Article Effects of Mineral-Solubilizing Microorganisms on Root Growth, Soil Nutrient Content, and Enzyme Activities in the Rhizosphere Soil of Robinia pseudoacacia Chong Li 1 , Zhaohui Jia 1, Lu Zhai 2, Bo Zhang 2, Xiaonan Peng 1, Xin Liu 1 and Jinchi Zhang 1,* 1 Co-Innovation Center for Sustainable Forestry in Southern China, Jiangsu Province Key Laboratory of Soil and Water Conservation and Ecological Restoration, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China; [email protected] (C.L.); [email protected] (Z.J.); [email protected] (X.P.); [email protected] (X.L.) 2 Department of Natural Ecology Resource and Management, Oklahoma State University, Stillwater, OK 74078, USA; [email protected] (L.Z.); [email protected] (B.Z.) * Correspondence: [email protected] Abstract: Background: Abandoned mining sites are becoming increasingly common due to anthro- pogenic activities. Consequently, external-soil spray seeding technology has attracted increasing attention as a strategy to remediate them. However, significant challenges remain that greatly in- hibit the efficacy of such technologies, such as insufficient nutrients available for plants. Methods: For this study, we designed an experiment, which involved the addition of mineral-solubilizing microorganisms and R. pseudoacacia seedlings to the external-soil spray seeding (ESSS) substrate, and measured the soil nutrients, enzyme activities, and root growth of R. pseudoacacia. Results: First, the combination of certain mineral-solubilizing microorganisms with ESSS advanced its ef- ficiency by increasing the availability of soil nutrients and soil enzymatic activities in association with R. pseudoacacia. Furthermore, the improvement of root growth of R. pseudoacacia was intimately related to soil nutrients, particularly for soil total nitrogen (TN) and total sulfur (TS). In general, the Citation: Li, C.; Jia, Z.; Zhai, L.; Zhang, B.; Peng, X.; Liu, X.; Zhang, J. effects of the J2 (combined Bacillus thuringiensis and Gongronella butleri) treatment for soil nutrients, Effects of Mineral-Solubilizing enzyme activities, and plant growth were the strongest. Conclusion: In summary, the results of Microorganisms on Root Growth, Soil our experiment revealed that these mineral-solubilizing microorganisms conveyed a promotional Nutrient Content, and Enzyme effect on R. pseudoacacia seedlings by increasing the soil nutrient content. These results provide Activities in the Rhizosphere Soil of basic data and microbial resources for the development and applications of mineral-solubilizing Robinia pseudoacacia. Forests 2021, 12, microorganisms for abandoned mine remediation. 60. https://doi.org/10.3390/ f12010060 Keywords: mineral-solubilizing microorganisms; ecological restoration technique; root growth; soil nutrient contents; soil enzyme activities Received: 3 November 2020 Accepted: 4 January 2021 Published: 6 January 2021 1. Introduction Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- The emergence of steep rocky slopes is increased rapidly in China due to the con- ms in published maps and institutio- struction of highways and railways, as well as the over-exploitation of rock mineral re- nal affiliations. sources [1,2]. These activities have severely damaged ecosystems [3,4]. At present, multiple strategies are employed for restoring the abandoned mine sites [5–7], where the external- soil spray seeding (ESSS) technique has become a common technique for their ecological restoration [8,9]. The ESSS (Figure1) technique contains the spraying of artificial mixed Copyright: © 2021 by the authors. Li- soil materials (soil, water-retaining agents, fertilizer, and plant seeds) onto rocky slopes censee MDPI, Basel, Switzerland. toward the ecological restoration of abandoned mine sites [10]. Soil comprises the core This article is an open access article element of this technique, which provides plants with water and nutrient sources [11]. distributed under the terms and con- However, major challenges remain, as the soils on steep slopes are inadequate and the ditions of the Creative Commons At- available nutrients absorbed by plants are unsatisfactory [12]; therefore, this technique tribution (CC BY) license (https:// creativecommons.org/licenses/by/ has been difficult to establish toward the promotion of long-term ecological restoration 4.0/). maintenance in abandoned mining areas [13]. Forests 2021, 12, 60. https://doi.org/10.3390/f12010060 https://www.mdpi.com/journal/forests Forests 2021, 12, x FOR PEER REVIEW 2 of 13 Forests 2021, 12, 60 2 of 12 this technique has been difficult to establish toward the promotion of long-term ecolog- ical restoration maintenance in abandoned mining areas [13]. Figure 1. External-soil spray seeding (ESSS) technique. The addition addition of of roots roots can can strengthen strengthen the the soil, soil, as the as theroot root growth growth is intimately is intimately related re- tolated the to soil the [14]. soil [14Soil]. Soilnutrient nutrient and and soil soil enzyme enzyme activity activity can can affect affect root root growth growth [15–17]. [15–17]. Therefore, it it is is very very important important to to study study the the ch changesanges in inroot root growth, growth, soil soil nutrients, nutrients, and andsoil enzymesoil enzyme activity activity for the for thepromotion promotion of long-term of long-term ecological ecological restoration restoration maintenance maintenance in abandonedin abandoned mining mining areas. areas. The The application application of of mineral-solubilizing mineral-solubilizing microorganisms microorganisms is is a novel attempt attempt for for abandoned abandoned mine mine remediation, remediation, which which can can accelerate accelerate mineral mineral weathering weather- [18–20].ing [18– 20In]. recent In recent years, years, the research the research into intomineral-solubilizing mineral-solubilizing microorganisms microorganisms have havebeen morebeen morein the in scope the scope of Plant of Plant Growth-Promoti Growth-Promotingng Rhizobacteria Rhizobacteria (PGPR); (PGPR); the thefunctions functions of mineral-solubilizingof mineral-solubilizing microorganisms microorganisms also also have have many many similarities similarities with with PGPR. PGPR. Firstly, Firstly, in- creasedincreased essential essential minerals minerals solubilized solubilized by by microorganisms microorganisms (e.g., (e.g., P, P, K, K, S, S, Mg, Mg, Cu, Fe, and Zn) contribute to the promotion of plantplant growthgrowth [[21].21]. Secondly, thesethese microorganismsmicroorganisms can also promote plant development through the generation of plant-growth-promotingplant-growth-promoting substances and siderophore productionproduction [[22,23].22,23]. Moreover, several microorganisms have the capacity to improve plant growth through the stimulation of plant hormones and root elongation (including the formation of root hairs and lateral roots) [[24–27].24–27]. Previous studies have reported that root and plant growth are intimately correlated with the availability of soil nutrients [[28–3028–30] andand soilsoil enzymeenzyme activitiesactivities [[31–33].31–33]. Rhizo- Rhizo- spheric microorganisms microorganisms are are crucial crucial in in nutrient nutrient cycling, cycling, they they can can increase increase soil soil enzyme enzyme ac- tivitiesactivities and and the the mineralization mineralization of of available available nu nutrientstrients in in the the soil; soil; thus, accelerating root growth [[21,23,26,34].21,23,26,34]. ForFor instance,instance, the the combination combination of ofAdvenella Advenella incenata incenataP4, P4,Serratia Serratia ply- plymuthicamuthica P35, P35,Providencia Providencia rettgeri rettgeriP2, P2, and andAcinetobacter Acinetobacter calcoaceticus calcoaceticus P19P19 microorganismsmicroorganisms increased soilsoil urease,urease, phosphatase, phosphatase, catalase, catalase invertase, invertase activity, activity, available available phosphorus phosphorus (AP), (AP),available available potassium potassium (AK), (AK), and available and available nitrogen nitrogen in the in rhizosphere the rhizosphere of Avena of Avena sativa sativa[35]. [35].The additionThe addition of Glomus of Glomus etunicatum etunicatumalso increased also increased the invertase, the invertase, urease, proteinase,urease, proteinase, catalase, catalase,and phosphatase and phosphatase activities inactivities the rhizosphere in the rh soilizosphere of maize soil [36 of]. maize Moreover, [36]. theMoreover, application the applicationof Bacillus sp of. strainBacillus UM15 sp. strain improved UM15 soilimproved available soil phosphorus available phosphorus and total nitrogen and total [ 37ni-]. trogenTherefore, [37]. microorganisms Therefore, microor wereganisms observed were to beobserved closely correlatedto be closely with correlated soil nutrients with andsoil enzyme activities. nutrients and enzyme activities. R. pseudoacacia is the dominant tree species in abandoned mine restoration due to R. pseudoacacia is the dominant tree species in abandoned mine restoration due to its its capacity for tolerating dry environments and nitrogen-fixing abilities [38,39]. The capacity for tolerating dry environments and nitrogen-fixing abilities [38,39]. The micro- microorganisms in this study were mineral-solubilizing microorganisms isolated from the soil surrounding weathered dolostones, which secreted organic acid to enhance the release of Ca and Mg [40,41]. In our previous study, these mineral