Arbuscular mycorrhizal fungi enhanced the growth, phosphorus uptake and Pht expression of olive (Olea europaea L.) plantlets

Plant materials and cultivation conditions

Olive cultivar ‘Koroneiki’ was selected since it is one of the most common olive cultivars in Yunnan province with promising performance (Li et al., 2020). The plantlets used in this study were obtained by mist propagation. The semi-woody cuttings of approximately 10 cm long and two pairs of leaves at the top end were selected for the study. The bottom basal end of each cutting was immersed in an ethanol solution containing 5 g of indol-3-butyric acid/L to promote root development. Propagation was performed in an arch tunnel with a controlled glass greenhouse under an air temperature of 20 ± 2 °C, a relative humidity of 55–70%, a range of irradiance of 400–650 µmol photon m⁻² s⁻¹ (Martín et al., 2006). After three months, the rooted cuttings were transferred to 7.5 cm × 11.5 cm plastic pots filled with growing medium.

The growing medium was a mixture of 50% peat moss and 50% vermiculite (v/v) with a pH of 6.85, organic matter of 251.83 g/kg, hydrolyzable nitrogen of 406.76 mg/kg, available phosphorus of 43.12 mg/kg, available potassium of 469.38 mg/kg, total nitrogen, phosphorus and potassium of 5.08 g/kg, 0.62 g/kg and 16.57 g/kg, respectively. The growing medium was autoclaved twice for a period of 2 h at 121 °C, with a 24 h interval between the two sterilizations.

AMF and fertilization treatments

The native soil inocula were collected from five orchards located in the representative olive introduction and cultivation areas in Yunnan Province. The five growing sites sampled were coded AMF1∼AMF 5. AMF1 was sampled from the orchard of Olive Cultivation Technology Extension Base of Diqing Forest Institute of Yunnan Province. AMF2 from the orchard of Yunnan Lvyuan Industrial Development Company Limited. AMF3 from the orchard of Chuxiong Xinyuan Biotechnology Company Limited. AMF4 from the orchard of Lijiang Tianyuan Olive Technology Development Company Limited. AMF5 from the orchard of Lijiang Sanquan Olive Industrial Development Company Limited. For specific location information, please refer to Table S1. The spore numbers of AMF1 to AMF5 were 350, 121, 82, 198 and 116 in 20 grams of soil. The dominant AMF species of AMF1, AMF2, AMF3 and AMF5 were Funneliformis geosporum, that of AMF4 was Septoglomus constrictum (see the Table S1), the detailed AMF communities characterization was reported in our previous work (Jing, Mao & Li, 2022). A commercial AMF inoculant (coded AMF6) was assayed as a reference alongside the native soil inocula. AMF6 was produced in the form of granules, spores and roots, viz. Rhizophagus intraradices, F. mosseae, R. aggregatus and Claroideoglomus etunicatum (without other additives). Three fertilizer treatments were used here to compare their effects with AMF inocula, namely, Fert1 (3 gram/pot of compound (15:15:15) fertilizer), Fert2 (3 gram/pot of earthworm manure, whose main components include 56.2% organic matter, 1.78% total nitrogen, 1.67% total phosphorus, 1.02% total potassium, pH 7.66) and Fert3 (Fert1 + Fert2). Plantlets with neither inoculation nor fertilizer application were used as the control (CK). Six repetitions were performed for each treatment. Inoculation was carried out at the time of transferring the rooted cuttings into the containers. Three grams of inoculum was deposited directly below the roots of the rooted cuttings.

To expose the cuttings in every treatment to an equal nutritional level, for the AMF1 treatment, 3 g of sterilized rhizosphere soil from AMF2 to AMF5 were added to the pot, and for AMF2, 3 g of sterilized rhizosphere soil from AMF1, AMF3 to AMF5 were added to the pot, and so on. For the AMF6, Fert1, Fert2, Fert3 and CK treatments, the rhizosphere sterilized soil of AMF1 to AMF5 was added to the pot, at the rate of 3 g each.

The plantlets were grown in the greenhouse of the Yunnan Academy of Forestry and Grassland and watered manually with tap water The frequency of watering depended on the environmental conditions prevailing inside the greenhouse, generally once a week. After six months of growth, the shoots and roots of the olive plantlets were harvested separately.

Detection of AMF colonization

The roots were first washed with tap water, and randomly collected root segments were then cleared with 10% (w/v) KOH at 90 °C in a water bath for approximately 60 min. After cooling to room temperature, root samples were thoroughly washed with tap water, stained with blue ink (Hero^® 203; Hero, Shanghai, China), mounted on microscope slides and then examined under a compound light microscope (Olympus-BX53; Olympus Corporation, Tokyo, Japan) for the presence of AM fungal structures. The percentage of root length occupied by hyphae, arbuscules and vesicles was quantified on each sample by a modified line intersection method (McGonigle et al., 1990). At least 200 intersections per root sample were examined. Four replicates were performed for each treatment and control.

Plantlet growth measurements

Growth parameters were measured for all plantlets, including height, root collar diameter, and above- and belowground biomass. The dry weights of the shoots and roots were recorded after oven-drying at 70 °C until they reached a constant mass. The foliar nutrient concentration was determined on dried material in the tested plantlets. Dried leaves were milled and passed through a 0.25 mm sieve, and a sample of 0.5 g of leaf powder was taken for digestion with H₂SO₄ and H₂O₂ (v/v) 1:4. Nitrogen, P and K contents were determined by the Kjeldahl Method (Foss Kjeltec 8400; Foss Analytics, Hillerød, Hovedstaden, Denmark), ultraviolet–visible spectrophotometry (Hitachi U-5100; Hitachi, Tokyo, Japan) and atomic absorption spectrophotometry (Hitachi Polarized Zeeman Atomic Absorption Spectrophotometer ZA3000; Hitachi, Tokyo, Japan), respectively. In addition, the ratio of root:shoot growth was calculated to reflect the robustness of the plantlets and the efficiency of AMF inoculation (Tobar, Azcón & Barea, 1994). Six replicates were performed for each treatment and control.

qRT–PCR of phosphate transporter genes

The wild olive assembly and annotated genome (Unver et al., 2017) were used as queries for phosphate transporter protein genes (Pht). Firstly, we use “phosphate transporter” as the keyword to search target genes in the wild olive genome of Olea europaea var. sylvestris (RefSeq assembly accession: GCF_002742605.1), then obtained the name and location information of target genes, found the corresponding protein sequence. Secondly, we got 30 gene members of the three phosphate transporter gene families based on the verifying of sequence length and function annotation information. Thirdly, we randomly selected several genes from each gene family as representatives, synthesized primers and amplified them. Finally, four olive Pht genes with good effect were selected to analyze their gene expression level in this study. According to the annotation and retrieval results of the wild olive genome, they are named OePht1;11, OePht1;2, OePht1;4 and OePho3 respectively. The four olive Pht studied here refer to Table S2.

To understand the homology of the four olive Pht genes with other known crop ones, a phylogenetic tree was constructed using their DNA sequences together with 11 known Pht genes ones in other crops, MtPHT2;1, MtPT1 and MtPT4 from alfalfa [Medicago truncatula] (Harrison, Dewbre & Liu, 2002; Versaw & Harrison, 2002), LePT3 and LePT4 from tomato [Lycopersicon esculentum] (Xu et al., 2007), StPT1, StPT3 and StPT4 from potato [Solanum tuberosum] (Nagy et al., 2005), OsPT11 and OsPT13 from rice [Oryza sativa] (Paszkowski et al., 2002; Yang et al., 2012) and ZmPT6 from maize [Zea mays] (Wright et al., 2005). All 15 nucleotide sequences representing 6 species were aligned in MAFFT program, then the phylogenetic tree was constructed with MrBayes under the General Time Reversible (GTR) nucleotide substitution model and 10,000 burn-in length in Geneious Prime 2020.0.5 software.

Total RNA of olive roots was isolated using an RNAprep Pure Plant Kit (Tiangen, China). DNase was used to eliminate the potential trace of genomic DNA in RNA samples. Then, 1.0% agarose gel and a NanoDrop ND-2000 spectrophotometer (Thermo Fisher, USA) were used to evaluate and quantify RNA, respectively. RNA samples were reverse-transcribed into cDNA with the FastKing RT Kit (Tiangen, Beijing, China), and synthesized cDNAs were used as templates for qRT–PCR with the SuperReal PreMix Plus Kit (Tiangen, China). O. europaea translation elongation factor-1 alpha (OeEF1α) served as the internal control for the qRT–PCR (Ray & Johnson, 2014). All the primer sequences are listed in Table 1.

A 20 µL reaction solution containing 1 µL cDNA (20 ng), 1 µL of each primer (10 µM), 10 µL SYBR Green I Master mix reagent (Tiangen) and 7 µL ddH₂O was amplified with a LightCycler96 (Roche, Basel, Switzerland). The qRT–PCR program was as follows: 95 °C for 15 min, followed by 45 cycles at 95 °C for 10 s, 55 °C for 10 s and 72 °C for 10 s. The qRT–PCR was performed with three biological replicates, and the data are shown as the mean ± SD. The relative transcript level was calculated using the 2-^ΔΔCt method (Ray & Johnson, 2014). Three biological replicates were performed for each treatment and control.

Statistical analysis

Values of mycorrhizal parameters were summarized for each native and commercial AMF using some descriptive statistics viz. mean, standard error (SE). Data related to olive plantlet growth parameters per treatment were visualized using boxplots. Differences in AMF colonization intensities, plant growth parameters, the leaf concentrations of N, P and K between treatments, the expression level of Pht genes, were analyzed using one-way analysis of variance (ANOVA), comparisons among mean values in each treatment were made using the Tukey’s test (HSD) (p < 0.05). The significance level adopted for all statistical tests was of 5% and the SPSS Statistics 24 (SPSS Inc., Chicago, IL, USA) was utilized for the statistical analysis. Boxplot diagrams were drawn using the R package (R Core Team, 2019). All values were reported as means and standard errors.

Root colonization by AMF

No mycorrhizal colonization was detected, as expected, in the roots of plantlets under fertilizer application (treatment Fert1, Fert2 and Fert3) and noninoculated ones (treatment CK). Except for AMF4, all other treatments showed similar or higher root AMF colonization percentages compared with the commercial AMF inoculum (AMF6). The percentage of AMF hypha colonization in roots of olive plantlets ranged from 40.20% in AMF4 to 93.78% in AMF3, 3.32% (AMF4) to 42.46% (AMF5) for the arbuscule colonization, 2.07% (AMF4) to 49.56% (AMF5) for the abundance of vesicles (Table 2). Some typical structures of AMF colonizing the roots of olive plantlets are presented in Fig. 1.

Effect of AMF inoculation on the growth of olive plantlets

Six months after inoculation, all AMF inoculation treatments had a positive influence on plant growth in terms of height, diameter, biomass and leaf area (Fig. 2). Compared to the noninoculated CK, the commercial AMF inoculum increased the shoot and root fresh weights of plantlets by 66.27% and 91.90%, respectively. However, the native AMF inocula achieved higher effects (Fig. 2).

AMF3 showed the highest effect on aboveground growth, and the shoot dry weights of inoculated plantlets were 2.16 times, 1.41 times, 2.02 times, 1.38 times and 1.24 times those of the CK, Fert1, Fert2, Fert3 and AMF6 treatments, respectively. Whereas AMF1 showed the highest effect on enhancing the belowground growth of the plantlets, the plantlets inoculated with AMF1 had root dry weights 2.84 times, 2.17 times, 2.71 times, 2.19 times and 1.35 times those of treatment CK, Fert1, Fert2, Fert3 and AMF6 treatments, respectively. The root to shoot ratios of olive plantlets inoculated with AMF1, AMF2, AMF3, AMF4, AMF5, AMF6, Fert1, Fert2, Fert3 and CK were 0.64, 0.52, 0.33, 0.48, 0.49, 0.44, 0.34, 0.35, 0.37 and 0.39, respectively.

Effect of AMF inoculation on the N, P, and K contents in olive plantlets

The leaf nitrogen (N) contents of all the AMF inoculated plantlets were lower than the CK, except AMF2, which had a similar value to the control. Fertilization significantly increased the foliar N content compared with AMF and CK (Fig. 3A). The foliar P contents of olive plantlets inoculated with AMF1 and AMF2 were significantly higher than those of all other treatments (Fig. 3B). AMF inoculation marginally enhanced the potassium (K) uptake of the plantlets, and the leaf K contents of plantlets treated with AMF1 to AMF6 were 8.34%, 15.54%, 9.33%, 12.07%, 9.20% and 16.29% higher than those of the control; however, the differences were not significant (Fig. 3C).

Effect of AMF inoculation on expression levels of phosphate transporter genes

A phylogenetic tree was constructed using a multiple DNA sequence alignment of four olive Pht genes and ones of other plants (Fig. 4). The four olive Pht genes were clustered into three main groups, OePht1;11 and OePht1;2 were clustered together with PT4 genes of tomato (LePT4), potato (StPT4) and alfalfa (MtPT4); OePho3 and OePht1;4 were individually clustered into a separate group.

We characterized the expression level of the four Pht genes in olive roots inoculated with AMF or fertilized with compound fertilizer and earthworm manure (Fig. 5). One member, Pht1;11, exhibited strong expression in olive roots inoculated with AMF, especially in the plants treated with AMF6, which of expression level jumped as much as 3400-fold compared to the noninoculated control. Pht1;4 was only expressed at high levels in the AMF1 treatment. The low levels of Pht1;2 transcripts in both the AMF and fertilizer treatments were completely different from those in the control. The expression level of the Pho3 gene responded positively to inoculation and fertilization, but fertilization seemed more effective.