The maize seeds (Zea mays L.) were Zheng Dan 958 and were surface sterilized with a 10% (v/v) solution of H2O2 for 10 min and incubated at 25 °C for 24 h. The earthworms (Eisenia fetida) were washed with distilled water and kept in sterilized glass vessels for 24 h to reduce the occurrence of naturally occurring mycorrhizal propagules. The AM inoculum (Funneliformis mosseae, BGC HEB02) contained approximately 1000 infectious AM spores per 10 g of soil. The propagule infectivity was tested according to the method of Sharma et al.
There were four treatments, each consisting of four replicates as follows: CK, no addition of earthworms or sterilized AM fungi; EW, addition of earthworms with a sterilized AM fungi inoculation; AM, no addition of earthworms but with AM fungi inoculation; and AE, addition of both earthworms and AM fungi. Plastic pots (23 cm in diameter and 20 cm in height) were filled with 2.7 kg soil (dry weight) mixed with 8.0 g of oven-dried wheat straw. One germinated maize seed was sown in each pot. The mycorrhizal inoculum was placed approximately 2 cm under the soil surface at 2.5 g kg−1, and ten earthworms of similar fresh weights were added when the third leaf emerged. Each pot received a 10 ml soil suspension filtered through 0.45 μm filters (to eliminate mycorrhizal spores) to reintroduce fresh microorganisms. The pots were placed randomly in a greenhouse and incubated at 20/30 °C (day/night) with a photoperiod of 12/12 h of light/dark. Throughout the incubation period, deionized water was added to maintain the soil moisture content at 60% of the soil water holding capacity.
Simulated acid rain treatment: Concentrated sulfuric acid and concentrated nitric acid were mixed at a volume ratio of 3:1, distilled water was adjusted to pH 1.0 as the mother liquor, and then the mother liquor was diluted to pH 3 as the simulated acid rain. The simulated acid rain spraying period was during the seedling stage of the maize. Twenty days after the seeds were sown, acid rain stress was simulated for each treatment at 4 PM every day. The entire plant was sprayed with a spray pot to simulate acid rain. During spraying, the soil surface was covered with kraft paper to prevent the simulated acid rain solution from entering the soil. Deionized water was replenished every two days during the leaf drop limited water test to maintain the water content of the pot soil at 20%. The stress treatment lasted for 15 d, and samples were collected for measurement.
Sampling and analysis
The maize shoots were harvested by cutting at the soil surface. Maize shoot and root biomass were recorded for the different treatments. Aliquots were stored at -80 °C for physiological analysis and root colonization or oven-dried at 65 °C for 72 h for nutrient concentration measurements. The collected earthworms were washed and weighed. The root was carefully removed from each pot, and 200 g undisturbed soil was stored separately in a rigid box and air-dried for soil aggregate analyses. The remaining aliquots were mixed and sieved through 2 mm mesh and were stored at -80 °C for DNA analysis or air-dried for nutrient concentration measurements.
Assays for soil physical and chemical properties
The pH of the soil was determined in a 5:1 water-soil ratio. The soil organic matter was determined using the concentrated sulfuric acid-potassium dichromate oxidation method and titrated with a ferrous sulfate standard solution. The soil Olsen-P was extracted with 0.5 mol L-1 NaHCO3 and determined using the molybdenum-antimony colorimetric method. The soil available potassium was extracted with 1 mol L-1 NH4OAc and determined using flame photometry.
Assays for mycorrhizal colonization rate and mycorrhizal colonization density
The roots of each sample were cut into root segments of 1 cm and digested for 60 min in a water bath of 10% KOH at 90 °C. After cooling, the KOH solution was poured off, and the roots were washed with water and acidified with 2% hydrochloric acid for 5 min. After the hydrochloric acid was poured off, a lactic acid glycerin solution containing 0.05% trypan blue was directly added to the water bath for 30 min; the dye was poured off and the solution of lactate glycerin was added to decolorize the roots. Finally, 30 root segments were randomly selected from the dyed root segments, and the infection rate was measured under a microscope. The calculation method was conducted according to Trouvelot et al (Trouvelot et al., 1986).
Determination of high-throughput microbial community structure
Bacterial 16S rDNA V3-V4 regions were selected for microbial diversity detection, and the DNA samples were sent to Beijing Baimaike Gene Technology Co., Ltd. for sequencing using the Illumina MiSeq PE2500 high-throughput sequencing platform. A PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) was used to extract the DNA. The primers for the amplification of bacterial 16S rDNA V3-V4 were 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). The PCR system included 12.5 μL 2×Taq PCR MasterMix, 3 μL BSA (2 ng/L), 2 μL primer (5 μM), 2 μL primer, and 5.5 μL ddH2O. The reaction parameters were predenaturation at 95 °C for 5 min; denaturation at 95 °C for 45 s, annealing at 55 °C for 50 s, and elongation at 72 °C for 45 s, repeated for 32 cycles. The original sequence was uploaded to the NCBI SRA database after 10 min extension at 72 °C.
Photosynthesis and transpiration assays
The photosynthetic and transpiration rates of the maize leaves were measured using a portable photosynthesis system (model 6400; Li-Cor, Lincoln, NE) at a light intensity of 1800 μmol m-2 s -1 PAR and a constant 350 μbar CO2 partial pressure in the sample chamber. All measurements were performed between 08:00 and 11:00 h. During the measurements, the relative humidity of the air was approximately 75%, the leaf temperature ranged from 25 to 27 °C, and the ambient CO2 concentration was approximately 355 mmol.
Plant hormones and antioxidant enzymes assays
Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IAA, ABA, GA3 and ZR. A 0.50 g sample was weighed and ground into a homogenate in an ice bath with phosphate buffer. The sample was extracted at 4 °C for 4 h and centrifuged at 3500 r for 8 min, and the supernatant was collected. One milliliter of the extract was added for repeated extraction for 1 h and centrifuged. The supernatants were combined and purified using a C18 solid phase extraction column. The product was dried under nitrogen to a constant volume. Standards, samples, and antibodies were added and quantified using an enzyme-linked plague spectral photometer (490 nm). The POD and SOD activities of plant antioxidant enzymes were measured with an antioxidant enzyme kit (Jancheng, Nanjing).
SPSS 17.0 software (SPSS Institute, Inc., Cary, NC, USA) was used to test the homogeneity of variance for all data (Levene's test). Differences between treatments were compared using a one-way ANOVA (P <0.05). The significance of earthworm and mycorrhizal influences on each measurement index was compared using a two-factor analysis of variance.
Paired-end sequencing was performed on an Illumina MiSeq platform. The debarking data were filtered, spliced, and chimeras were removed using QIIME (V1.8.0) software; paired sequences with scores less than 20, base ambiguity, primer mismatch, or a sequencing length less than 150 bp were removed. Based on barcodes, the sequence information from each treatment was grouped into OTUs (operational taxonomic units) for species classification, and OTU similarity was set at 97%. The species classification information corresponding to each OTU was obtained by comparison with the SILVA database.
AMOS 21.0 was used to construct a structural equation model (SEM) to analyze the causal relationship among biological or nonbiological indicators and biomass.