3.1 Plant growth, MDA, and soluble protein content in leaf / pinna
The plant growth was positively affected by intercropping treatment (Fig. 1). Compared with monoculture treatment (PM), the rhizoid and frond biomass of P. vittata was enhanced by 20.1% and 13.2% for PS treatment, 34.1% and 24.3% for PH treatment, consequently, the total biomass was significantly increased by 19.9% and 34.1%, respectively (P < 0.05). The shoot biomass of S. alfredii in PS treatment was significantly improved by 21.1%, and that of H. spectabile from PH treatment improved by 11.5% as compared with SM and HM, respectively (P < 0.05). This is consistent with previous results that intercropping could protect neighboring companion plants and enhance plant growing on soil contaminated with heavy metals (Wan et al., 2017; Zeng et al., 2019b; Cao et al., 2020). This may be contributed to the improvement of the rhizosphere environment where the soil As efficiently extracted by P. vittata (Wan et al., 2017). Hong et al (2017) confirmed that different plant species show different temporal and spatial resource requirements, so intercropping can obtain essential growth resources more conveniently than monoculture plant.
The content of MDA in pinna of P. vittata from the intercropping system of PS and PH was significantly dropped by 15.3% and 17.3% (P < 0.05) compared with PM, respectively (Table 1). The result was in consistent with former findings that intercropping with Solanum nigrum or Solanum photeinocarpum significantly decreased the MDA content in eggplant in comparison to monoculture (Tang et al., 2017). The reason may be related to that intercropping alleviates the oxidative stress of metal(loid)s to decrease the degree of leaf lipid peroxidation and promote the P. vittata growth (Du et al., 2020). The soluble protein is beneficial to reduce water loss and maintain the main function of cellular membranes stressed by heavy metals (Pan et al., 2018). Intercropping treatments of PS and PH significantly increased the soluble protein content of P. vittata compared with PM (P < 0.05) (Table 1), which was consistent with the result of higher P. vittata biomass from the intercropping system (Fig. 1). The difference in the leaf MDA and protein content of S. alfredii and H. spectabile between intercropping and monoculture treatment was not significant, indicating that intercropping has no obvious influence on their physiological response.
3.2 Arsenic and Cd uptake by plants
3.2.1 Uptake and transport of As and Cd
The As content in rhizoid and frond of P. vittata from intercropping treatments was significantly increased by 10.9 ~ 28.3% (P < 0.05) in contrast with PM, and the PS treatment showed the highest As content in rhizoid (245 mg∙kg− 1) and frond (1339 mg∙kg− 1) of P. vittata. The Cd content in root and shoot of S. alfredii in PS treatment was also significantly enhanced by 13.3% and 25.2% in comparison to monoculture, respectively (P < 0.05) (Table 2). Similar studies have been confirmed that As content in P. vittata rhizoid was significantly improved when intercropping with Morus alba and Broussonetia papyrifera L. (Wan and Lei, 2018; Zeng et al., 2019b), and Cd content in S. alfredii intercropped with pakchoi was significantly improved (Ma et al., 2020). These results may be due to the interactions of root exudates which increased the heavy metal phytoavailability in rhizosphere soil (Yang et al., 2006). Unfortunately, the content of Cd in P. vittata frond and that of As in S. alfredii shoot was very low with the maximum value of 2.00 mg∙kg− 1and 19.0 mg∙kg− 1, respectively. This may be related to that most hyperaccumulators strongly accumulate specific metals (Mahar et al., 2016). The difference in the TFshoot value of As for P. vittata and Cd for S. alfredii between intercropping and corresponding monoculture treatment was not significant. The content of Cd in root and shoot of H. spectabile from intercropping treatment were sightly decreased, while shoot As content was significantly increased compared with HM (Table 2). Though the content of Cd and As in H. spectabile was low, the TFshoot value of Cd was high than 1.0, suggesting that it was effective in Cd uptake. Our previous study has shown that the maximum Cd content in shoots of H. spectabile grown in 5 mgCd∙L− 1 solution reached up to 603 mg/kg with the TFshoot value of 5.62 (Zhou et al., 2020). This may be owing to low soil Cd content in this study (Yang et al., 2018).
3.2.2 Arsenic and Cd accumulation in plants
The As accumulation in fronds of P. vittata was significantly enhanced by 27.5% and 23.4% (P < 0.05) after intercropping with S. alfredii and H. spectabile compared with PM, respectively (Table 3). Similarly, the Cd accumulation in S. alfredii from PS treatment was significantly increased by 14.6% (P < 0.05) in comparison to monoculture. Previous studies also have been reported that intercropping can enhance As accumulation in P. vittata intercropped with wood species of Morus alba L. or Broussonetia papyrifera L. (Zeng et al., 2019b). Cao et al. (2020) have demonstrated that co-planting with oilseed rape can improve Cd phytoextraction of S. alfredii due to reducing intra-species competition for nutrients and water. The As accumulation of H. spectabile from PH treatment was significantly higher than monoculture (P < 0.05). It is possible that more root exudates (organic acids) secreted between intercropped plants, which could increase the phytoavailable As and Cd contents in rhizosphere soil and promote heavy metal extraction by corresponding plant (Kim et al., 2013, Li et al., 2019). Generally, the As accumulation from PS and PH treatments were close with 2065 and 1988 µg∙pot− 1, respectively, and the Cd accumulation for PS (397 µg∙pot− 1) was far higher than that from PH (19.0 µg∙pot− 1) treatment, suggesting that the Cd removal was more effective when P. vittata intercropped with S. alfredii than H. spectabile. Thus, intercropping of P. vittata and S. alfredii may be more effective in simultaneous phytoextraction As and Cd in co-contaminated soil.
3.3 Soil pH, available contents of As and Cd after remediation
The rhizosphere soil pH was slightly varied from 7.84 to 7.99 (Fig. 2), which was coincided with previous findings that soil pH under the intercropping of P. vittata and castor bean slightly altered (Yang et al., 2017), suggesting that acidification is not the mobilization mechanism of soil metals in present study (Liang et al., 2019). The available As content in PS-S rhizosphere soil was significantly higher than that from unplanted soil (CK) and other planting treatments except for PS-P and PH-H rhizosphere soil. The available Cd content in SM and PS-P rhizosphere soil was higher than that in HM, PH-P and PH-H rhizosphere soil, indicating that intercropping P. vittata and S. alfredii could enhance As and Cd mobility in soil (Fig. 2). Organic acids secreted by hyperaccumulators could facilitate the transformation of heavy metals to an exchangeable fraction thus indirectly resulting in high phytoremediation efficiency (Zu et al., 2020). Previous researches have been confirmed that organic acids like oxalic acid as a predominant root exudate of both S. alfredii and P. vittata could trigger soil As and Cd availability and enhance uptake by plant (Tao et al., 2016; Das et al., 2017; Liang et al., 2021). Also, Xia et al. (2018) have reported that the secretion components in soil from intercropping systems with Conyzacanadensis, Cardaminehirsuta, and Cerastiumglomeratum are significantly more complex than those from monoculture treatments, which have effects on heavy metal accumulation. Therefore, further research is needed to investigate organic acids in the intercropping system of P. vittata and S. alfredii. Additionally, the available As content in HM rhizosphere soil and that of Cd in PH-H rhizosphere was lower than that from other treatments, which might explain the results of lower As and Cd content in H. spectabile (Table 3).
3.4 Soil microbial diversity and community structure
3.4.1 soil bacterial community diversity
Generally, intercropping enhanced the soil bacterial community diversity. The bacterial α-diversity indices of ACE, Chao 1 and Shannon in intercropping treatments were significantly more than in SM and HM (Table S2), which could help to maintain the stability of soil microbial structure, indicating that these corresponding species could be intercropped. There were 590 OTUs common to CK and planting treatments (Fig. S1a). The PM, SM and PS treatments shared higher OTUs of 646, and 632 OTUs common to PM, HM, and PH treatments was found (Fig. S1b, S1c). Though the unique OTUs in CK and planting treatments was very low, which from intercropping treatments were higher than monoculture. This agreed with former research that the microbial composition from the intercropping system of P. vittata with M. alba or B. papyrifera was more complex (Zeng et al., 2019a). Different plant has distinct rhizosphere soil condition due to its own spectrum and specificity of root exudates (Deng et al., 2018). Bian et al. (2021) have found that acetic acid, malic acid, and n-hexadecanoic acid were closely correlated with multiple bacterial species in rhizosphere for intercropping system of Moso bamboo with Sedum plumbizincicola.
3.4.2 Bacterial community structure
The phylum Proteobacteria was the dominant bacterial community in all treatments, accounting for 31.0-36.4% of total phyla in soil (Fig. 3a). The abundance of Proteobacteria was increased in intercropping system compared with monoculture, and that in S. alfredii rhizosphere soil of PS treatment was highest. Similar result has been reported that Proteobacteria predominated in bacterial phyla with more than 31% relative abundances from intercropping treatment with Moso bamboo and Sedum plumbizincicola (Bian et al., 2021). Proteobacteria was important in resistance to metal(loid)s toxicity and significantly correlated with soil nutrients such as carbon, nitrogen, phosphorus contents (Zhang et al., 2016; Zhao et al., 2019). These results suggested that Proteobacteria could help plants increase the environmental quality and intensify the biological function of metal(loid) contaminated soil, in turn the presence of plants could provide a better living condition for microorganisms. Additionally, the other predominant phyla included Actinobacteria, Chloroflexi, Acidobacteria, Bacteroidetes and Gemmatimonadete, which was in consistent with prior research reported by Chen et al. (2018), and they are important for the microbial community reconstruction in metal(loid)-contaminated soil (Zhai et al., 2020).
The genera Massilia, Lysobacter and Sphingomonas, belonging to the phylum Proteobacteria, could adapt to extreme soil conditions (Yang et al., 2019; Jiao et al., 2019). Massilia, Sphingomonas, Lysobacter, arthrobacter and norank_c__Subgroup_6 with an average abundance more than 1% were the main genera in soil after phytoremediation (Fig. 3b). Lysobacter can resist the pathogens by generating extracellular enzymes and affect the bacterial behaviors (Expósito et al., 2015; Feng et al., 2019), and Massilia could effectively enhance soil P mobilization (Zheng et al., 2017). In the present study, Massilia in S. alfredii rhizosphere soil and Lysobacter in P. vittata rhizosphere soil of PS intercropping system was the richest genus. Zhang et al. (2018) have found that intercropping of Morus alba L. and Medicago sativa L. had a positive impact on bacterial taxa with soil nutrients cycling such as Bacillus, Bradyrhizobium and Sphingomonas as compared to monoculture. The results may be related to that root exudates of different cropping treatments may alter the bacterial community (Li et al., 2016).
3.5 Relationship between rhizosphere microecological characteristics and phytoextraction efficiency
The redundancy analysis (RDA) showed that the abundance of Massilia, Arthrobacter and Lysobacter were positively correlated with NaHCO3-As and DTPA-Cd content in rhizosphere soil (Fig. 4a). Moreover, significant correlations were found between the abundance of Massilia and Arthrobacte and Cd content, the abundance of Lysobacter and As content in plant tissues, indicating that they may effectively promote plant Cd and As uptake (Fig. 4b). This was in accordance with the findings reported by Rojjanateeranaj et al. (2017). Previous studies also have confirmed that some types of microorganisms such as Arthrobater and Bacillus could enhance the phytoremediation efficiency through alleviating metal toxicity to plant (Ma et al., 2016). Therefore, rhizosphere associated microorganisms could play a crucial in regulating phytoremediation, and further studies on the application of the critical genus of microorganisms in phytoremediation with intercropping of P. vittata and S. alfredii/H. spectabile are warranted.