761bc 1.032 ± 0.089 cd 0.432 ± 0.051b 160 nm 2.289 ± 0.206e 4.009 ± 0.267f 0.489 ± 0.047e 0.147 ± 0.031d am 5.752 ± 0.766d 9.522 ± 1.031de 0.733 ± 0.105de 0.292 ± 0.035c Zhang et al. Rice (2023) 16:18 Page 9 of 18 inoculants at different salt concentrations significantly the OsHBP1b, Os10g21268, Os05g25850, Os06g51150, + + increased the ratio of K /Na in the shoots and roots Os10g38229, Os9g17740, and Os10g41780 genes were + + of rice (P < 0.05). The K /Na of am 0, am 80, am 120 significantly higher in the am-treated group than in the and am 160 under salt stress was 2.26-fold to 1.33-fold, nm-treated group (P < 0.001). The relative expression 1.55-fold to 1.43-fold, 3.95-fold to 1.91-fold and 2.51-fold levels of the Os10g21268, Os08g35420, Os05g25850, to 2.38-fold that of nm 0, nm 80, nm 120 and nm 160, Os06g51150, Os10g38229, Os9g17740, and Os10g41780 respectively. genes in the am group were 22.56-, 57.03-, 17.02-, 2+ + Salt stress caused negative effects on the Ca /Na 28.47-, 3.82-, 10.29-, 9.95- and 5.11-fold, respectively. ratio of the shoots and roots of rice (Tables 1, 2). How- ever, the shoot ratio of rice increased slightly under Expression of Ion Transport‑Related Genes in Rice 80 mM, and the difference was not significant (P > 0.05). In this experiment, the relative expression differences of 2+ + The Ca /Na of rice shoots in the am and nm groups genes related to cation exchange in rice were analysed. We produced significant differences from 0 mM at salt con - selected nine genes, namely OsNCX1, OsNCX2, OsNCX3, 2+ + centrations above 120 mM (P < 0.05). Ca /Na in the OsNCX4, OsNCX5, OsNCX6, OsNCX7, OsNCX10 and rice roots of the am and nm groups was significantly dif - OsNCX15. We carried out qRT‒PCR verification of rice ferent from 0 mM at salt concentrations above 80 mM samples at 80 mM salt concentration. There no significant (P < 0.05). Compared to the nm group, the am group difference in the relative expression of OsNCX1, OsNCX3, 2+/ + under salt stress showed shoot and root Ca Na ratios and OsNCX5 at the transcriptional level between the am- of rice that were significantly increased (P < 0.05). The treated group and the nm-treated group (P > 0.05). There 2+ + shoot and root C a /Na in rice were 2.33 to 1.18, 2.12 to was a highly significant difference in the relative expression 1.65, 1.42 to 2.19, and 1.50 to 1.99 times higher in the am of OsNCX2, OsNCX4, OsNCX6, OsNCX7, and OsNCX15 0, am 80, am120, and am160 groups than in the nm 0, nm transcript levels between the am-treated and nm-treated 80, nm120, and nm160 groups, respectively. groups (P < 0.001). The relative expression levels of OsNCX2, OsNCX4, OsNCX6, OsNCX7, and OsNCX15 transcript levels in the am group were 1.97-, 2.01-, 1.57-, Relative Gene Expression 1.72-, and 3.37-fold of those in the nm group (Fig. 6). Relative Expression of Salt Tolerance Genes in Rice In this experiment, the am 80-treated and nm 80-treated Principal Component Analysis (PCA) groups with the most significant growth and physi - Principal component analysis was carried out on the data ological differences were selected for qRT‒PCR analy - of rice biomass, mineral ion absorption, homeostasis, ROS sis. We selected eight rice salt tolerance-related genes, scavenging enzymes, light, and parameters. The X-axis namely OsQHB, OsPRX2, OsPRX4, OsPRX9, OsPRX72, and Y-axis denote the first (PC1) and second (PC2) prin - OsPRX112, OsPRX-A2, and OsABSRP5. We carried out cipal components, respectively (Fig. 7), with a total PCA qRT‒PCR verification of rice samples at 80 mM salt explanation of approximately 79.88%. The results of this concentration. The relative expression of the OsQHB, study show that there is a clear distinction between differ - OsPRX2, OsPRX9, and OsPRX72 genes at the transcrip- ent samples. The differences between salinity levels were tional level in the am-treated group was significantly dif - distinguished by PC1, while PC2 tended to distinguish the ferent from that in the nm-treated group (P < 0.001) and mycorrhizal inoculum status of the samples. In the scatter they were 13.86-, 3.33-, 15.25-, 4.72-, 4.40-, and 1.95- fold plot of PC1 and PC2, a smaller distance between the sam- that in the nm group, respectively. The relative expres - ples indicates similarity. The am treatment group and the sion of OsQHB, OsPRX2, OsPRX9, OsPRX72, OsPRX112, nm treatment group are obviously separated. Under the OsPRX-A2, and OsABSRP5 transcript levels was signifi - same salt concentration, the difference between the am 80 cantly higher in the am-treated group than in the nm- group and nm 80 group was the most significant. There treated group (P < 0.05). was the most significant difference between the am 0 group and nm 160 group under different salt concentrations. Expression of Photosynthesis‑Related Genes in Rice The OsHBP1b, Os10g21268, Os08g35420, Os05g25850, Discussion Os06g51150, Os10g38229, Os9g17740, and Os10g41780 Eec ff t of Inoculation with AMF Compound Inoculants genes can regulate the intensity of photosynthesis in on the Infection and Biomass of Rice Seedlings Under rice. We carried out qRT‒PCR verification of rice sam - Sodium Chloride Stress ples at 80 mM salt concentration. No significant dif - Agrobacterium rhizogenes infecting plants can induce ference in Os08g35420 expression was found between the root system to differentiate many fast-growing hairy the am-treated and nm-treated groups under salt roots and increase the root area and biomass (Wang et al. treatment (P > 0.05). The relative transcript levels of Zhang et al. Rice (2023) 16:18 Page 10 of 18 Fig. 6 The relative gene expression of the am 80 treatment group and nm 80 treatment group. Note: a Relative expression of genes related to salt tolerance, b Relative expression of genes related to photosynthesis, c Relative expression of genes related to ion transport; * indicates the difference between different inoculation treatments when the same gene is treated (*P < 0.05; * *P < 0.01; ***P < 0.001) 2020a, b). It has been shown that Agrobacterium can act higher than that of other treatments (Zhang et al. 2022). as a mycorrhizal auxotroph to directly promote AMF Additionally, AMF can promote plants to release root spore germination, mycelial growth and colonization of secretions. In turn, root secretions are the main source plant roots by AMF and Indian pear-shaped spores (Frey of nutrients for plant rhizosphere bacteria, which pro- et al. 2007). Previous studies found that the mycorrhi- mote the infection of root-emitting Agrobacterium (Paul zal infection rate of the AMF complex was significantly et al. 2011). Piriformospora indica ecological functions are similar to those of AMF and can act synergistically. The combined application of AMF and Pi promotes the growth and photosynthetic efficiency of plant seedlings. In the present study, the AMF compound inoculants increased the biomass of rice seedlings under salt stress and positively affected the light and gas exchange param - eters and proline content of rice seedlings. This is consist - ent with the findings of Moreira et al. (2015). However, whether Ar can be used as an active agent to improve AMF infection and the compound infection mechanism of AMF and Pi need to be further investigated. Rice is most sensitive to salt stress at the seedling stages. The salt tolerance of rice seedlings is an important indicator to for evaluating the salt tolerance of rice dur- ing the whole reproductive period (Das et al. 2019; Singh et al. 2015). NaCl stress may negatively affect fungal growth, spore formation, and infection (Peng 2019; Wang et al. 2020a, b). This study found that rice seedlings can establish a good symbiosis with AMF compound inocu- lants. In contrast, at a salt concentration of 80 mM, both mycorrhizal infection rates were elevated, but the spore number in rhizosphere soil was significantly reduced. Fig. 7 Principal component analysis (PCA) in Oryza sativa L. This may be because low salt stress promoted the release under salt and AMF compound inoculant treatments. Note: nm: of AMF secretions and the formation of mycorrhizal non-mycorrhizal; am: Fm + Pi + Ar.0: 0 mM NaCl; 80: 80 mM NaCl; 120: symbiosis in the rice root system. However, the spore 120 mM NaCl; 160: 160 mM NaCl Zhang et al. Rice (2023) 16:18 Page 11 of 18 reproduction capacity of AMF was reduced due to the Photosynthesis is an important physiological process inhibition of salt stress. The mycorrhizal infection rate that affects plant growth. Previous studies have shown decreased significantly as the salt concentrations contin - that inoculation with AMF can improve the intensity of ued to increase, which may be due to the negative effect photosynthesis in plants(Frosi et al. 2018). The results of of high salt concentration on mycelial growth and spor- the study also showed that the photosynthetic capacity of ulation (Zhou et al. 2022). Compared to the nm-treated leaves of mycorrhizalized beech Zelkova serrata (unb.) Th group, the am-treated group did not show significant Makino was higher than that of non-mycorrhizalized improvements in some indices, such as A, E, GH O, root Zelkova serrata (unb.) Th Makino at a salt concentra - length, fresh weight, and POD, under high concentra- tion of 100 mM(Peng et al.2020; Wang et al. 2019). In tions of salt stress. This may indicate that there is some this experiment, we found that the E, GH O, A, and Ci of positive correlation between the infection rate of AMF rice leaves in the am treatment group were higher than compound inoculants on rice and the improvement in those in the nm treatment group. Mycorrhizal rice may salt tolerance in rice. We speculate that there is a greater improve photosynthetic capacity by increasing photo- potential for effective infection of AMF compound inoc - synthetic gas exchange capacity and water absorption, to ulants in alleviating salt stress in rice plants. reduce the toxicity caused by salt stress. This is consist - Salt stress affects many metabolic processes in plants, ent with the findings of Chang et al. (2018). The results resulting in slow growth and development (Lutts et al. showed that the inoculation of AMF compound inocu- 1995). Many studies have shown that mycorrhizal plants lants improved the water use and light energy efficiency can mitigate the adverse effects of salt stress by activat - of rice under salt stress, and effectively improved the salt ing antioxidant enzyme systems(Shrivastava et al. 2015; tolerance of rice seedlings. Kakar et al. 2019). Mycorrhizal plants can absorb more mineral nutrients and water from the soil, increasing Eec ff t of AMF Compound Inoculants on the Antioxidant the osmotic potential of the soil solution and allowing Enzyme System, MDA, and Pro Content of Rice the plants to grow and develop properly. Evelin et al. Plants mitigate damage from salt stress by secreting (2012) found that the biomass of mycorrhizal Trigonella antioxidant enzymes and synthesizing osmoregulatory foenum-graecum increased significantly under different substances (Garcia-Garrido and Ocampo 2002). The concentrations of salt stress treatment. Similar results experimental results showed that under salt stress con- were obtained in the present study. Under salt stress con- ditions, the MDA content of rice leaves increased under ditions, the plant height, root length, root fresh weight, increasing salt stress and was significantly higher than and aboveground fresh weight of mycorrhizal rice seed- that of non-salt stress treatments. This indicates that salt lings were significantly increased. Mycorrhizal plants stress induces increased levels of intracellular ROS in rice may attenuate the toxic effects of salt stress on plants plants, which subsequently causes oxidative damage to by maintaining high biomass (Parvin et al. 2020). In this cell membrane structures. Under salt stress, the MDA study, salt stress significantly reduced the biomass of rice content of mycorrhizal rice seedlings was significantly seedlings, while inoculation with the AMF compound lower than that of non-mycorrhizal rice seedlings. The inoculants improved rice growth and physiological sta- results showed that inoculation with AMF compound tus. The decrease in mycorrhizalized rice biomass was inoculants could reduce the degree of membrane lipid smaller than that of the control treatment, which facili- peroxidation in rice seedlings under salt stress. Chen tated the better adaptation of rice to salt stress. et al. (2022) found that inoculation with AMF could sig- nificantly increase the activity of antioxidant enzymes in Eec ff t of Inoculating AMF Compound Inoculants Alhagi sparsifolia seedlings under salt stress. It also pro- on Photosynthetic gas Exchange Parameters and SPAD moted the accumulation of osmoregulatory substances in Values of Rice Under NaCl Stress seedlings of Alhagi sparsifolia, and significantly reduced Salt stress reduces the photosynthetic capacity of plants the content of MDA. Combined with the results of previ- mainly by destroying photosynthetic organs and reduc- ous studies, it was further clarified that AMF compound ing photosynthetic pigment content. A chloroplast is an inoculants could improve the resistance of plants to salt important site of photosynthesis, and its chlorophyll con- stress. tent is among the main indices of plant photosynthetic Excess salt causes a decrease in soil osmotic poten- capacity(Pitman et al. 2002). The SPAD value represents tial, preventing plants from absorbing water from the the relative content of chlorophyll in plants. The results soil and causing physiological drought in plants. A large showed that salt stress decreased the chlorophyll content. amount of osmoregulatory substances such as free AMF compound inoculants can increase the SPAD value, proline, are produced and accumulated in plant cells; thus promoting the photosynthesis of rice seedlings. these substances can reduce the intracellular osmotic Zhang et al. Rice (2023) 16:18 Page 12 of 18 Ion Content and Dynamic Equilibrium potential and ensure normal water uptake and utiliza- The sensitivity of plants to salt mainly depends on the tion by plants. Plants utilize this strategy to cope with absorption, accumulation, and distribution of Na . Salt physiological drought caused by salt stress (Ahmed stress increased the content of Na in the roots and et al. 2020). This physiological change ensures normal shoots of rice seedlings, and gradually increased with water uptake and utilization by plants, thus alleviating the increasing salt stress. In this study, inoculation with the physiological drought caused by salt stress. In addi- AMF compound inoculants effectively reduced the con - tion, it has been suggested that proline in plants also tent of Na in the shoots and roots of rice. The content plays a variety of important functions, such as scaveng- of Na in the shoots was lower than that in the roots. ing ROS, and stabilizing proteins and cell membrane The results showed that inoculation with AMF com - structures(Deinlein et al. 2014). In the present experi- pound inoculants inhibited the transport of Na from ment, we found that the proline content of rice seed- rice roots to leaves. This may be a method for mycor - lings significantly increased under salt stress at 80 mM, rhizal plants to inhibit the accumulation of toxic ions in 120 mM, and 160 mM. Inoculation with AMF com- photosynthetic tissues (Porcel et al. 2016). pound inoculants further increased the proline content Potassium is one of many mineral elements neces- in rice seedlings. The results showed that inoculation sary for plant growth. In the present study, salt stress with AMF compound inoculants could induce the showed the same pattern for the accumulation of K accumulation of proline in rice under salt stress, reduce in all parts of rice. Salt stress (80 mM salt stress could the cellular osmotic potential and improve the salt tol- increase) increased the content of K in the shoots and erance of rice seedlings. Similar results were obtained roots of rice. However, an excess of salt reduces the in the salt tolerance test of mycorrhizalized cucumber content of K . Compared with the nm group, inocu- conducted by Han (2012). lation with AMF compound inoculants increased the Under salt stress, plants produce more harmful oxy- content of K in rice and maintained a higher level gen radicals, which interfere with the normal metabolic + + of K /Na . The results showed that AMF compound activities of the plant body and inhibit plant growth and inoculants promoted the accumulation of K in rice. development (Didi et al. 2022). In plants, SOD catalyses 2− Mycorrhizal plants regulate the ion homeostasis of the conversion from O to O and H O . Then POD 2 2 2 plants. This is consistent with the results obtained by and others reduce the damage caused by salt stress by Wang et al. (2022a, b, c). catalysing H O to H O and O (Wang et al. 2023). It 2 2 2 2 2+ In plants, Ca is involved in many important biologi- was previously found that AMF can effectively reduce cal processes, such as protecting the structural and func- the level of membrane lipid peroxidation and enhance tional integrity of plant cell membranes, stabilizing cell the tolerance of plants to weak light and salt stress by wall structure, and regulating ion transport and selec- promoting the growth and antioxidant enzyme activity tion. In this study, 80 mM salt stress slightly increased the of Cucumis melo L. under weak light and salt stress (Xu 2+ content of Ca in rice plants. However, with the increase 2017). The results of Cao showed that inoculation of 2+ in salt concentration, the content of Ca decreased, AMF under salt stress could effectively reduce the level 2+ + and the content of Ca /Na decreased with salt stress. of cell membrane lipid peroxidation by promoting the 2+ This suggests that salt stress inhibits Ca transport by accumulation of osmotic regulators and increasing the plants. Inoculation with AMF compound inoculants can activity of antioxidant enzymes in Asparagus officinalis 2+ increase the Ca content in rice shoots and roots, which L. This alleviates the damage of salt stress to the plant 2+ can promote Ca uptake by rice and reduce ion loss (Cao 2017). In this experiment, salt stress increased caused by salt stress. the antioxidant enzyme activities in rice. However, Inoculation with AMF compound inoculants increased under high salt stress, some antioxidant enzyme activi- + 2+ the contents of K and Ca in rice plants under both ties decreased. This indicates that the resistance of salt-stressed and nonsalt-stressed conditions. This may rice to salt stress is limited. The activity of antioxidant occur because, mycorrhizal symbionts increase root enzymes in rice was significantly increased by inocula - length and root area through the action of hyphae and tion with AMF compound inoculants at all concentra- hairy roots, which effectively improves the ability of tions. It is suggested that AMF compound inoculants plants to absorb mineral nutrients. On the other hand, can improve the salt tolerance of rice by increasing the + 2+ K and Ca , as osmotic substances, can be selectively activities of antioxidant enzymes. This result is simi - absorbed by mycorrhizal symbionts and transported to lar to those of previous studies, indicating that AMF plant organs and tissues to prevent plants from absorbing compound inoculants can improve plant salt tolerance more Na (Hammer et al. 2011). These results suggest by increasing the activities of antioxidant enzymes in that inoculation with AMF compound inoculants can plants. Zhang et al. Rice (2023) 16:18 Page 13 of 18 regulate ion homeostasis in plants and help rice adapt to greatly affect the stress resistance of plants. It was pre - different concentrations of salt stress. viously found that the OsHBP1b gene can regulate and maintain high chlorophyll concentrations in plants Relative Gene Expression (Lakra et al. 2015). The overexpression of this gene can AMF contains multiple SOD-encoding genes and improve rice growth and photosynthesis activity. Overex- induces an increase in plant SOD isoforms (Cu–Zn SOD, pression of OsHBP1b in rice signifies its own role in stress Mn-SOD and Fe-SOD). The symbiotic mechanism of management by minimizing the level of ROS, increasing AMF increases the content and concentration of antioxi- antioxidant enzyme activity, maintaining organelle struc- dant enzymes in plants and upregulates the expression of ture, increasing photosynthesis and modulating the level related genes (Evelin et al. 2012). AMF induces upregu- of photosynthesis and stress-related transcripts. In addi- lated expression of ASA-GSH (ascorbate–glutathione) tion, the OsHBP1b gene enhanced stress tolerance in rice cycle-related enzymes in the host plant, maintains high and was associated with the ABA (abscisic acid) signal- antioxidant content, and prevents free radicals and mem- ling pathway (Das et al. 2019). In this experiment, the brane fatty acid interactions, thus increasing membrane OsHBP1b gene was analysed by qRT‒PCR. The results stability and preventing protein denaturation (Hashem showed that the expression of OsHBP1b was higher in et al.2015). Chang et al. (2023) investigated the effect of mycorrhizal rice under 80 mM salt stress. It was also ver- salt stress on mycorrhizal Elaeagnus angustifolia L. by ified that mycorrhizal rice exhibited stronger photosyn - LFQ proteomics (label-free quantification). Mycorrhizal thetic gas exchange parameters and higher SPAD values symbiosis helps the host plant Elaeagnus angustifolia L. under salt stress. respond positively to salt stress and improve its salt tol- Salt tolerance in rice is a quantitative trait controlled erance by regulating the activity of some key proteins by multiple genes. Salt stress can activate some genes related to amino acid metabolism, lipid metabolism related to salt tolerance in rice to maintain a high level of + + and glutathione metabolism in root tissues. AMF can K /Na and maintain the integrity of the cell membrane increase the content and activity of antioxidant enzymes (Wang et al. 2020a, b). The dynamic balance of Na , + 2+ by regulating the expression of plant-related genes and K , and Ca is the key factor affecting plant growth can regulate the activity of certain metabolic key enzymes and development. Singh studies have shown that NCX in plants to improve salt tolerance in host plants. protein can change the level of intracellular ions, espe- 2+ Excessive accumulation of ROS can cause oxidative cially Ca . Differential expression of NCX genes plays damage to cells. To alleviate this damage, plants have an important role in the physiological processes of rice evolved a defense system for scavenging ROS to increase (Singh et al. 2015). In this experiment, the OsNCX gene the activity of ROS scavenging enzymes and enhance salt was analysed by qRT‒PCR. The results showed that the tolerance. Genes such as PRX and APX can regulate the expression of the OsNCX gene was higher in the am 80 activity of ROS scavengers (Liu et al. 2021b; Savina et al. treated group under salt stress at 80 mM. It was also veri- 2020). Zhou et al. (2022) found that overexpression of the fied that mycorrhizal rice exhibited higher K concentra- OsQHB gene enhanced salt tolerance in rice. Multiple tions and lower N a concentrations, maintained a higher + + peroxidase (PRX) genes are regulated by OsQHB, result- K /Na and could adapt to salt stress. ing in higher ROS scavenging enzyme activity and lower This study shows that mycorrhizal symbionts play an MDA accumulation in rice. In this study, OsQHB and important role in improving the salt tolerance of rice. its related PRX genes were analysed by qRT‒PCR. The This study provides a theoretical basis for further reveal - results showed that mycorrhizal rice exhibited higher ing the regulatory mechanism of mycorrhizal symbi- gene expression under salt stress at 80 mM. This is con - onts on rice growth under salt stress. The salt tolerance sistent with the results of the study on the activity of genes, photosynthesis-related genes, and cationic regula- ROS-scavenging enzymes mentioned in this paper. Myc- tory genes of rice seedlings were verified by qRT‒PCR. orrhizal symbionts may induce overexpression of OsQHB However, the corresponding metabolic pathways and and regulate related PRX genes, thus increasing the activ- upstream and downstream related regulatory genes are ity of ROS scavenging enzymes. It is helpful to improve still unclear and need to be further studied. the salt tolerance of rice, but the mechanism of overex- pression behind the related genes induced by mycorrhi- Conclusion zal symbionts is unclear. AMF compound inoculants significantly alleviated the Salt stress has a serious negative impact on plant pro- damage caused by different concentrations of salt stress. ductivity, and plant stress resistance and yield are regu- The effect is the most significant at a salt concentration lated by transcription factors (Mbodj et al. 2018). In of 80 mM. At 80 mM salt, the AMF compound inocu- addition, activating related transcription factors can lants significantly increased the activities of antioxidant Zhang et al. Rice (2023) 16:18 Page 14 of 18 enzymes in rice leaves. AMF compound inoculants rice seedling soil and used as the rice seedling substrate; increased the content of osmotic regulatory substances the germinated rice seeds were soaked in the A. rhizo- + + 2+ + and the contents of K /Na and Ca /Na . AMF com- genes bacteria liquid with a rice seed to bacterial liquid pound inoculants reduced the damage to plasma mem- ratio of 1:1 (w/v), and a soaking time is 8–16 h; the soaked brane peroxidation caused by salt stress and increased seeds were sown in the seedling substrate; and after dilut- the biomass of rice seedlings. The relative expression of ing 100 mL of Pi fungal liquid into 500 mL of bacteria liq- the OsQHB, OsHBP1b, and OsNCX genes increased sig- uid with sterile water, the solution was sprayed evenly on nificantly. This increase provides favorable conditions for the surface of the seeds(one seedling tray needed 500 mL rice to survive in salinized soil. AMF compound inocu- of Pi fungal liquid, i.e., 15 L/hm ).u Th s the inoculation of lants have the potential to improve the production of rice the AMF compound inoculants was completed (Fig. 8). in salinized soil and have certain application prospects. Experimental Design and Methods Materials and Methods Experimental Design Material Preparation The pot experiment began in June 2022; rice seeds with Rice (Oryza sativa L.) seeds were selected from Longjing full grains were selected, sterilized with 5% NaClO for 31, a salt-intolerant variety mainly planted in Northeast 10 min, and washed five times with sterile water. The seeds China, and were purchased from the Jiansanjiang Farm were placed into a constant temperature incubator at 30 °C Management Bureau, Heilongjiang Province. to accelerate germination, and when the germination rate The following strains of AMF compound inoculants reached more than 80%, we performed the above inocula- were included: (1) Funneliformis mosseae (Fm, isolate tion process with AMF compound inoculants, cultivated number: CGMCC No. 3012): Obtained by pot expansion them in seedling trays (length: 45 cm, width: 30 cm, height: using sorghum as host (spore number ~ 39/g); (2) Pirifor- 5 cm) and covered them with plastic film to maintain high mospora indica (Pi): P. indica fungal liquid (Spore content humidity. On the 20th day of growth, rice seedlings were 2.5 × 10 CFU/mL); (3) Agrobacterium rhizogenes (Ar): transplanted into rectangular plastic pots (upper aper- (Bacteria liquid 3.0 × 10 CFU/mL). The above strains ture 33 cm × 16 cm; lower aperture 25 cm × 10 cm; height were preserved by the Restoration Ecology Research Lab- 14 cm) to simulate paddy culture; the pots were lined with oratory of Hei-Longjing University. double waterproof plastic bags to prevent water loss. Each The AMF compound inoculants were inoculated as fol - pot was filled with 3 kg of sterilized soil (the soil was auto - lows: the Fm inoculants (5%, w/w) were mixed with the claved at 121 °C for 120 min), exogenous salt was added to Fig. 8 Nursery process of AMF compound inoculants Zhang et al. Rice (2023) 16:18 Page 15 of 18 the soil with NaCl (analytically pure) solution, and 2.5 L of (LI-6400XT, LI-COR Corporate, Lincoln, Nebraska NaCl solution with different concentrations was added to USA, USA). On August 11, 2022 (42 days of rice seed- each pot. ling cultivation), from 9:00 to 11:00 a.m., plants with The experimental design included the following experi - consistent growth were selected in each treatment mental factors: AMF compound inoculants and NaCl to measure the relevant indices, and each treatment stress. The following levels of treatment were used for the was measured three times. The indicators measured AMF compound inoculants treatment: AMF compound include net photosynthetic rate (A), stomatal conduct- inoculants inoculum and sterilized inoculum, and four ance (GH O), intercellular CO concentration (Ci), 2 2 levels of treatment were used for NaCl treatment (0 mM, and transpiration rate (E). The SPAD value of rice 80 mM, 120 mM, 160 mM NaCl). In this experiment, a leaves was measured with a chlorophyll analyser (FK- total of eight treatment combinations were used, includ- YL01, Shandong Fangke Instrument Co., Ltd., Weifang, ing nm 0 (sterilized inoculum + 0 mM NaCl), am 0 (AMF China). compound inoculants inoculum + 0 mM NaCl), nm 80 (sterilized inoculum + 80 mM NaCl), am 80 (AMF com- Determination of Rice Biomass pound inoculants inoculum + 80 mM NaCl), nm 120 On the 42nd day of rice growth, five rice plants were (sterilized inoculum + 120 mM NaCl), am 120 (AMF taken from each treatment. First, the aboveground and compound inoculants inoculum + 120 mM NaCl), and root parts of the plant, were separated, the roots were nm 160 (sterilized inoculum + 160 mM NaCl), am 160 cleaned with tap water and washed it with deionized (AMF compound inoculants inoculum + 160 mM NaCl). water three times, and the surface water was dried with Six replicates of each treatment were randomly arranged filter paper. Finally, the plant height and shoot and root with 5 seedlings per pot, for a total of 48 pots. fresh weight were measured. Determination of the Rice Mycorrhizal Infection Rate Determination of MDA Contents, Osmotic Regulatory and Spore Number in Rhizosphere Soil Substances, and Antioxidant Enzyme Activities of Rice The 42 days of rice seedling transplanting coincided with the The Superoxide Dismutase Kit (Solarbio Life Sci - tillering stage of the rice sample. This period is the most sen - ences, BC0175), Peroxidase Kit (Solarbio Life Sciences, sitive period of salt stress for rice seedlings and can reflect BC0095), Glutathione Reductase Kit (Solarbio Life Sci- the salt tolerance of rice throughout its life cycle. And in this ences, BC1165), Malondialdehyde Kit (Solarbio Life period, rice seedlings under high concentrations of salt stress Sciences, BC0025), Proline Kit (Solarbio Life Sciences, showed signs of death, so it was representative. Therefore, we BC0295), and Phenylalnine Ammonialyase (PAL) Activ- collected samples at this time for measurements. Fifty to one ity Assay Kit (Solarbio Life Sciences, BC0215) were hundred root segments in each treatment were randomly used to determine the enzyme activity of rice according selected, stained with the Trypan blue staining method, to the manufacturer’s instructions. transparent, stained, decolorized, sliced, and observed under a 10 × 40 microscope. The mycorrhizal infection rate was calculated as follows (Feng and Song 2020). Number of arbuscular mycorrhiza − positive segments root colonization (%) = ×100% Total number of segments studied Soil spore counts were performed by the wet sieve Determination of Ion Concentration method (Sun et al. 2022). Five grams of air-dried soil was Rice samples were dried at 105 °C for 30 min and then weighed and placed on a soil sieve with a pore size of 0.5– dried at 70 °C until constant weight. Plant sample parts 0.0385 mm. The washing solution was collected and fil - were ground and sieved (80 mesh). The sample was tered in a funnel, the filter paper loaded with spores were placed in a digestion tube, 10 mL nitric acid was added placed on a clean Petri dish, and the spores were observed and the samples were soaked overnight. The next day, and counted with a stereomicroscope. the samples were placed it in an electrothermal diges- tion apparatus for digestion. When 1 mL of solution Determination of Photosynthetic Gas Exchange Parameters remained left in the tube, it was removed and cooled. + + 2+ and SPAD Values of Rice The volume was set to 25 mL and Na, K , and Ca Photosynthetic gas exchange parameters were deter- concentrations were determined using a flame atomic mined using an LI-6400 photosynthesis instrument absorption spectrophotometer (AA-1800H; Shanghai Zhang et al. Rice (2023) 16:18 Page 16 of 18 Ar Agrobacterium rhizogenes Meixi Instrument Co., Ltd.); the standard solution con- ROS Reactive oxygen species figuration refers to the DB22/T 2344–2015 standard, Pro Proline and the linear correlation coefficient of the standard SOD Superoxide dismutase POD Peroxidase curve is 0.9998. qRT-PCR Quantitative reverse transcription-Polymerase chain reaction SPAD Soil and plant analyzer develotrnent A Net photosynthetic rate RNA Extraction, cDNA Synthesis, and Quantitative Reverse E Transpiration rate Transcription Ci Intercellular CO concentration The am 80 and nm 80 treatment groups showed the most GH O Stomatal conductance MDA Malondialdehyde significant differences in growth and physiology, so we Gr Glutathione reductase selected these two treatment groups for qRT‒PCR analy- PAL Phenylalanine ammonia lyase sis; fresh rice leaves were ground in liquid nitrogen, and PCA Principal Component Analysis LFQ Label-free quantification RNA was extracted with an Omega Plant RNA Extraction ABA Abscisic Acid Kit (Plant RNA Kit R6827). The integrity and purity were ASA-GSH Ascorbate–glutathione verified with a NanoDrop 2000c system (Thermo Sci - entific, Pittsburgh, PA, United States), and then reverse Supplementary Information transcription was performed with a TAKARA-RR036A The online version contains supplementary material available at https:// doi. Reverse Transcription Kit (TAKARA RR036A Prime- org/ 10. 1186/ s12284- 023- 00635-2. Script RT Master Mix). Additional file 1. Supplementary Tables and Figures. The primer sequences of related genes were cited from previous research results (Additional file 1: Table 1), and primers were synthesized by Sangon Biotech (Bei- Acknowledgements Xu Zheng: Providing language help. Hongyang Pan: Providing language jing). The reaction system was configured according to help. Yuqiang Wen: Providing language help. Fuqiang Song: Proofreading the the template tracer-type dye quantification PCR assay article. kit (ChamQ SYBR Colour qPCR Master Mix (Low ROX AUTHOR CONTRIBUTIONS Premixed), Vazyme). Each real-time fluorescence quan - Bo Zhang and Feng Shi: Conceptualization, Methodology, Data curation, titative polymerase chain reaction mixture consisted of Writing-original draft, Formal analysis, Visualization, Writing-review & editing. 15 μL of 2 × ChamQ SYBR qPCR Master Mix, 1.2 μL Xu Zheng: Data curation. Hongyang Pan: Help with qRT-PCR experiments. Yuqiang Wen: Supervision. Fuqiang Song: Resources, Supervision, Funding of primers, 2 μL of cDNA diluted 1:10, and 12.3 μL of acquisition. ddH O. The total reaction system was 30 μL. qRT‒PCR were conducted using a REFA40425 fluorescent quanti - Funding This work is supported by the team project of the Heilongjiang Natural Sci- tative PCR apparatus (Cottage Technologies Holdings ence Foundation ( TD2019C002) and; the Key R&D plan guidance projects in Ltd.). The reaction conditions were selected accord - Heilongjiang Province (GZ20210009). ing to the manufacturer’s instructions (Additional file 1: Availability of Data and Materials Table 2) and the temperature required for the relevant All data generated or analyzed during this study are included in this published primers. The relative expression level of transcript sam - article and its Additional files. −ΔΔct ples was analysed by the 2 method; each treatment was repeated 3 times. Declarations Ethics Approval and Consent to Participate Statistical Analysis Not applicable. Microsoft Excel and SPSS 27 were used for data process- Consent for Publication ing and statistical analysis. Plots were generated using Not applicable. Origin 2021b, PowerPoint, GraphPad Prism9, ggplot2, and ggbiplot2 packages in R software (4.2.1). The LSD Competing interests The authors declare that they have no competing interests. test in one-way ANOVA was used to analyse the signif- icance of the difference between the groups at the 0.05 Author details level. In addition, a two-way ANOVA of salt, AMF com- Engineering Research Center of Agricultural Microbiology Technology, Minis- try of Education & Heilongjiang Provincial Key Laboratory of Ecological Resto- pound inoculants, and their interaction was performed ration and Resource Utilization for Cold Region & Key Laboratory of Microbiol- on the data. ogy, College of Heilongjiang Province & School of Life Sciences, Heilongjiang University, Harbin 150080, China. Jiaxiang Industrial Technology Research Institute of Heilongjiang University, Jiaxiang 272400, Shandong, China. Abbreviations AMF Arbuscular mycorrhizal fungi Received: 31 December 2022 Accepted: 4 April 2023 Fm Funneliformis mosseae Pi Piriformospora indica Zhang et al. Rice (2023) 16:18 Page 17 of 18 References turgidumForssk by altering photosynthetic and antioxidant pathways. Abdelhameed R, Metwally R (2018) Mitigation of salt stress by dual applica- J Plant Interact 10(1):230–242. https:// doi. org/ 10. 1080/ 17429 145. 2015. tion of arbuscular mycorrhizal fungi and salicylic acid. 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Rice – Springer Journals
Published: Dec 1, 2023
Keywords: Rice; AMF compound inoculants; Salt stress; Ion homeostasis; Gene expression
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