Cd tolerance of T. pubescens MB 89 strain
Growth inhibition is a common response of plants to heavy metal stress. Cadmium is one of the most toxic heavy metals that can affect soil quality and reduce plant productivity. Furthermore, the high cadmium rice is a serious threat to food safety and human health. In this work, we used a cadmium-tolerant T. pubescens to investigate its resistance against cadmium stress. The radius growth diameter of the strain in PDA plate were measured every 24 h with different concentration gradient of Cd2+, as shown in Fig. 1. It can be seen that T. pubescens MB 89 grew well at the Cd2+ concentration of 10 mg/L. With the increase of cadmium concentration, the growth of T. pubescens was gradually inhibited. When Cd2+ concentration reached 200 mg/L there was no hypha observed. Cadmium tolerance assay showed that strain T. pubescens MB 89 has remarkable tolerance to cadmium. Accoring to the literature, under solid cultivation, the MICs for Cd2+ of Aspergillus sp. 2 and Penicillium sp. isolated from metal-contaminated soils were 35.59 and 44.48 mM, respectively(Zafar et al., 2007). The MIC of Rhizopus sp. for Cd2+ was 17.8 mM(IQBAL AHMAD, 2005). Figure 1.
Cadmium removal by T. pubescens mycelium
As shown in Fig. 2, the cadmium removal by T. pubescens MB 89 from the aqueous solution was investigated. It is observed that with the initial Cd2+ concentration of 100 mg/L, the Cd adsorption of T. pubescens living mycelium was 6.565 mg/g which was the highest in this experiment. The maximum removal rate of T. pubescens MB 89 was 53.13% when the initial cadmium concentration was 10 mg/L. while a literature reported the removal efficiency of A. aculeatus reached the maximum (46.8%) at 10 mg/L Cd2+ concentrations(Xie et al., 2019). Since this process was affected by the mycelium's cell surface substances and metabolism, the cadmium removal by mycelia in aqueous solution depends on complex factors (Arivalagan et al., 2014). From Fig. 2 it can be seen that biosorption capacity increased with increased initial concentration of Cd2+ before it was saturated. This result was in agreement with the literature in which Cd2+ biosorption capacity of living fungus Pseudomonas sp. 375 was studied (Xu et al., 2020). Figure 2.
SEM analysis
Jacob et al. (Jacob et al., 2017) have reported the alteration of cell wall morphology due to heavy metal stress. Figure 3 Shows the SEM micrograph of Cd treated and untreated (control) T. pubescens MB 89 cells. It confirms that heavy metals were able to inhibit various physiological processes such as cell membrane distribution (Karthik et al., 2016; Khan et al., 2009; Yuan et al., 2015), inhibited cell division, depressed enzyme activity, and caused denaturation of protein (Khan et al., 2009; Wyszkowska et al., 2014). As shown in Fig. 3b, a large number of mycelia were deformed and became fragmented when the cadmium concentration in the medium was too high for the fungus to bear. Even in the shaking bottle, the mycelia originally growing together had fragments falling down. Nevertheless, dense nanoscale particles on the cell surface of mycelium were observed in the low Cd experimental groups (Fig. 3d), while these particles were not seen in the control group (Fig. 3a). This means the dense particles were Cd (0) sediments formed after T. pubescens had adsorbed and reduced Cd (II). The phenomenon is similar to the work with Pseudomonas chengduensis strain MBR (Wang et al., 2020b). The fungal detoxification of heavy metal contaminated environment includes valence transformation, intra and extracellular precipitations as well as active uptake (Thatoi et al., 2014). Typically, the high content of carboxyl groups in the mannuronic and guluronic acids of the cell wall polysaccharides and the protosufficiency enhance heavy metal biosorption (Raja et al., 2016). Figure 3.
FTIR analysis
The FTIR analysis was implemented to verify the metal ion interacting with the functional groups existing on the fungal surface in the wavelength of 500–4000 cm− 1. The FTIR spectra of T. pubescens MB 89 exposed to Cd showed varying asymmetrical stretching bands and peaks in Fig. 4. The major variations in peaks are shown in Table 1. Among them, the stretching vibration peaks of amino and hydroxyl groups were found to shift from 3487.32 cm− 1(control group) to 3419.93 cm− 1 (5 mg/L Cd), 3405.40 cm− 1 (10 mg/L Cd), 3390.31 cm− 1 (50 mg/L Cd) and 3402.99 cm− 1 (100 mg/L Cd), indicating that these hydroxyl groups and amino groups from polysaccharide, fatty acid and protein components were participated in the adsorption process (Zhou et al., 2016). In addition, there is a C-H stretching vibration peak near 2928 cm− 1, and the slight shift of the spectrum after adsorption indicate that C-H of methyl groups was participated in the cadmium adsorption process (Khan et al., 2018). The carbonyl stretching vibration of amide and –NH distortion bands was observed at 1651 cm− 1 (Kuyucak and Volesky, 1989). The spectrum shows the low-intensity vibration deviation of band 1077 cm− 1, the peak that shifted from 1084 cm− 1 to 1075 cm− 1 could be attributed to the C-O stretching of carboxyl groups and S = O groups (Loukidou et al., 2003). To sum up, it can be inferred that the functional groups of –OH, –NH, C-H, C = O, C-O, S = O existing on the surface of T. pubescens MB 89 might participate in the Cd2+ biosorption process. Table 1.
Table 1
The shift of absorption peak band in FTIR spectra of T. pubescenceat different cadmium concentrations
IR peaks
|
Wavenumber(cm-1)
|
Association
|
control
|
5 mg/L
|
10 mg/L
|
50 mg/L
|
100 mg/L
|
1
|
3487.32
|
3419.93
|
3405.4
|
3390.31
|
3402.99
|
bonded-OH, -NH Stretching
|
2
|
2928.13
|
2928.29
|
2928.96
|
2928.30
|
2928.74
|
C-H Stretching
|
3
|
1651.29
|
1651.78
|
1653.14
|
1651.94
|
1644.45
|
C = O Stretch of COOH
|
4
|
1403.71
|
1401.84
|
1402.42
|
1404.51
|
1413.96
|
C-H Stretching
|
5
|
1077.98
|
1079.33
|
1079.75
|
1077.86
|
1076.57
|
C-O and S = O Stretching
|
1 Effect of T. pubescens colonization on growth of rice plants under Cd stress
Rice seedlings were grown in a plant tissue culture chamber exposed to a specific temperature, humidity, and light conditions. Cd stress experiments were performed by adding CdCl2 to a final concentration of 10 mg/L. Under Cd stress, rice seedling growth was significantly inhibited, which was characterized by short plant height and yellow withered leaves (Fig. 5a). However, there was remarkable difference observed between the growth of rice seedlings colonized with T. pubescens and CONT group without Cd stress. Further measurement results (Fig. 5b, d, e) indicated that the addition of Cd2+ had inhibited growth of rice seedlings with both the height of shoots and the length/diameter of root decreased significantly. It was found that the shoot height was almost 38.46% less in Cd2+ treated seedlings than non-treated ones. On the other hand, the root length was almost 52.5% less in Cd2+ treated seedlings than non-treated seedlings. Furthermore, the root diameter was almost 49.2% less in Cd2+ treated seedlings than non-treated seedlings. Therefore, Cd2+ had an apparent toxic effect on rice seedlings, especially on the roots, this results were in close agreement with the work in literature (Wang et al., 2019). Figure 5.
In this study, we were surprised to find that the colonization of T. pubescens had remarkably reduced the cadmium toxicity and relieved its inhibition on rice seedlings. Moreover, it showed to promote the growth of rice seedlings, showing healthier growth state and larger roots. Therefore, T. pubescens rhizosphere was seen to possess the ability to counteract Cd-stress.
2 T. pubescens colonization on plant tissues cadmium accumulation against Cd toxicity
Regardless of the presence or absence of microorganism, roots usually accumulated more Cd than that in culms, leaves and grains (Shan et al., 2020). After 15 days of growth in Cd-containing (10 mg/mL) aqueous culture medium, the Cd accumulation in roots and shoots of the rice plants are showed in Fig. 6. For seedlings colonized by T. pubescens, Cd concentrations in roots and shoots were significantly reduced by 53.54% and 86.38%, respectively. These results clearly show that T. pubescens was able to suppress the accumulation of Cd in rice. It thus has positive potential on heavy metal removal for more sustainable agriculture. Our experimental outputs were consistent with previous studies by other workers (Lin et al., 2016; Zhou et al., 2016), in which some other Cd-tolerant microorganisms were used. Figure 6.
3 T. pubescens colonization on rice roots against Cd toxicity
Evidences has suggested that active growth of the roots accelerated the absorption of nutrients and thereby facilitated shoot growth(Cai et al., 2020). As shown in Fig. 7 (with root cross section slices g, h, and i), cadmium contents in rice plants have caused serious damage to the cell morphology of roots, and further resulted in cell deformation and senescence. The T. pubescens colonization has protected root cells from disrupture. As a result of cadmium adsorption by T. pubescens, the residual cadmium concentration in the medium was significantly reduced, which alleviated the toxicity of cadmium to roots. It is seen from the vertical section of rice roots (Fig. 7a, b, c, d, e, f) that the CONT(Cd) with cadmium showed retarded plant development with cell degeneration of the root tips, root thinning. Obviously, cadmium in excess in the soil interfered with the uptake and transport of mineral nutrients by roots, resulting in nutrient deficiency of plants. Furthermore, cadmium ion might degrade root tip cells, switched down water absorption and transport system, it thus resulted in reduced nutrient supply. In comparison, development of the root tips for the rice plants in (Tp(Cd)) group were healthy and similar to that for CONT group. As can be seen from the comparison between Fig. 7b and c, T. pubescens colonization on rice plants could significantly prevent the rice roots from the damage by environmental cadmium, protect the rice root tip cells, and enable absorption and transportation of nutrients from the rhizosphere micro-environment. The results suggested that T. pubescens has played an important roles in protecting rice plants from Cd-induced damages. Figure 7.