Improved Salt Tolerance of Lamtoro ( Leucaena leucocephala ) through the Application of Indigenous Mycorrhiza

Salt stress is one of the serious abiotic stressors which limit the growth and development of important crops in agricultural lands. Arbuscular mycorrhizal fungi (AMF) have been implemented as a strategy to mitigate the adverse eﬀects due to an impact of salt stress through the structural and physiological adjustment. This study aimed to determine a relationship between salinity levels (0, 150, 300, and 450mM NaCl) and AMF treatments ( Glomus manihotis, Glomus etunicatum, and G. manihotis + G. etunicatum ) to the salt tolerance of Leucaena leucocephala seedlings in a greenhouse. Salinity reduced the plant height, biomass, and root colonization by AMF. However, the inoculation of AMF, especially the consortium, ameliorated the negative eﬀects by stabilizing the growth performance and supporting the photosynthetic outputs through optimum nutrient and mineral absorptions. These results were indicative through a signiﬁcant interaction between salinity levels and the types of AMF treatment in all parameters except in the total leaf protein and proline contents from the two-way ANOVA results. Root colonization was highly correlated with the plant height, biomass, and total carbohydrate content with a maximum contribution conferred by the AMF consortium, based on Pearson’s correlation coeﬃcient test and PCA analysis. Our study then showed the positive impact of AMF toward salt tolerance by L. leucocephala with potential application and cultivation in salt-stressed ecosystems.


Introduction
Conversion of agriculture land into human settlement and industry has led to the decrease territory of arable lands. Upcoming climate change with consequences on the rise of sea level, sea water intrusion, and high evaporation was regarded as a major environmental issue which also posed some challenges in the cultivation of economical crops [1]. e consequence of this land conversion has directed farmers to utilize marginal lands such as saline soils [2]. Saline soils are characterized by their high salt content (NaCl, Na 2 CO 3 , Na 2 SO 4 ) with electric conductivity >4 dS/ m ≈40 mM NaCl which deter the optimum growth and development of many horticultural crops around the world [3]. Saline soils in Indonesia cover an area of 27,4 million ha with potential being utilized for cultivation of salt tolerant crops [4]. However, excessive salt ions will limit native plants to thrive in the environment by decreasing plant height, lowering photosynthetic capacity and nutrient absorption with death penalty [5]. Some strategies have been employed to maximize the use of saline soils, one of which through the application of soil-borne microorganisms, such as bioamelioration by arbuscular mycorrhizal fungi (AMF) [6,7].
Lamtoro (Leucaena leucocephala (Lam.) de Wit) from family Fabaceae is a pioneer legume species from Central America known for its notable nitrogen fixation capacity and fast-growing woody plant. e species has been utilized as biofertilizer or green leaf manuring to improve soil fertility, as forage for animal feed, and a prominent source of high quality timber [8][9][10]. In Indonesia, the species has been cultivated since 1800 with some of its cultivars being recognized as important timber producers due to its valuable traits [11,12]. erefore, the species is well-adapted towards abiotic stress in marginal lands while being proved useful for afforestation and landscaping [13]. Studies have also revealed the significant contribution of AMF to the improved growth performance of L. leucocephala seedlings under stress condition especially in heavy metal contaminated sites [14][15][16].
Plants maintain their growth and development under salinity stress through biological adjustment to thrive in the harsh environments. e adjustment may vary such as accumulation of compatible solutes, production of ROSscavenging enzymes, induction of phytohormones, and ionhomeostasis balancing [17]. However, the performance may be limited due to the rapidly changing environment and high influx of salt ions which demand a more stable adjustment from the plant [18]. Symbiotic establishment between AMF and plant species was initiated 400 million years ago and considered as stable mutualism which form inter-regulation and enhancement on the survivability of terrestrial plants [19]. e salt resistance and growth improvement conferred by AMF may be different across species and strain origins. Claroideoglomus etunicatum colonizing the roots of rice plant (Oryza sativa L.) showed enhancements in terms of photosynthetic productivity and stomatal conductance under salinity stress [20]. Another strain of C. etunicatum colonizing the roots of a halophytic C 4 grass species Aeluropus littoralis has been reported to increase the shoot and root biomass of its host following other adaptive traits such as improved stomatal conductance, synthesis of compatible solutes, and balanced ion uptakes [21]. Another example from legume species alfalfa (Medicago sativa L.), the coinoculation of AMF and rhizobia improved the yield through increases in mycorrhizal colonization, rhizobia nodulation, root Ca content, and shoot proline content [22].
Based on our understanding, the information of AMF colonization in L. leucocephala under saline conditions is still limited. e present study then investigates the role of two indigenous AMF following their biological outputs in maintaining the normal growth of L. leucocephala under salinity stress by examining physiological adjustment by the plant. is study will also discriminate which fungal species is the better option as an AM fungal inoculant or AMF consortium for improving salt tolerance in L. leucocephala.

AMF Identification and Inoculum Preparation.
Two indigenous AMF isolates, Glomus sp1 and Glomus sp2, were isolated in a previous study using Pueraria javanica as host through trap cultures and single spore isolation. Spore suspension was prepared for molecular identification. Genomic DNA extraction and nested polymerase chain reaction (PCR) were performed commercially by Macrogen, Inc. (Singapore). Molecular identification of AMF was based on the amplified nuclear rDNA fragments using a pair of SSUmAf/LSUmAr and SSUmCf/LSUmBr primers [23]. Crude inoculum was prepared in sterilized zeolite filled with colonized root segments of P. javanica maintained in dry state. e AMF inoculum was then preserved for further experiment.

Plant and Soil
Treatments. Seeds of L. leucocephala, provided by the Research Institute of Haurbentes (Bogor, Indonesia), were disinfected with 1% (v/v) NaOCl solution for 20 min, washed three times with distilled water, and soaked in sterile water for 24 h. After that, the seeds were put on a seed tray covered with river sand and rapidly germinated in a greenhouse. e seedlings were exposed to sunlight for 12 h every day, and the water was given conditionally. Once the seedlings had grown to 10 cm and produced two leaves, they were put into a plastic cup (93.4 mm × 65.82 mm, 5 holes) filled with sterilized zeolite (particle size � 1 mm) + AMF inoculum in a ratio of 1 : 1 (w/w) or in a ratio of 2 : 1 : 1 for AMF consortium (Glomus sp1 + Glomus sp2) and maintained in a greenhouse for salinity treatment.

Experimental Design.
e experiment was arranged in a Randomized Complete Block Design (RCBD) with two factors ( Figure 1): AMF inoculation (Glomus sp1, Glomus sp2, and Glomus sp1 + Glomus sp2) and salt stress (0, 150, 300, and 450 mM) and five replications for each, totaling 4 × 4 × 5 � 80 pots. e 60 seedlings inside the plastic cups were transplanted in contact with the top layer of 15 cm × 14 cm pots (no hole) filled with saline waters (150, 300, and 450 mM) and grown in a greenhouse. Pots without AMF inoculum were prepared as nonmycorrhizal controls. e seedlings were watered with distilled water every day.
Supplementation of Hyponex ® (N, P, K � 25%, 5%, 20%) was given once in two weeks with a concentration of 2 g/L. e treatments were maintained for 1 month.

AMF Colonization.
After 1 month of saline treatment, the seedlings were harvested, and the fresh roots were collected. e roots were washed, cut into 1 cm segments, and fixed with 10% KOH at 90°C until being colorless. e root segments were fixed with 10% HCl and stained with 0.05% trypan blue at 90°C for 15 min [24]. e AM colonization rate was determined by the gridline intersection method [25]. Data were recorded as the proportion of root length colonized.

Plant Height and Total
Biomass. Measurement of growth response under saline treatment was expressed as the average growth in plant height over one month, determined from the beginning and at the end of a 1-month saline treatment. e seedlings were dried in an oven at 60°C until constant weight (g) to obtain the total biomass of L. leucocephala.

Total Protein Content.
Fresh leaves from each treatment were sampled and washed with running tap water. One g of leaves was crushed and diluted with 20 mL of phosphate buffer saline (pH 7.4). e solution was centrifuged at 10,000 rpm for 10 min and then supernatants were collected.
Five mL of Quick Start ™ Bradford Protein Assay (Bio-Rad, US) containing Coomassie Brilliant Blue G-250 was mixed with 0.1 mL of sample solution [26]. Samples were incubated at room temperature for 5 min and then read at A 595 . Estimation of leaf protein (mg/g) content was compared to the standard solution using bovine serum albumin (BSA).

Total Carbohydrate Content.
Total carbohydrate containing polysaccharide and free sugars was estimated from the leaf sample solution as prepared previously using anthrone method [27]. e solution was acid-hydrolyzed using HCN and added with anthrone reagent. Samples were read at A 630 and estimated for their carbohydrate content (mg/g) with the standard solution using glucose.

Proline Content.
Leaf proline content was estimated from the leaf sample solution as prepared previously using the method described by Monneveux and Nemmar [28]. Samples were read at A 520 and estimated for their proline content (µmol/g) with the standard solution using proline.

Data Analysis.
e data were analyzed using a twofactor analysis of variance (ANOVA) obtained from salinity level and AMF treatment at the α level of 5%, followed by a pairwise comparison with Tukey test using Minitab ver. 16.0. Graphical images were generated using GraphPad Prism ver. 8.0.2.

Identification of Indigenous AMF.
Two samples of indigenous AMF were sent for molecular identification. DNA sequencing result showed the sequence of amplified region (partial 18S (SSU), 5.8S (ITS), and partial 28S (LSU)) rDNA of two Glomus isolates, each with a size of 1,796 bp for Glomus sp1 and 1,764 bp for Glomus sp2. Based on the bioinformatic analysis and phylogenetic construction among members of Glomeromycota, it was revealed that Glomus sp1 was identified as Glomus manihotis while Glomus sp2 was identified as Glomus etunicatum compared to the DNA sequence of Archaeospora trappei as an outgroup ( Figure 2).

Effect of AMF Inoculation on Height and Biomass of L. leucocephala under Salt Stress.
Results obtained after 1 month of growing L. leucocephala in greenhouse showed that the increasing NaCl concentration caused significant reduction to plant height of L. leucocephala seedlings from 21.5% (150 mM) to 44.6% (450 mM) compared to controls at 0 mM ( Figure 3). e biomass of L. leucocephala also experienced significant reduction from 20.96% (150 mM) to 61.35% (450 mM) compared to controls at 0 mM ( Figure 4). In the presence of AMF, the height and biomass of L. leucocephala seedlings were higher and significant compared to controls at all levels of salinity stress. e application of AMF consortium (G. manihotis + G. etunicatum) was observed to significantly alleviate the salt stress better than the application of single AMF species even matching the biomass in control plants at 150 mM and 300 mM treatments. In addition, the AMF consortium also promoted the best growth performance (height, biomass) of L. leucocephala seedlings in this study.

Effect of AMF Inoculation on the Total Protein, Carbohydrate, and Proline Contents of L. leucocephala Leaves under Salt Stress.
e total protein, total carbohydrate, and total and proline contents in L. leucocephala fresh leaves were quantified after 1 month of salt stress and the results showed a significant increase in AMF-inoculated plants compared to control plants at all concentrations ( Figures 5 and 6). e interaction between salinity and AMF treatment was not significant for total protein and proline content indicating that the salinity did not affect the AMF performance in International Journal of Forestry Research elevating or lowering the total protein and proline contents in L. leucocephala. Meanwhile, the total carbohydrate content in L. leucocephala leaves was significant in both treatment and its interactions between salinity and AMF treatment (Figure 7). e total protein and carbohydrate contents in control plants were observed for its maximum value at 150 mM NaCl, although insignificant for total carbohydrate parameter. e highest proline content in control plants was observed at 300 mM and insignificant among the group. e inoculation of AMF consortium increased the total protein, total carbohydrate, and proline content of L. leucocephala seedlings which almost was matched by the single inoculation of G. manihotis. Based on these results, it was revealed that the inoculation of G. etunicatum did not play an important role in the accumulation of the proteins and sugars in L. leucocephala leaves compared to control plants.

Effect of Salt
Stress on the Root Colonization of L. leucocephala. Root colonization of L. leucocephala by AMF isolates was observed microscopically after 1 month of salt stress. In control plants, no colonization was observed by other indigenous AMF due to the application of sterilized zeolites as growth medium. e microscopic images showed that each AMF treatment was successful as indicated by the presence of hyphal structures in all samples with some vesicles and few arbuscules (Figure 8). Soil salinity decreased the root colonization of L. leucocephala in all treatments. e AMF colonization decreased by 30%, 29%, and 32% (in the G. manihotis, G. etunicatum, and AMF consortium, respectively) in the 450 mM treatments compared to control plants (0 mM NaCl) (Figure 9). ere was a significant difference between salinity and AMF treatments. e application of single AMF isolate, G. manihotis, almost matched the root colonization by the AMF consortium in the 150, 300, and 450 mM treatments while the colonization by G. etunicatum was recorded the lowest in our study.

PCA Analysis.
Multivariate analysis of all datasets was meant to depict the physiological response patterns of L. leucocephala to salt stress and AMF inoculation. Correlation among parameters was priorly analyzed using Pearson's correlation coefficient test (Table 1). e height of L. leucocephala was negatively correlated with the biomass which may indicate the limit of growing the seedlings in greenhouse. e carbohydrate content in L. leucocephala leaves was negatively correlated with the plant height and positively correlated with the biomass. e proline content was negatively correlated with the biomass indicating a stress response to the salt stress. Root colonization by AMF showed a high correlation with plant height, biomass, and carbohydrate content which indicated a growth promotion through better nutrient absorption facilitated by the fungi. In addition, the proline content in L. leucocephala leaves was less likely correlated with the root colonization by AMF which gave us a clue on other tolerance mechanisms under salt stress. All Total protein (mg/g) Figure 5: Effects of AMF inoculation on the total protein content of L. leucocephala leaves at different NaCl stress. AMF: arbuscular mycorrhizal fungi. * : significant at 0.01 ≤ P ≤ 0.05; * * : significant at P ≤ 0.01. Bars that do not share a letter are significantly different at P ≤ 0.05. parameters were standardized and reduced to the most representative dimensions. e distance between treatments in the score plot of PCA showed the similarity of tested parameters. e PCA components, PC1 and PC2, accounted for 57% and 25% of the variance, respectively, yet were categorized as optimum in depicting the correlation among parameters (PC 1+2 >70%). Based on the plot, it can be seen that control plants and AMF-inoculated plants were separated in different quadrants showing distinct results of individual parameters ( Figure 10). e growth response of L. leucocephala seedlings also tends to be grouped together under type of AMF treatment rather than at different salt levels. e application of either single AMF species or consortium could enhance the salt tolerance of L. leucocephala seedlings, especially at 150 and 300 mM NaCl.

Discussion
Salinity stress is a global limiting factor that reduces the growth and viability of valuable plants cultivated in the agricultural fields [29]. Excessive salt ions in soil environment impose serious physiological stressors to the plants with the biological consequences like reduced photosynthesis capacity, cellular dehydration, and nutrient deficiencies [7,18,30]. e effect of AMF inoculation to L. leucocephala plants was studied through an experiment of different NaCl concentrations (0, 150, 300, and 450 mM) for 1 month under greenhouse. Inoculation of AMF into L. leucocephala seedlings has been reported previously and studied mostly under heavy metal toxicity experiments. AMF species such as Glomus aggregatum, Glomus etunicatum (syn. Claroideoglomus etunicatum), Acaulospora longula, and Acaulospora scrobiculata have been implemented successfully as bioameliorants in planta [15,31,32]. Our finding on G. manihotis (syn. Rhizophagus manihotis) as one of the indigenous AMF was considered unique since the species was known to be abundant in cassava [33,34]. In addition, there is still no recent record on the application of G. etunicatum, especially G. manihotis on L. leucocephala c (c) Figure 9: Effects of AMF inoculation on the root colonization of L. leucocephala seedlings at different NaCl stress. AMF: arbuscular mycorrhizal fungi. * : significant at 0.01 ≤ P ≤ 0.05; * * : significant at P ≤ 0.01. Bars that do not share a letter are significantly different at P ≤ 0.05. under salinity stress. Hence, our results may be beneficial for future investigation and possible formulation as a product. e AMF colonization rate in L. leucocephala roots was considered as stable under severe salt stress, although the highest percentage of colonization only reached >30% for G. manihotis and AMF consortium in the 450 mM NaCl treatment. Salt stress may inhibit the mycelial growth and formation of vesicles in the mycelial network due to the toxicity of excessive sodium ions [35]. A significant decline in root colonization also indicated that the protective effect by preinoculation of AMF was less potent at higher salinity levels and diminished hereafter. In this study, the excessive salt ions reduced the root colonization capacity by AMF in the 150 and 300 mM NaCl. e early response of L. leucocephala towards salt stress may be regulated in the form of delayed growth. Reduced cell growth of L. leucocephala manifested in the form of shorter plant and low biomass under salinity stress is normal but the application of AMF also proved to promote growth better than control plants (0 mM NaCl) in this study. Application of AMF is effective to support the vegetative growth of plants through improved root absorption especially phosphorus (P) under abiotic stress which helps to maintain the structural integrity or biomass of the plants [36]. Measurement of total soluble protein in NaCl-affected L. leucocephala seedlings was meant to estimate the stability of dissolved membrane and pigment proteins. In this study, the interaction between salinity levels and AMF inoculation was not significant which means that there are other factors affecting the result. A study on a tall perennial cane species, Arundo donax, showed that the leaf protein content was lower in salt-stressed plants than control plants and the interaction was more likely by nutrition than AMF treatment. In addition, the chlorophyll content was not significant in the moderate salt stress as supplied by adequate C and P nutrition [37]. Another possible mechanism as revealed from the correlation test between root colonization and leaf protein content is that AMF may also stabilize the S2 S1 S1 S1 S1 S1 S1 S1 S1 S1 S1 S1 S1 S0 S0  International Journal of Forestry Research Na + translocation from the root system to the shoot system by maintaining internal Na+ concentration in the mycelial network, thus preventing its accumulation and translocation into the photosynthetic tissues [38,39]. Salinity may also induce the accumulation of glomalin, a typical heat shock protein produced by AMF in the soil environment [40]. However, the distribution of this protein was more likely to improve the soil integrity to maximize mineral and nutrient absorption for the host. It would be interesting for further study to evaluate these parameters in salt-stressed L. leucocephala. Tissue dehydration under salt stress may be prevented by the accumulation of osmolytes such as proline, sugars, organic acids, amino acids, and trehalose [41]. ese molecules serve as osmoprotectants to stabilize protein and membrane integrity and scavenging of outgoing ROS incidence [42]. Here, the leaf proline content of salt-stressed L. leucocephala was quantified and showed that there was no significant interaction between salinity levels and AMF treatments. e reports on the accumulation of proline have been inconsistent between mycorrhizal and nonmycorrhizal plants [18]. However, it is generally accepted that under salt stress the accumulation of proline as a stress marker by plants is stable due to the presence of AMF involving other possible mitigation mechanisms [43]. Accumulation of carbohydrates or soluble sugars by plants is also considered as another salt-induced mechanism in adjusting the osmotic balance and as carbon storage [44]. Here, the sugar content in L. leucocephala leaves was considerably similar in control plants; however, there was a significant interaction between salinity levels and AMF treatments with the highest sugar content in the 150 mM NaCl compared to control plants and then decreasing hereafter. e relationship between root colonization and carbohydrate content was strongly correlated, which indicated that there was a significant contribution by AMF in the accumulation of soluble sugars in L. leucocephala. By considering the possible supportive feature by AMF in enhancing a nutrient absorption through root colonization, the higher quantity of soluble sugars under salt stress may be a result of an uninterrupted photosynthesis and rapid production of sucrose as carbon storage for further substrate decomposition and utilization for energy balance [45]. Consistent with the previous reports, the application of single AMF and its consortium could mitigate the output of salt stress and improve salt tolerance of L. leucocephala seedlings, although the parameters are finite and still need further investigation.

Conclusions
Salt stress induced several adjustments by L. leucocephala in terms of structural and physiological adjustments. Salt stress also reduced the root colonization of both AMF species, G. manihotis and G. etunicatum, in the 150, 300, and 450 mM NaCl treatments. However, the inoculation of AMF showed a significant relationship with the plant's ability to thrive in stressful conditions as being revealed from a steady height growth, biomass production, and total carbohydrate content. e application of AMF, especially the consortium, produced the highest result in enhancing a nutrient uptake, as indicated by a strong relationship between the root colonization and height, biomass, and carbohydrate content and multivariate analysis through PCA projection. Based on these results, the tolerance of L. leucocephala was improved to some degree, while also opening up the possibilities to further study in acquiring a deeper understanding of other possible mechanisms facilitated through an AMF symbiosis with the plants.
Data Availability e data are available upon request provided by the corresponding author's laboratory record at the Department of Forestry, Faculty of Forestry, Universitas Sumatera Utara, Indonesia.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this work.