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Introduction
Plants require both macronutrients and micronutrients, with the former being very important in determining the quality of plant growth and development. In general, adding macronutrients to plants can increase crop yields and the overall quality of plant production (Monib et al., 2023). Unlike macronutrients, plants require only a small amount of micronutrients, but these nutrients are essential for vital cell function (Denton-Thompson & Sayer, 2022). The low availability of essential plant nutrients and the toxicity of nutrients in the soil can hurt plant production (Hashimi et al., 2023). The yield and quality of horticultural plant species can also be limited by excess uptake of potentially toxic metal cations in soil, such as when plants are grown in polluted substrates.
Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with the roots of nearly all land-dwelling plants, increasing growth and productivity, especially during abiotic stress. AMF improve plant development by improving nutrient acquisition and mineral uptake. AMF improve plant tolerance and resilience to abiotic stressors such as drought, salt, and heavy metal toxicity (Wahab et al., 2023).
Generally, the response in quality of plant growth to AMF colonization differs markedly with host plant species. Some species usually show positive plant growth, while others respond with decreased growth or show no effect. For example, in carrot (Daucus carota subsp. sativus), a positive effect on the growth of the host was shown due to the inoculation of Funneliformis mosseae, Acaulospora longula, and Scutellospora heterogama (Kim et al., 2017).
AMF have negative and positive impacts on plants depending on environmental conditions. In low water environmental conditions, plant height was surprisingly repressed by AMF inoculation of the nursery shrub species Artemisia ordosica. The adverse effects of AMF on plant growth at low water and nutrient levels may indicate that, under these conditions, AMF inoculation does not strongly contribute to nutrient and water acquisition (Wang et al., 2023). A positive response of host plants to mycorrhizal colonization regarding metal uptake has also been previously reported. AMF can form a mutualistic symbiotic relationship with many plants. Application of AMF to plants can increase nutrient uptake for the host plant. In this case, AMF can mobilize insoluble nutrients in the soil, especially phosphorus (Zhang et al., 2022; Lu et al., 2023).
The response of plants to AMF inoculation varies depending on the length of the plant roots and the genes in each plant species and also depends on soil conditions such as water stress, the amount of metal in the soil, and other environmental conditions (Yang et al., 2023b). Yang et al. (2023a) reported a significant correlation between mycorrhizal colonization rate and Zn absorption efficiency in winter wheat roots during the post-anthesis period. However, there was no significant correlation during the pre-anthesis period. Additionally, AMF application reduced the adverse effects of salinity stress by increasing the uptake of micronutrients such as Zn, Fe, Cu, and Mn while regulating the uptake of Na (decrease) and K (increase) (Huang et al., 2023). Glomus fasciculatum, an AMF, alleviates the adverse effects of Pb, As, and Ni and improves the growth parameters of Monarda citriodora Cerv. ex Lag (Luxmi et al., 2023). The presence of fungal symbionts can increase the tolerance of A. vulneraria to heavy metal stress and help plants adapt to extreme conditions in calamine tailings (Sujkowska-Rybkowska et al., 2023).
On the other hand, phosphate-solubilizing bacteria (PSB) have been shown to increase the availability of soil P fractions, thereby promoting plant growth (Long & Wasaki, 2023). Phosphate-solubilizing soil microorganisms can make soil-insoluble phosphate bioavailable for plants through solubilization and mineralization (Timofeeva et al., 2022). In addition, applying organic amendments with PSB could further ameliorate soil conditions for sustainable management of immobilized nutrients in calcareous soils (Adnan et al., 2022). PSB can also improve plant growth by supplying P for the plant and improving heavy metal mobility in soils and enhancing phytoextraction. Even PSB, with the peripheral direct oxidation pathway, was likely to have a high Cd-mobilizing ability (Yang et al., 2018).
PSB-assisted phytoextraction can enhance the removal of toxic metals by plants from contaminated soil (Yuan et al., 2022). A recent review stated that a combination of rock phosphate with PSB inoculum might be preferable to minimize the risk of long-term total P soil deficit (Kalayu, 2019).
It is expected that the combination of vesicular arbuscular mycorrhizae fungi (VAMF) and PSB in heavy metal-contaminated soil will increase the survival of plants that grow in such soils. The objective of this study was to compare the response of vegetable host plants (tomato, red chili, and hot chili) to a different type of inoculant (control, VAMF, PSB, and co-inoculation VAMF-PSB) under heavy metal-contaminated soil conditions. Three vegetable plant species were also compared in the uptake of the essential elements (Zn and Cu) and the non-essential elements (Cd and Ni) and the overall effect on plant growth or survival. These findings will be essential to finding alternative solutions to the excessive heavy metal content in the soil problem.
Materials and methods
Soil treatment
An ultisol soil with low P (H2O-extractable P = 1.9 mg∙kg-1) from a garden area in Ketintang, Surabaya, East Java - Indonesia, was pasteurized twice at 80 °C for 1,440 min to eliminate the indigenous mycorrhizal fungi, with cooling at room temperature for one day between the two periods of heating. Two weeks before the experiment, every metal and nutrient was combined with the soil, with its moisture kept at 15 %, then incubated at room temperature until the experiment commenced.
Nutrients were applied in the form and concentration as follows (per 1 kg of soil): 200 mg N (NH4NO3), 20 mg P [Ca (H2PO4)2], 100 mg Mg (MgSO4), 200 mg K (K2SO4), and 10 mg Fe (NH4-Fe-citrate). In addition, four different metals were supplied (per 1 kg of soil): 2 mg Zn (ZnSO4), 2 mg Cu (CuSO4), 2 mg Ni (NiSO4), and 0.2 mg Cd (CdSO4) (Kloke, 1980; Lee, 2005). Tomato (Solanum lycopersicum L. cv. Ratna), red chili (Capsicum annuum L. cv. Gada), and hot chili (Capsicum frutescens L. cv. Pusaka Brengolo) were used. The VAMF were from G. mosseae isolate retrieved from SEAMEO-BIOTROP, Bogor, Indonesia. The fungi were applied in the soil as spores combined with soil. As much as 0.01 kg of soil with 20-25 spores of VAMF were applied to each experiment pot before plant treatment.
Plant treatment and experimental design
Seeds were sterilized with 10 % H2O2 for 5 min and then pregerminated for 240 min in saturated CaSO4 (Lee, 2005), and then 20 mL of PSB B. subtilis (Universitas Negeri Surabaya Laboratory collection), at a concentration of 109 CFU∙mL-1, was applied before the seed was sown directly into the pot. The pots had a 5 L volume and were filled with 3 kg of soil. After germination, plants were thinned to two.
The experiment was set up in a one-factor, randomized complete block design. Experimental treatments included the following: 1) control (non-inoculation), 2) inoculated with VAMF, 3) inoculated with PSB, and 4) co-inoculated (VAMF-PSB). Four treatments with four replicates gave 16 pots for each vegetable plant species. Plants were grown in a greenhouse (day min/max temperatures of 28/32 °C; max light intensity of approximately 34,000 lux).
Plant analysis
After eight weeks of growth, roots and shoots were harvested to determine the root colonization by mycorrhizae and to analyze the plant dry weight (Chandrasekaran, 2022) and nutrient (N, P, Zn, Cu, Cd, y Ni) concentrations. The percentage of root length colonized by VAMF was determined on roots stained in trypan blue using the gridline-intersect method (Kumar & Tapwal, 2022). P and N concentrations were determined by UV-Vis spectrophotometry and by the Kjeldahl method, respectively. The metal (Zn, Cu, Ni, and Cd) concentrations were measured using an atomic absorption spectrophotometer (Bhandari, 2018).
Statistical analysis
Data were shown as the mean ± standard deviation of replicates. The data were checked for normality and homogeneity of variance before performing the analysis of variance using SPSS (IBM SPSS Statistics 23). One-way analysis of variance followed by Duncan’s multiple-range test (P ≤ 0.05) was performed to compare the means of root colonization, plant biomass, P and N concentrations, and the metal (Cu, Zn, Cd, and Ni) concentrations for each vegetable plant species.
Results
Root colonization
Table 1 shows that co-inoculation (VAMF-PSB) resulted in significantly higher root colonization than the rest of the treatments in all crops.
Table 1 Root colonization in vegetable plants grown in heavy metal-contaminated soil.
| Treatments | Root colonization in vegetable plants (%) | |||||||
|---|---|---|---|---|---|---|---|---|
| Tomato | Red chili | Hot chili | ||||||
| Mean ± SD | CV | Mean ± SD | CV | Mean ± SD | CV | |||
| Control | 4.9 ± 0.52 cz | 10.5 | 5.9 ± 0.28 d | 16.9 | 5.3 ± 0.36 d | 6.9 | ||
| VAMF | 31.1 ± 0.70 b | 2.3 | 43.0 ± 1.27 c | 3.0 | 41.7 ± 1.73 b | 4.2 | ||
| PSB | 29.8 ± 0.99 b | 2.6 | 51.7 ± 0.56 b | 0.7 | 38.7 ± 1.56 c | 4.0 | ||
| VAMF-PSB | 43.1 ± 1.41 a | 3.3 | 57.8 ± 2.05 a | 3.6 | 51.9 ± 2.01 a | 3.9 | ||
VAMF = vesicular arbuscular mycorrhizae fungi; PSB = phosphate-solubilizing bacteria; SD = standard deviation; CV = coefficient of variation. zMeans with the same letters within each column do not differ statistically (Duncan, P ≤ 0.05).
Plant biomass
Root colonization by VAMF significantly increased the plant biomass of the three vegetable species compared to control plants (Table 2).
Table 2 Plant biomass in vegetable plants grown in heavy metal-contaminated soil.
| Treatments | Plant biomass in vegetable plant (g) | |||||||
|---|---|---|---|---|---|---|---|---|
| Tomato | Red Chili | Hot Chili | ||||||
| Mean ± SD | CV | Mean ± SD | CV | Mean ± SD | CV | |||
| Control | 3.10 ± 0.29 cz | 9.5 | 2.92 ± 0.22 c | 7.6 | 2.72 ± 0.25 b | 9.2 | ||
| VAMF | 4.10 ± 0.18 b | 4.5 | 4.57 ± 0.25 b | 5.5 | 3.90 ± 0.21 a | 5.5 | ||
| PSB | 4.28 ± 0.21 b | 5.0 | 4.80 ± 0.18 b | 3.8 | 3.87 ± 0.18 a | 4.9 | ||
| VAMF-PSB | 5.65 ± 0.31 a | 5.5 | 5.87 ± 0.29 a | 5.1 | 4.10 ± 0.36 a | 8.9 | ||
VAMF = vesicular arbuscular mycorrhizae fungi; PSB = phosphate-solubilizing bacteria; SD = standard deviation; CV = coefficient of variation. zMeans with the same letters within each column do not differ statistically (Duncan, P ≤ 0.05).
P and N concentrations
P and N concentrations in VAMF plants, PSB plants, and plants co-inoculated were increased compared to the control (Figures 1 and 2). In all plant species, plant P concentrations in the shoot and root indicated a sufficient supply of P to plants (Figure 1). However, N concentrations in the shoot and root (Figure 2) indicated a deficient status compared to standard values of adequate nutrition (Bergmann, 1992; Balemi & Negisho, 2012; Frydenvang et al., 2015).
Figure 1 P concentrations for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Figure 2 N concentrations for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Although the N concentration increased for the VAMF, PSB, and VAMF-PSB combination treatments, the N concentration was still relatively low compared to the standard value of N concentration (Figure 2).
Heavy metal concentration
Although the application of VAMF, PSB, or VAMF-PSB could decrease the Cd and Ni concentrations in the shoot of three plant species, the concentrations of Cu, Zn, Cd, and Ni were higher in the root than shoot of three plant species (Figures 3, 4, 5, and 6). Inoculation of VAMF-PSB significantly increased Zn concentration in the root compared to the other treatments (Figure 4a). It led to a significant increase in Zn concentration in the shoot in tomato. VAMF-PSB co-inoculation did not have any effect on Cd content in roots (Figure 5a). However, each co-inoculation treatment decreased the Cd content in the shoots of every plant compared to the control (Figure 5b).
Figure 3 Cu concentration for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Figure 4 Zn concentrations for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Figure 5 Cd concentrations for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Figure 6 Ni concentrations for three vegetable species treated without inoculation (control), inoculated with vesicular arbuscular mycorrhizae fungi (VAMF), inoculated with phosphorus-solubilizing bacteria (PSB) and co-inoculated (VAMF-PSB): a) roots and b) shoots. dw = dry weight; bars represent standard deviation. Columns with the same letters within each vegetable species do not differ statistically (Duncan, P ≤ 0.05).
Discussion
Sun et al. (2018) reported that Cd concentrations in roots were higher than those in shoots of all cultivars, regardless of inoculation. However, the AMF function has a different effect on Cd accumulation in upland and lowland cultivation. Zhao et al. (2023) reported that inoculation with two AMF species (G. intraradices and G. mosseae) significantly elevated Cd concentrations in the contaminated land areas due to the collapse of tailings dams. However, they reduced the Cd concentration in the upper layers of the tailings pond, while in the downwind area around the smelter, which was contaminated by serious smoke and dust pollution, inoculation with G. mosseae significantly decreased the Cd concentration. AMF promoted plant Cd tolerance and detoxification by enhancing P uptake, Cd passivation, Cd retention in the cell wall, and functional group modulation (You et al., 2022). Gao et al. (2023) evaluated the effect of AMF on cereals established under controlled greenhouse conditions, observing a significant reduction (between 5.14 and 33.6 %) in Cd stress. This was achieved by enhancing osmotic regulators (soluble proteins, sugars and total prolines, from 14.8 to 36.0 %) and by reducing the membrane lipid peroxidation product (MDA, 12.9 %).
Narwal et al. (2018) reported a 32.4 % higher total uptake of Zn in rice plants treated with AMF than in untreated control plants. The colonization of AMF significantly increased Zn uptake. A similar outcome was observed in a study by Zaheer et al. (2019), where applying PSB increased Zn uptake in plants. These findings are consistent with the results of our study, as shown in Figure 4a, where the Zn content in roots was elevated with VAMF and PSB inoculation, leading to increased Zn content in tomato shoots.
Brunetto et al. (2023) revealed that pre-inoculation with selected AMF (Rhizophagus clarus X UFSC 14 and Rhizophagus intraradices X UFSC 32) significantly reduced the Cu concentration after a long period of exposure. In addition to AMF, PSB has a similar ability to improve critical metal concentrations, such as the concentration of Cu. Lin et al. (2018) also reported that the removal efficiency of Cu from the soil by Wedelia trilobata increased with an increase in the concentration of PSB, and the translocation factors of Cu (i.e., leaf:root and stem:root) were both significantly upregulated by PSB.
In line with these reports, we also found an increased concentration of Zn and Cu due to mycorrhizal colonization and PSB in all plant species tested under heavy metal-contaminated soil compared with inoculation with VAMF or PSB. For Cd, however, many contradictory results have been reported. Some reports found that mycorrhizal infection increases root Cd2+ uptake (Han et al., 2021). AMF, in turn, increase corn Cd uptake, which is closely related to the influence of AMF on root morphology and LMWOA (low molecular weight organic acid) secretion, with varying effects at different levels of Cd pollution (Chen et al., 2022). Others found that AMF reduces Cd leaching from sand columns by reducing availability and enhancing uptake by maize roots (Yu et al., 2022).
Yang et al. (2018) stated that PSB administered to contaminated soil enhanced Cd uptake by plants. You et al. (2021) reported that AMF alleviate the toxic effects of Cd on P. australis by increasing plant biomass and through different regulatory patterns under different toxic trace elements concentrations. As is known, Cd can potentially be a hazardous heavy metal that can be toxic for plants even at deficient levels (0.5 μg Cd∙g-1 soil) (Zhi et al., 2020). The generation of reactive oxygen species (ROS) could be induced by Cd and cause peroxidation of lipids and, consequently, increase the permeability of membranes (Su et al., 2019). Nafady and Elgharably (2018) reported that Cd concentrations in the shoot decreased with inoculation of native AMF G. aggregatum; however, the Cd concentration increased with an injection of G. intraradices.
The availability of additional P can reduce the Cd concentration in plant shoots due to the interaction between P and Cd in the uptake process by plant roots (Zhao et al., 2020). Figure 1 shows that the P concentration increased significantly due to co-inoculation with VAMF-PSB compared to the other treatments, coinciding with a decreasing Cd concentration in the shoot. From these results, it can be suggested that the increase in P availability due to VAMF-PSB application in heavy metal-contaminated soil could minimize the uptake of heavy metals except in hot pepper, positively affecting plant growth and, as a consequence, plant survival.
In the present study, we found a higher Cd concentration in the control plants than in the inoculated ones. We also found higher Cd and Ni concentrations in the root compared with the concentration of Cd and Ni in the shoot (Figures 5 and 6). Kuang et al. (2023) reported that AMF inoculation significantly increased the root’s Cd concentration while significantly reducing the shoot’s Cd concentration.
Generally, the inoculant type affects the N concentration in roots and shoots with a similar pattern to the Cd concentration (Figure 5). The VAMF function and PSB regulation were influential because there was a significant increase in root Ni (Figure 6a). On the other hand, Ni cannot be accumulated in the shoot (Figure 6b). The protective effects of VAMF colonization against metal toxicity may be due to heavy metal accumulation in mycorrhizal roots and reduced translocation from root to shoot by immobilizing metals in fungal hyphae in roots. VAMF can enhance the uptake of nutrients effectively since they significantly increase the P concentration (Table 2) in many crops in low-phosphorus soil. The combination of VAMF and PSB can improve P uptake by a plant that grows in heavy metal-contaminated soil (Figure 1). Wu et al. (2019) stated that PSB inoculation treatments could improve leaf N and P content and had positive effects on the available N, P, and K content of rhizosphere soil. Consequently, PSB inoculation could improve the acquisition of essential elements (P, Cu, and Zn) and, at the same time, protect the host plant from Cd and Ni toxicity by reducing shoot or root concentration when these metals exist at a high level in the soil.
VAMF colonization resulted in preferential uptake and transfer of some metals depending on the essentiality of the element, e.g., improvement of essential metal acquisition and protection of VAMF against toxic metals, suggesting a possible use in the metal-containing substrate. Tan et al. (2023) stated that AMF mycelium plays a vital role as a metal adsorbent, reducing local metal concentrations in the soil and creating a more suitable environment for plants growing in metal-contaminated soil.
The effect of AMF colonization on metal uptake was independent of the growth-promoting influence in the VAMF plant. Growth response to VAMF colonization differed from plant species irrespective of metal concentration in VAMF plants. However, the metal uptake was similar in the species tested. These different growth responses of host plants were also related to the amount of metal supplied. All plant species tested had an effective response to VAMF colonization for the growth response. This difference in the growth response of host plants to VAMF colonization may be due to differences in colonization rate and growth of hyphae of VAMF by interaction with host plant species, consequently related to the ability of P acquisition. A better colonization rate and development of hyphae in some VAMF and host plant combinations could improve successful P acquisition from the soil and result in higher biomass in host plants.
Under heavy metal-contaminated soil conditions, VAMF association increased the biomass of all three host plant species tested. VAMF improve plant development by enhancing the acquisition of nutrients, such as P, as well as water and mineral uptake. VAMF improve plant tolerance and resilience to abiotic stressors, such as heavy metal toxicity. These benefits come from the arbuscular mycorrhizal interface, which lets fungal and plant partners exchange nutrients, signaling molecules, and protective chemical compounds (Wahab et al., 2023).
VAMF could enhance plant growth (survival) on severely disturbed sites, including ones with heavy metal contamination. VAMF were reported to play an important role in metal tolerance and accumulation.
Conclusions
Plants co-inoculated with VAMF-PSB had significantly higher root colonization by VAMF, and greater P and N concentrations in shoot and root than plants inoculated with only VAMF or PSB. Consequently, the plant biomass also increased.
Cu, Zn, Cd, and Ni concentrations were higher in the roots than in the shoots of the three plant species tested. The type of inoculant used in this study selectively discriminated between plant essential (Cu, Zn) and non-essential elements (Cd, Ni). These findings recommend using VAMF and PSB application in horticultural production in heavy metal-contaminated soils to increase plant survival.
