**3.2 Plant growth performance under different treatments**

Throughout the experimental period (90 days), regardless of the treatment applied, no visible heavy metal morphological toxicity symptoms, such as leaf chlorosis and root browning, appeared when A. mangium was planted in heavy metalpolluted dumping soil under greenhouse conditions (**Figure 1**). This result revealed that *A. mangium* is able to grow in metal-contaminated soils and is a metal-tolerant plant species, as suggested by [3, 4].

The significantly (P < 0.05) lowest height (**Figure 2a**) and total dry weight biomass (**Figure 2b**) were obtained under the non-inoculated (T0) treatment, with 25.7 cm and 11 g, respectively (**Figure 2a** and **b**). The greatest height and total dry weight biomass were observed when *A. mangium* was co-inoculated with *P.*


### **Table 2.**

*Evolution of number and weight of earthworm during 90 days*. *n.d: none determined.*

*Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

### **Figure 1.**

A. mangium *growth performance (number of leaves, length of stem, root system development) under different treatments: non-inoculated, control (T0); inoculated with earthworms (T1); inoculated with Bradyrhizobium ORS (T2); co-inoculated with Bradyrhizobium ORS and earthworms (T3).*

*corethrurus* and *Bradyrhizobium* (T3)*,* at 54.5 cm and 141.7 g, followed by T1 (*A. mangium* inoculated with earthworm) at 51.5 cm and 101 g, by T2 (A. mangium inoculated with *Bradyrhizobium*) at 45.8 cm and 77 g (**Figure 2a** and **b**). Ours results indicated, for respective effect of *P. corethrurus* earthworms, *Bradyrhizobium* and of both inoculants, a growth stimulation of *A. mangium* by approximately twofold and 10-fold for the biomass under T1 treatment, by approximately 1.5-fold and sevenfold for the biomass under T2 treatment and by approximately twofold and 14-fold for the biomass under T3 treatment. This phenomenon was probably due to the action of *P. corethrurus* earthworms, which have the potential to modify edaphic parameters such as soil structure, organic matter decomposition and indirectly improve soil microorganisms proliferation and activities, facilitate the uptake of many important nutrients by plant, and consequently promote plant growth [26, 27]. Our results are consistent with the well-known fact that earthworms enhance plant growth and biomass [28]. Because, by bioturbation, earthworms stabilize organic matter in soil, form soil aggregates, modify the structure and chemical composition of soil [29]. Such changes generally increase soil water holding capacity, soil nutrient content, and plant productivity [28–30]. Most previous studies justified this better enhancement of acacia growth performance in the presence of earthworm by the fact that in

### **Figure 2.**

*Effect of different treatments (non-inoculated, control (T0); inoculated with earthworms (T1); inoculated with* Bradyrhizobium *(T2); co-inoculated with* Bradyrhizobium *and earthworms (T3) on average plant height (a), total dry weight biomass (roots and shoots) (b), number of nodules (c), concentrations (mg.kg<sup>1</sup> dry weight) of Chromium (Cr) (d), Nickel (Ni) (e), and Lead (Pb) (f), in* Acacia manguim *total biomass. Histograms with the same letters (a, b, c) indicated no significant differences between treatments at 0.05 probability level according to Student–Newman–Keuls test. \*\*very highly significant at 0.01 probability level, \*significant at 0.05 probability level according to Student–Newman–Keuls test.*

metal-contaminated soil, some earthworms species (*Eisenia fetida*, *Lumbricus terrestris, P. corethrurus*) can decrease the content of potential toxic elements (PTEs) in metal contaminated soil through the accumulation potential toxic elements (PTEs) in their tissues and consequently promote plant growth [31–33]*.* A similar finding has been documented by [16], who showed that the presence of *P. corethrurus* could enhance the biomass of *Lantana camara L.* by approximately 1.5–2-fold under Pb stress.

The lower increase of acacia growth and biomass under inoculated with *Bradyrhizobium* treatment, compared with inoculated with *P. corethrurus* treatment (T1), may be due to the competitive effects that may occur between autochthonous soil microorganisms and exogenous strains (*Bradyrhizobium*). Because, several studies have demonstrated that inoculation of seedlings such as *A. mangium* with rhizobial strains results in the change of root morphology, that is, increases in nodules, lateral

### *Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

roots, root hairs, root surface area, and total root length [34] and thus improve plant growth [22, 35] in unpolluted soil and in metal-contaminated sites.

In comparison to the control treatment (T0) or single inoculated (T1 or T2), the presence of *P. corethrurus* earthworms and *Bradyrhizobium* strain significantly (P < 0.05) increases better plant growth stimulation. This positive effect might be due to the additive action of the two bioinoculants, which are recognized to promote plant growth and biomass production in metal-contaminated soil [36, 37]. So, ours findings showed that *A. mangium* exhibited better growth and high biomass production when both bioinoculants were present.

However, the greatest number of nodules per plant was obtain when *A. mangium* was inoculated with *Bradyrhizobium* (T2)*,* at 12 nodules/plant, followed by T1 (A. mangium inoculated with earthworm) at 9 nodules/plant, by T0 (*A. mangium* noninoculated) at 5 nodules/plant (**Figure 2c**). The lowest number of nodules (two nodules/plant) was observed when *A. mangium* was inoculated with the two bioinoculants. The presence of nodules in all the treatments, especially under noninoculated control treatment, suggested that the soil contained autochthonous strains that were able to colonize the root system of *A. mangium* and to form symbiotic structures (nodules). Moreover, the lowest rate of nodules noted when *A. mangium* was co-inoculated with the two bioinoculants might be due to the interactions between the activities of *Bradyrhizobium* strain and *P. corethrurus* earthworms. In fact, by ingesting soil, *P. corethrurus* earthworms increased organic matter mineralization and nutrient availability, which indirectly stimulated the soil microorganisms. Therefore, the competitive action between autochthonous soil microorganisms and exogenous strains (*Bradyrhizobium*) could affect the capacity of exogenous symbionts (*Bradyrhizobium*) to colonize plant roots and to form symbiotic structures (nodules).

The presence of *P. corethrurus* appeared to reduce the positive effect of *Bradyrhizobium* on *A. mangium* nodulation. This result was in agreement with the findings of [38], who noted that the presence of earthworms (*Allolobophora chlorotica*) can reduce the positive effect of *Glomus intraradices* on the *Allium porrum* L roots biomass.

It was concluded that the interaction between *P. corethrurus* and *Bradyrhizobium* could promote growth and biomass production, but not nodulation, of *A. mangium*.

### **3.3 Effect of inoculation on metal uptake by** *A. mangium*

In the control treatment (T0), when *A. mangium* was non-inoculated, the concentrations of chromium, nickel, and lead were 1.33 mg.kg<sup>1</sup> ; 1.98 mg.kg<sup>1</sup> , and 3.8 mg. kg<sup>1</sup> , respectively (**Table 3**). In addition, Cr and Ni contents were very highly significant (P < 0.001), three to fourfold greater in roots tissue, with 1.04 mg.kg<sup>1</sup> for Cr and 1.6 mg.kg<sup>1</sup> for Ni, than in shoot tissue, with 0.3 mg.kg<sup>1</sup> for Cr and 0.44 mg.kg<sup>1</sup> for Ni (**Figure 3**). Whereas, the concentration for Pb in roots tissue was lower (1.5 mg. kg<sup>1</sup> ) than in shoots tissue (2.3 Cr mg.kg<sup>1</sup> ) (**Figure 3**). Our results indicated that in the absence of inoculation, acacia preferentially uptake Cr and Ni in its roots and Pb in its shoots (**Figure 3**).

Moreover, the translocation factors ([metal]shoot/[metal]root), indicator of the effectiveness of the plant to translocate metals from roots to shoots of *Acacia* specie, were TF < 1 for Cr and Ni, and TF > 1 for Pb under non-inoculated treatment but under inoculated treatment, whatever metal dosed TF < 1 (**Table 4**). This emphasizes that acacia may possess metal exclusion strategy, which probably depended to the nature of the metal. The bioconcentration factors (BCF) ([metal]plant biomass/


### **Table 3.**

*Content of chromium, nickel, and lead (mg.kg<sup>1</sup> dry material) in different compartments: Rhizosphere Soil (RS), Drilosphere Soil (DS), and in Acacia biomass after pot experiment under different treatments: non-inoculated, control (T0); inoculated with earthworms (T1); inoculated with* Bradyrhizobium *(T2); co-inoculated with* Bradyrhizobium *and earthworms (T3). n.d (none determined).*

[metal]soil) were BCF <0.1 under non-inoculated and inoculated treatments (**Table 4**), which were indicated that acacia are not hyperaccumulator plant as demonstrated [39]. Our findings did not differ from those of various studies that observed a higher accumulation of Pb in the shoots of *A. mangium* compared with the roots, indicating that acacia is able to tolerate and uptake heavy metal in its tissues and therefore could be suitable for phytostabilization of metal-contaminated sites [3, 4, 40]. Furthermore, Cr, Ni, and Pb phytoextraction efficiency (PEE) of *A. mangium* non-inoculated was PEE <1 (**Table 4**) whatever the nature of metal, which could be attributed to the form of the metal in the soil rhizosphere. Thus, it appeared that, according to the nature of the metal in soil, acacia could have different phytoremediation processes (phytoimmobilization and phytoextraction) when it was non-inoculated. But, this phytoremediation process of acacia seems to depend on the nature and the mobile form of metal in the rhizosphere soil.

However, the inoculation of *A. mangium* with *P. corethrurus* earthworms, *Bradyrhizobium* or with both inoculants, significantly (P < 0.05) increased the concentrations of chromium, nickel, and lead taken up in the plant biomass, which ranged from 2.4 to 11.2 mg.kg<sup>1</sup> for Cr, 2.5 to 7 mg.kg<sup>1</sup> for Ni, and 3.4 to 12.7 mg.kg<sup>1</sup> for Pb compared to the control treatment with 1.98 mg.kg<sup>1</sup> , for Ni, 1.33 mg.kg<sup>1</sup> for Cr, and 3.8 mg.kg<sup>1</sup> for Pb (**Table 3**). The respective effect of *P. corethrurus* earthworms, *Bradyrhizobium* or of both inoculants, for Cr uptake by plant, was increased around twofold under T1 treatment (2.41 mg.kg<sup>1</sup> ), four-fold under T2 treatment (5.23 mg.kg<sup>1</sup> ) and 10-fold under T3 treatment (11.2 mg.kg<sup>1</sup> ) (**Figure 2d, e**, and **f**). For Ni uptake by plant, the effect of *P. corethrurus* earthworms, *Bradyrhizobium* or both inoculants was enhanced by 1.3-fold under T1 (2.48 mg.kg<sup>1</sup> ), threefold under T2 treatment (5.24 mg.kg<sup>1</sup> ), and fourfold under T3 treatments (7 mg.kg<sup>1</sup> ) (**Figure 2d, e**, and **f**). *P. corethrurus* earthworms decreased the Pb uptake by plant ranging from 3.8 to 3.4 mg.kg<sup>1</sup> . *Bradyrhizobium* individually or the combined *P. corethrurus* earthworms and *Bradyrhizobium* enhanced twofold (7.2 mg.kg<sup>1</sup> ) and fourfold (12.7 mg. kg<sup>1</sup> ), respectively. Pb uptake by *A. mangium* as compared with non-inoculated plants (**Figure 2d, e** and **f**). In addition, under inoculated with earthworm treatment (T1),

*Combined Effects of Earthworms and Plant Growth-Promoting Rhizobacteria (PGPR)… DOI: http://dx.doi.org/10.5772/intechopen.108825*

### **Figure 3.**

*Accumulation of Cr, Ni and Pb in* Acacia mangium *shoot and root tissues under different treatments: noninoculated, control (T0); inoculated with earthworms (T1); inoculated with* Bradyrhizobium *(T2); co-inoculated with* Bradyrhizobium *and earthworms (T3). Histogram with the same letters (a, b) indicated no significant differences between Cr, Ni, or Pb concentrations in shoot and root tissues under. \*\*\* very highly significant at 0.001 probability level, \*\*highly significant at 0.01 probability level, \* significant at 0.05 probability level according to Student–Newman–Keuls test.*

Cr, Ni, and Pb contents were very highly significant (P < 0.001), 2–10-fold greater in roots tissue, with 1.6 mg.kg<sup>1</sup> for Cr, 1.9 mg.kg<sup>1</sup> for Ni and 3.1 mg.kg<sup>1</sup> for Pb, than in shoot tissue, with 0.8 mg.kg<sup>1</sup> , 0.63 mg.kg<sup>1</sup> , and 0.3 mg.kg<sup>1</sup> , respectively (**Figure 3**). Furthermore, under inoculated with *Bradyrhizobium* treatment (T2), Cr, Ni, and Pb contents were very highly significant (P < 0.001), 8–15-fold greater in roots tissue, with 4.6 mg.kg<sup>1</sup> for Cr, 4.7 mg.kg<sup>1</sup> for Ni, and 6.8 mg.kg<sup>1</sup> for Pb, than in shoot tissue, with 0.6 mg.kg<sup>1</sup> ; 0.5 mg.kg<sup>1</sup> , and 0.4 mg.kg<sup>1</sup> , respectively (**Figure 3**). In the presence of *P. corethrurus* earthworms and *Bradyrhizobium*, Cr, Ni, and Pb contents were very highly significant (P < 0.001), 4–20-fold greater in roots tissue, with 10.4 mg.kg<sup>1</sup> for Cr, 5.7 mg.kg<sup>1</sup> for Ni, and 12 mg.kg<sup>1</sup> for Pb, than in shoot tissue, with 0.8 mg.kg<sup>1</sup> ; 1.3 mg.kg<sup>1</sup> , and 0.7 mg.kg<sup>1</sup> , respectively (**Figure 3**). The phytoextraction efficiency (PEE) of *A. mangium* was much greater under inoculation treatments with 1–9% for Cr, 5–13% for Ni, and 7–18% for Pb (**Table 3**). Irrespective of the heavy metal dosed (**Table 4**), the significantly higher PEE (P < 0.05) was obtained when *A. mangium* was inoculated with *P. corethrurus* earthworms and *Bradyrhizobium* with PEE >9% (**Table 4**). This finding indicated that the


### **Table 4.**

*Bioaccumulator (BCF), translocation factors (TF) and phytoextraction efficiency (PEE) of Cr, Ni and Pb in Acacia mangium biomass in metal-contaminated soil under different treatments: non-inoculated, control (T0); inoculated with earthworms (T1); inoculated with* Bradyrhizobium *(T2); co-inoculated with* Bradyrhizobium *and earthworms (T3). Values with the same letters (a, b, c, d) indicated no significant differences between treatments according to Student–Newman–Keuls test.*

inoculation of *A. mangium* with *P. corethrurus* earthworms, *Bradyrhizobium,* or with both inoculants significantly increased the bioavailability of Cr, Pb and Ni in soil, then their uptake by *A. mangium* in biomass particularly in its roots tissue. In the presence of these organisms, the phytoextraction efficiency of *A.mangium* was significantly (P < 0.05) improved. The accumulation of potential toxic elements in acacia biomass may have been caused by the different soil organisms (*Bradyrhizobium* and/or earthworm), as demonstrated in previous studies in the presence of earthworms [5, 27, 31, 33] and of *Bradyrhizobium* [36, 41] only and also in presence of combined soil organisms such as earthworm and PGPR [42]. The metal uptake-stimulating effect of both inoculants was much greater than that of individual inoculated organism.

Moreover, under earthworm treatment, only the content of Cr at 29 mg.kg<sup>1</sup> dry soil and Pb at 4.9 mg.kg<sup>1</sup> dry soil was higher in the rhizosphere soil (RS) than in Drilosphere soil (DS) at 18 mg.kg<sup>1</sup> Cr dry soil and 4.7 mg.kg<sup>1</sup> Pb dry soil (**Table 3**). The lower content of Cr in the DS than in the RS compartment with earthworm treatment and the highest content of Cr (68 μg Cr/plant) in plant shoots suggested that Cr mobilized by earthworm in their structures (burrows and casts) was temporarily stored in these structure, which acted as sinks for the element [43], and transferred Cr to the RS compartment and subsequently to plant tissue. In contrast, despite the highest content of lead in the RS compartment, lead content was lowest in the plant shoots (25.2 μg Pb/plant). This phenomenon could be linked to the physiological behavior of *A. mangium,* which behaves as a Pb-excluder plant in the presence of bioinoculants. Likewise, despite the highest content of Ni in the DS (5.6 mg.kg<sup>1</sup> Ni dry soil) under earthworm treatment, the content of Ni was higher in the shoot parts than in the root part, which suggested that Ni mobilized in the DS compartment was transferred to plants. Here, the DS compartment was used by the plant as a sink for the element [43].
