Maize P uptake regulated by earthworms and mycorrhizae in saline soil
It is well known that high salinity decreases plant P uptake and adversely affects crop growth (Sima et al., 2019). In addition to being involved in membrane formation and intracellular metabolism, the increased plant inorganic phosphate (H2PO4−) could enhance salt tolerance by strengthening the protoplasm’s buffering capacity to alkaline pH (KH2PO4 + KOH→K2HPO4 + H2O) (Bental et al., 1988). Previous studies demonstrated that plant growth was significantly inhibited by salt stress, with less biomass under deficient P than sufficient P (Talbi Zribi et al., 2011; Sima et al., 2019). In this study, earthworms, mycorrhizae and their combination significantly increased maize biomass and plant P contents, indicating enhancement of maize P uptake in saline soil. There was a significant positive correlation between maize biomass and plant P content (r = 0.678, p < 0.01), suggesting that earthworms and mycorrhizae could increase plant P uptake to promote maize growth. Our previous study demonstrated that earthworms and AMFs could decrease the salt content, which could be attributed to the decrease in predominant salt ions (i.e., Cl− and Na+) induced by AMF inoculation (Table 1). Additionally, earthworms and AMFs could improve soil structure, which promotes salt leaching (Zhang et al., 2018; Oo et al., 2015). AMF inoculation significantly increased the amount of soil HCO3− along with the salt reduction. This may have been due to the release of CO2 by mycorrhizal roots, resulting in the solubilization of CaCO3 (CaCO3 + CO2 + H2O ⇋ 2Ca2++ 2HCO3−) during the desalting process (Akhter et al., 2003). Earthworms and AMFs increased the maize P content, which was significantly negatively correlated with the soil salt concentration (r = -0.547, p < 0.05, Fig. 7). These results indicated that the decrease in soil salt content induced by earthworms and AMFs could contribute to the P uptake by maize.
Magnesium plays a major role in plant phosphorus absorption and transport and enhances P use efficiency (Grzebis et al., 2018). Calcium could stabilize cell wall structure, regulate ion (K+, Na+ and Mg2+) transport and selectivity and improve plant osmoregulation (Elkelish et al., 2019). Earthworms, mycorrhizae and their combination significantly increased the shoot Mg and Ca contents, which were significantly positively correlated with maize P content (Fig. 8, p < 0.01). Thus, we speculated that earthworms and mycorrhizae probably enhance maize P uptake by increasing Mg and Ca utilization. Tuffen et al. (2002) demonstrated that earthworm activity could enhance 32P transfer by AMF mycelium to leek (Allium porrum L.) in non-saline soil. In this study, the E treatment resulted in the greatest magnitude of increase in maize P, Mg and Ca contents (Fig. 2), indicating that the addition of earthworms did not enhance the capacity of AMFs to transfer these soil nutrients to their host maize. However, earthworms addition increased the hyphal length density by 17.3% compared to that under AMF inoculation alone (Zhang et al., 2018). Therefore, we considered earthworm presence to help AMFs obtain P, Mg and Ca for their own growth instead of providing them to their host plant under salt stress. Consequently, earthworms played a predominant role in improving the P uptake and growth of AMF hyphae and their host plants by enhancing Mg and Ca utilization in saline soil.
Improvement by earthworms and mycorrhizae of soil P availability by activating stable inorganic P
The predominant form of soil P taken up for plant growth and productivity is free phosphate ions (HPO42− and H2PO4−) (Bucher, 2007). However, when applied to the soil, chemical phosphate fertilizer is largely immobilized into hard dissolved forms with low plant utilization efficiency, such as CaHPO4, Ca3(PO4)2, FePO4 and AlPO4(Peak et al. 2012). Soil inorganic P fraction distribution analysis showed that the largest proportions of soil inorganic P across all treatments were Ca-P (48–55%) and Al-P (18–21%) (Fig. 3). This indicates that soil phosphates predominantly existed in the form of calcium phosphate in the high-salinity soil (Khan et al. 2009). During precipitation with Ca, soil P initially generates dicalcium phosphate (Ca2-P), which can be easily absorbed by plants, and Ca2-P can then be transformed into more stable forms, such as octocalcium phosphate (Ca8-P) and hydroxyapatite (Ca10-P), which have limited availability to plants (Shen et al., 2011). Earthworms and AMFs significantly decreased the proportion of soil hydroxyapatite (Ca10-P) and increased the proportion of soil dicalcium phosphate (Ca2-P) (Fig. 3).Simultaneously, the soil Ca10-P proportion was significantly negatively correlated with the soil Ca2-P proportion (Fig. 8, p < 0.05). These results indicate a transformation of the soil stable P form (Ca10-P) into active or available P (Ca2-P) driven by earthworm and AMF combination. Additionally, AMF inoculation significantly enhanced soil phosphatase activity and increased the soil inorganic P fractions (Ca2-P, nonoccluded Al-P, Ca8-P, nonoccluded Fe-P and Oc-P) (Table 1 and Table S1). Soil phosphatase activity was negatively correlated with the soil Ca10-P proportion (Fig. 8, p < 0.01) and positively related to the increased soil inorganic P fractions (r > 0.640, p < 0.01). Therefore, we suggest that mycorrhizae could strengthen soil phosphatase activity to promote stable P (Ca10-P) dissolution and mineralization. Thus, AMF appears to have a dominant role in activating hard dissolved P by increasing soil phosphatase activity under salt stress.
Enhancement of soil phosphatase activity by earthworms and mycorrhizae via altering soil bacteria
To overcome the limitation of bioavailable inorganic orthophosphate, plants and bacteria secrete numerous phosphatases to cleave orthophosphate from complex organic P substrates (Lidbury et al., 2017). In this study, the separate and combined addition of earthworms and AMFs significantly increased the Chao1 and phylogenetic diversity indices (Table 1), which were significantly positively correlated with soil alkaline phosphatase (r > 0.656, p < 0.01). These results suggested that earthworms and AMFs probably increased soil bacterial diversity to enhance soil phosphatase activity. To understand the drivers of soil P availability, an SEM was constructed to jointly investigate the multiple disparate pathways associated with P activation. The SEM provided evidence that soil bacterial diversity was the most important driver of soil P availability, followed by soil Ca2+ concentration and salt concentration (Fig. 9b). This indicated that low bacterial diversity was the key factor limiting soil P activation, while the increase in soil bacterial diversity induced by earthworms and AMFs resulted in increased soil P availability (Zhang et al., 2018). Network analysis could provide new insights into the relationships between soil nutrients and their associated bacteria under complex conditions (Zheng et al., 2018). Our network analysis showed that the change in bacterial diversity was largely dependent on the soil salt concentration and Ca2+ concentration (Fig. 6). Simultaneously, the response of bacteria toCa2+ concentration but not salt concentration had a significant positive correlation with soil P availability.
Identifying the physiological attributes of the dominant bacterial taxa is critical for understanding the microbial controls on some key soil processes, e.g., soil carbon and nutrient cycling (Manuel et al., 2018). Previous studies reported that abundant phyla, including Acidobacteria and Chloroflexi, harboured the phoX alkaline phosphatase gene and participated in organic phosphorus cycling in multiple soil environments (Ragot et al., 2017; Shao et al., 2020). Earthworms and AMFs significantly increased the abundance of the phylum Chloroflexi, and AMFs alone increased the abundance of the phylum Acidobacteria. The abundances of Chloroflexi and Acidobacteria were positively correlated with the soil phosphatase and soil Olsen-P contents (Fig. 7, p < 0.05). Thus, we speculated that earthworms and AMFs may stimulate the growth of Chloroflexi and Acidobacteria to increase soil phosphatase activity and thus enhance soil P availability. LEfSe analysis reveals some predominant phylotypes in smaller taxonomic categories. Xanthomonadales and Oceanospirillaceaespecies were significantly higher in the E treatment than in other treatments (Fig. 5), indicating that earthworms could stimulate these bacteria to participate in P degradation and accumulation (Martin et al., 2006; Sosa et al., 2017). Earthworms and AMF inoculation increased the abundance of Chloroflexi and Anaerolineales (Fig. 5), which are dominant in carbon-rich soil and associated with cellulose hydrolysis (Lian et al., 2017; Pinnell et al., 2014). Simultaneously, the earthworm and AMF combination decreased the abundance of Cytophagale, which is an efficient degrader of complex organic compounds in relatively low-carbon soil (Zhang et al., 2013). The shift inbacterial composition from the low-carbon type (Cytophagales) to the rich-carbon type (Chloroflexi and Anaerolineales) induced by earthworm and mycorrhizal activities suggested an improvement in saline soil nutrient conditions.