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

Improved Salt Tolerance of Lamtoro (Leucaena leucocephala) through the Application of Indigenous... Hindawi International Journal of Forestry Research Volume 2021, Article ID 8100480, 11 pages https://doi.org/10.1155/2021/8100480 Research Article Improved Salt Tolerance of Lamtoro (Leucaena leucocephala) through the Application of Indigenous Mycorrhiza 1 2 Delvian Delvian and Adrian Hartanto Department of Forestry, Faculty of Forestry, Universitas Sumatera Utara, Medan, North Sumatra 20155, Indonesia Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, North Sumatra 20155, Indonesia Correspondence should be addressed to Delvian Delvian; delvian@usu.ac.id Received 17 May 2021; Accepted 24 July 2021; Published 31 July 2021 Academic Editor: Monika Markovic´ Copyright © 2021 Delvian Delvian and Adrian Hartanto. (is is an open access article distributed under the Creative Commons AttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkis properly cited. 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 stressthroughthestructuralandphysiologicaladjustment.(isstudyaimedtodeterminearelationshipbetweensalinity 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. (ese 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. to thrive in the environment by decreasing plant height, 1. Introduction lowering photosynthetic capacity and nutrient absorption Conversion of agriculture land into human settlement and with death penalty [5]. Some strategies have been employed industry has led to the decrease territory of arable lands. to maximize the use of saline soils, one of which through the Upcoming climate change with consequences on the rise of application of soil-borne microorganisms, such as bio- sea level, sea water intrusion, and high evaporation was amelioration by arbuscular mycorrhizal fungi (AMF) [6, 7]. regarded as a major environmental issue which also posed Lamtoro (Leucaena leucocephala (Lam.) de Wit) from some challenges in the cultivation of economical crops [1]. family Fabaceae is a pioneer legume species from Central (e consequence of this land conversion has directed America known for its notable nitrogen fixation capacity farmers to utilize marginal lands such as saline soils [2]. and fast-growing woody plant. (e species has been utilized Saline soils are characterized by their high salt content as biofertilizer or green leaf manuring to improve soil fer- (NaCl, Na CO , Na SO ) with electric conductivity >4dS/ tility, as forage for animal feed, and a prominent source of 2 3 2 4 m≈40mM NaCl which deter the optimum growth and high quality timber [8–10]. In Indonesia, the species has development of many horticultural crops around the world been cultivated since 1800 with some of its cultivars being [3]. Saline soils in Indonesia cover an area of 27,4 million ha recognizedasimportanttimberproducersduetoitsvaluable with potential being utilized for cultivation of salt tolerant traits [11, 12]. (erefore, the species is well-adapted towards crops[4].However,excessivesaltionswilllimitnativeplants abioticstress in marginallands whilebeing proveduseful for 2 International Journal of Forestry Research afforestation and landscaping [13]. Studies have also 2.2. Plant and Soil Treatments. Seeds of L. leucocephala, revealed the significant contribution of AMF to the im- provided by the Research Institute of Haurbentes (Bogor, proved growth performance of L. leucocephala seedlings Indonesia), were disinfected with 1% (v/v) NaOCl solution under stress condition especially in heavy metal contami- for 20min, washed three times with distilled water, and nated sites [14–16]. soaked in sterile water for 24h. After that,the seedswere put Plants maintain their growth and development under on a seed tray covered with river sand and rapidly germi- salinity stress through biological adjustment to thrive in the nated in a greenhouse. (e seedlings were exposed to harsh environments. (e adjustment may vary such as ac- sunlight for 12h every day, and the water was given cumulation of compatible solutes, production of ROS- conditionally. Once the seedlings had grown to 10cm and scavenging enzymes, induction of phytohormones, and ion- produced two leaves, they were put into a plastic cup homeostasis balancing [17]. However, the performance may (93.4mm ×65.82mm, 5 holes) filled with sterilized zeolite belimitedduetotherapidlychangingenvironmentandhigh (particle size �1mm)+AMF inoculum in a ratio of 1:1 influx of salt ions which demand a more stable adjustment (w/w) or in a ratio of 2:1:1 for AMF consortium (Glomus sp1+ Glomus sp2) and maintained in a greenhouse for from the plant [18]. Symbiotic establishment between AMF and plant species was initiated 400 million years ago and salinity treatment. considered as stable mutualism which form inter-regulation and enhancement on the survivability of terrestrial plants 2.3. Experimental Design. (e experiment was arranged in a [19]. (e salt resistance and growth improvement conferred Randomized Complete Block Design (RCBD) with two by AMF may be different across species and strain origins. factors (Figure 1): AMF inoculation (Glomus sp1, Glomus Claroideoglomus etunicatum colonizing the roots of rice sp2, and Glomus sp1+ Glomus sp2) and salt stress (0, 150, plant (Oryza sativa L.) showed enhancements in terms of 300, and 450mM) and five replications for each, totaling photosynthetic productivity and stomatal conductance 4 ×4 ×5 �80 pots. (e 60 seedlings inside the plastic cups under salinity stress [20]. Another strain of C. etunicatum were transplanted in contact with the top layer of colonizing the roots of a halophytic C grass species 15cm ×14cm pots (no hole) filled with saline waters (150, Aeluropus littoralis has been reported to increase the shoot 300, and 450mM) and grown in a greenhouse. Pots without and root biomass of its host following other adaptive traits AMF inoculum were prepared as nonmycorrhizal controls. such as improved stomatal conductance, synthesis of (e seedlings were watered with distilled water every day. compatible solutes, and balanced ion uptakes [21]. Another Supplementation of Hyponex (N, P, K �25%, 5%, 20%) examplefromlegumespeciesalfalfa(Medicago sativaL.),the ® was given once in two weeks with a concentration of 2g/L. coinoculation of AMF and rhizobia improved the yield (e treatments were maintained for 1 month. through increases in mycorrhizal colonization, rhizobia nodulation, root Ca content, and shoot proline content [22]. Based on our understanding, the information of AMF 2.4. AMF Colonization. After 1 month of saline treatment, colonization in L. leucocephala under saline conditions is the seedlings were harvested, and the fresh roots were still limited. (e present study then investigates the role of collected. (e roots were washed, cut into 1cm segments, two indigenous AMF following their biological outputs in and fixed with 10% KOH at 90 C until being colorless. (e maintaining the normal growth of L. leucocephala under root segments were fixed with 10% HCl and stained with salinity stress by examining physiological adjustment by 0.05% trypan blue at 90 C for 15min [24]. (e AM colo- the plant. (is study will also discriminate which fungal nization rate was determined by the gridline intersection species is the better option as an AM fungal inoculant or method [25]. Data were recorded as the proportion of root AMF consortium for improving salt tolerance in length colonized. L. leucocephala. 2.5. Plant Height and Total Biomass. Measurementof growth 2. Materials and Methods response under saline treatment was expressed as the av- erage growth in plant height over one month, determined 2.1. AMF Identification and Inoculum Preparation. Two from the beginning and at the end of a 1-month saline indigenous AMF isolates, Glomus sp1 and Glomus sp2, were treatment. (e seedlings were dried in an oven at 60 C until isolated in a previous study using Pueraria javanica as host constant weight (g) to obtain the total biomass of through trap cultures and single spore isolation. Spore L. leucocephala. suspension was prepared for molecular identification. Ge- nomic DNA extraction and nested polymerase chain reac- tion (PCR) were performed commercially by Macrogen, Inc. 2.6. Total Protein Content. Fresh leaves from each treatment (Singapore). Molecular identification of AMF was based on were sampled and washed with running tap water. One g of the amplified nuclear rDNA fragments using a pair of leaves was crushed and diluted with 20mL of phosphate SSUmAf/LSUmAr and SSUmCf/LSUmBr primers [23]. buffer saline (pH 7.4). (e solution was centrifuged at Crude inoculum was prepared in sterilized zeolite filled with 10,000rpm for 10min and then supernatants were collected. colonized root segments of P. javanica maintained in dry Five mL of Quick Start Bradford Protein Assay (Bio-Rad, state. (e AMF inoculum was then preserved for further US) containing Coomassie Brilliant Blue G-250 was mixed experiment. with 0.1mL of sample solution [26]. Samples were incubated International Journal of Forestry Research 3 AMF AMF AMF Nonmycorrhizal Treatment 1 Treatment 2 Consortium (T0) (T1) (T2) (T3) AMF = Arbuscular mycorrhizal fungi 0 mM NaCl S0 = (–) NaCl (S0) T0 = (–) AMF T1 = Glomussp1 T2 = Glomussp2 150 mM NaCl T3 = Glomussp1 + Glomussp2 (S1) 300 mM NaCl (S2) 450 mM NaCl (S3) Figure1:Experimentallayout(4 ×4factorial)forthegreenhouseexperiment,includingsalinityandAMFtreatments.Eachtreatmenthad5 replications. at room temperature for 5min and then read at A . Es- Glomus sp1 was identified as Glomus manihotis while timation of leaf protein (mg/g) content was compared to the Glomus sp2 was identified as Glomus etunicatum compared standard solution using bovine serum albumin (BSA). to the DNA sequence of Archaeospora trappei as an out- group (Figure 2). 2.7. Total Carbohydrate Content. Total carbohydrate con- taining polysaccharide and free sugars was estimated from 3.2. Effect of AMF Inoculation on Height and Biomass of the leaf sample solution as prepared previously using L. leucocephala under Salt Stress. Results obtained after 1 anthrone method [27]. (e solution was acid-hydrolyzed month of growing L. leucocephala in greenhouse showed using HCN and added with anthrone reagent. Samples were that the increasing NaCl concentration caused significant read at A and estimated for their carbohydrate content reduction to plant height of L. leucocephala seedlings from (mg/g) with the standard solution using glucose. 21.5% (150mM) to44.6% (450mM) compared to controls at 0mM (Figure 3). (e biomass of L. leucocephala also ex- perienced significant reduction from 20.96% (150mM) to 2.8. Proline Content. Leaf proline content was estimated 61.35% (450mM) compared to controls at 0mM (Figure 4). from the leaf sample solution as prepared previously using In the presence of AMF, the height and biomass of the method described by Monneveux and Nemmar [28]. L. leucocephala seedlings were higher and significant com- Samples were read at A and estimated for their proline pared to controls at all levels of salinity stress. (e appli- content (µmol/g) with the standard solution using proline. cation of AMF consortium (G. manihotis+ G. etunicatum) was observed to significantly alleviate the salt stress better 2.9. Data Analysis. (e data were analyzed using a two- than the application of single AMF species even matching factor analysis of variance (ANOVA) obtained from salinity the biomass in control plants at 150mM and 300mM level and AMF treatment at the α level of 5%, followed by a treatments. In addition, the AMF consortium also promoted pairwisecomparisonwith TukeytestusingMinitabver.16.0. the best growth performance (height, biomass) of GraphicalimagesweregeneratedusingGraphPadPrismver. L. leucocephala seedlings in this study. 8.0.2. 3.3. Effect of AMF Inoculation on the Total Protein, Carbo- 3. Results hydrate, and Proline Contents of L. leucocephala Leaves under 3.1. Identification of Indigenous AMF. Two samples of in- Salt Stress. (e total protein, total carbohydrate, and total digenous AMF were sent for molecular identification. DNA and proline contents in L. leucocephala fresh leaves were sequencing result showed the sequence of amplified region quantified after 1 month of salt stress and the results showed (partial 18S (SSU), 5.8S (ITS), and partial 28S (LSU)) rDNA asignificantincrease inAMF-inoculated plants comparedto of two Glomus isolates, each with a size of 1,796bp for control plants at all concentrations (Figures 5 and 6). (e Glomus sp1 and 1,764bp for Glomus sp2. Based on the interaction between salinity and AMF treatment was not bioinformatic analysis and phylogenetic construction significant for total protein and proline content indicating among members of Glomeromycota, it was revealed that that the salinity did not affect the AMF performance in 4 International Journal of Forestry Research 97 Glomus sp1 Glomus manihotis (Y17648.3) Glomus fasciculatum (Y17640.2) Glomus intraradices (X58725.1) Glomus microcarpum (LC379062.1) Glomus geosporum (Y17643.1) Glomus fragilistratum (AJ276085.2) Glomus mosseae (U96139.1) Glomus coronatum (AJ276086.2) Glomus luteum (AJ276089.3) Glomus lamellosum (AJ276087.2) Glomus etunicatum (AJ852598.1) Glomus sp2 Archaeospora trappei (Y17634.3) 0.010 Figure 2: Phylogenetic tree of partial 18S (SSU), 5.8S (ITS), and partial 28S (LSU) rDNA sequences of 14 Glomeromycota representatives. Neighbor-joining (NJ) analysis including Archaeospora trappei as outgroup. Bootstrap values are given for each branch (BV>50). Scale bar indicates the number of substitutions per site. ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF gh fg i hi 40 c ef 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 3: Effects of AMF inoculation on the height growth of L. leucocephala seedlings at different NaCl stress. AMF: arbuscular my- ∗ ∗∗ corrhizal 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. elevating or lowering the total protein and proline contents G. etunicatum did not play an important role in the accu- in L. leucocephala. Meanwhile, the total carbohydrate mulation of the proteins and sugars in L. leucocephala leaves content in L. leucocephala leaves was significant in both compared to control plants. treatment and its interactions between salinity and AMF treatment (Figure 7). (e total protein and carbohydrate 3.4. Effect of Salt Stress on the Root Colonization of contents in control plants were observed for its maximum value at 150mM NaCl, although insignificant for total L. leucocephala. Root colonization of L. leucocephala by carbohydrate parameter. (e highest proline content in AMF isolates was observed microscopically after 1 month of control plants was observed at 300mM and insignificant saltstress.Incontrolplants,nocolonizationwasobservedby among the group. (e inoculation of AMF consortium other indigenous AMF due to the application of sterilized increased the total protein, total carbohydrate, and proline zeolites as growth medium. (e microscopic images showed content of L. leucocephala seedlings which almost was that each AMF treatment was successful as indicated by the matched by the single inoculation of G. manihotis. Based on presence of hyphal structures in all samples with some these results, it was revealed that the inoculation of vesicles andfewarbuscules (Figure8).Soil salinity decreased Plant height (cm) International Journal of Forestry Research 5 2.0 ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF 1.5 bb cd 1.0 d d ef ef fg gh hi 0.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 4: Effects of AMF inoculation on the biomass 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. 20.0 ∗∗ ∗∗ ns P P P Salt AMF Salt×AMF 17.5 15.0 a a ab ab 12.5 bc bc cd cd cd cde de def def 10.0 ef 7.5 5.0 2.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium 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. therootcolonizationof L. leucocephalainalltreatments.(e among parameters was priorly analyzed using Pearson’s AMF colonization decreased by 30%, 29%, and 32% (in the correlation coefficient test (Table 1). (e height of G. manihotis, G. etunicatum, and AMF consortium, re- L. leucocephala was negatively correlated with the biomass spectively) in the 450mM treatments compared to control which may indicate the limit of growing the seedlings in plants (0mM NaCl) (Figure 9). (ere was a significant greenhouse. (e carbohydrate content in L. leucocephala difference between salinity and AMF treatments. (e ap- leaves was negatively correlated with the plant height and positivelycorrelatedwiththebiomass.(eprolinecontentwas plication of single AMF isolate, G. manihotis, almost matched the root colonization by the AMF consortium in negatively correlated with the biomass indicating a stress the 150, 300, and 450mM treatments while the colonization responsetothesaltstress.RootcolonizationbyAMFshoweda by G. etunicatum was recorded the lowest in our study. 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 3.5. PCA Analysis. Multivariate analysis of all datasets was proline content in L. leucocephala leaves was less likely cor- meant to depict the physiological response patterns of relatedwiththerootcolonizationbyAMFwhichgaveusaclue L. leucocephalatosaltstressandAMFinoculation.Correlation on other tolerance mechanisms under salt stress. All Total protein (mg/g) Total biomass (g) 6 International Journal of Forestry Research 5.0 ∗∗ ∗∗ ns P P P Salt AMF Salt×AMF 4.5 4.0 abcd 3.5 abcd abc bcde bdef efgh cdef defg 3.0 fghi fghij ijk ghijk hijk jk 2.5 2.0 1.5 1.0 0.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 6: Effects of AMF inoculation on the proline 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. ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF bc 20 c ef de h def a c 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 7: Effects of AMF inoculation on the total carbohydrate 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 rep- 4. Discussion resentative dimensions. (e distance between treatments in the score plot of PCA showed the similarity of tested pa- Salinity stress is a global limiting factor that reduces the rameters. (e PCA components, PC1 and PC2, accounted for growth and viability of valuable plants cultivated in the agricultural fields [29]. Excessive salt ions in soil environ- 57%and25%ofthevariance,respectively,yetwerecategorized as optimum in depicting the correlation among parameters ment impose serious physiological stressors to the plants with the biological consequences like reduced photosyn- (PC >70%). Based on the plot, it can be seen that control 1+2 plants and AMF-inoculated plants were separated in different thesis capacity, cellular dehydration, and nutrient defi- quadrants showing distinct results of individual parameters ciencies [7, 18, 30]. (e effect of AMF inoculation to (Figure 10). (e growth response of L. leucocephala seedlings L. leucocephala plants was studied through an experiment of also tends to be grouped together under type of AMF treat- different NaCl concentrations (0, 150, 300, and 450mM) for ment rather than at different salt levels. (e application of 1 month under greenhouse. Inoculation of AMF into either single AMF species or consortium could enhance the L. leucocephala seedlings has been reported previously and studied mostly under heavy metal toxicity experiments. salttoleranceof L. leucocephalaseedlings,especiallyat150and 300mM NaCl. AMF species such as Glomus aggregatum, Glomus Total carbohydrate (mg/g) Proline content (µmol/g) International Journal of Forestry Research 7 ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF bc de def f ef h h h h 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 8: Examples of AMF structures as indicators of L. leucocephala colonization. A. Dissected root of L. leucocephala observed at 4x showing (a) external hyphae of AMF; B. Internal root tissue of L. leucocephala stained with trypan blue showing (a) hyphae and (b) vesicle observed at 400x; C. Root cell containing an (c) arbuscule observed at 1000x. (a) b c (b) (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. etunicatum (syn. Claroideoglomus etunicatum), Acaulospora one of the indigenous AMF was considered unique since the longula, and Acaulospora scrobiculata have been imple- species was known to be abundant in cassava [33, 34]. In mented successfully as bioameliorants in planta [15, 31, 32]. addition, there is still no recent record on the application of Ourfindingon G. manihotis(syn. Rhizophagus manihotis)as G. etunicatum, especially G. manihotis on L. leucocephala Root colonization (%) 8 International Journal of Forestry Research Table 1: Pearson’s correlation matrix (r) of all morphological and physiological parameters. Height Biomass Protein Carbohydrate Proline Colonization Height Biomass −0.820 Protein −0.461 0.329 Carbohydrate −0.672 0.595 0.431 Proline 0.135 −0.492 0.277 0.157 Colonization 0.762 0.595 0.431 0.815 0.157 S2 S2 2 S2 S2 S2 S2 S3 S3 S3 S3 S2 S2 S2 S3 S1 S3 S1 S2 S3 S3 S1 S1 S1 S2 S3 S3 S1 S3 S1 S1 S2 S3 S1 S1 S0 S1 –1 S0 S0 S0 S0 S1 S0 S0 S0 S0 S0 S0 –2 S0 –3 –4 –3 –2 –1 0 1 2 3 PC1 (57%) Control Glomus etunicatum Glomus manihotis Consortium Figure 10: PCA plots of the two principal components of L. leucocephala, S0: control/0mM NaCl, S1:150mM NaCl, S2: 300mM NaCl, S3: 450mM NaCl; PC1: principal component 1, PC2: principal component 2. undersalinity stress. Hence, our resultsmay be beneficial for control plants (0mM NaCl) in this study. Application of future investigation and possible formulation as a product. AMF is effective to support the vegetative growth of plants (e AMF colonization rate in L. leucocephala roots was through improved root absorption especially phosphorus considered as stable under severe salt stress, although the (P) under abiotic stress which helps to maintain the highest percentage of colonization only reached >30% for structural integrity or biomass of the plants [36]. Mea- G. manihotis and AMF consortium in the 450mM NaCl surement of total soluble protein in NaCl-affected treatment. Salt stress may inhibit the mycelial growth and L. leucocephala seedlings was meant to estimate the stability formation of vesicles in the mycelial network due to the of dissolved membrane and pigment proteins. In this study, the interaction between salinity levels and AMF inoculation toxicity of excessive sodium ions [35]. A significant decline in root colonization also indicated that the protective effect was not significant which means that there are other factors affecting the result. A study on a tall perennial cane species, by preinoculation of AMF was less potent at higher salinity levels and diminished hereafter. In this study, the excessive Arundo donax, showed that the leaf protein content was salt ions reduced the root colonization capacity by AMF in lower in salt-stressed plants than control plants and the the 150 and 300mM NaCl. (e early response of interaction was more likely by nutrition than AMF treat- L. leucocephala towards salt stress may be regulated in the ment. In addition, the chlorophyll content was not signif- form of delayed growth. Reduced cell growth of icant in the moderate salt stress as supplied by adequate C L. leucocephala manifested in the form of shorter plant and and P nutrition [37]. Another possible mechanism as low biomass under salinity stress is normal but the appli- revealed from the correlation test between root colonization cation of AMF also proved to promote growth better than and leaf protein content is that AMF may also stabilize the PC2 (25%) International Journal of Forestry Research 9 Na translocation from the root system to the shoot system as indicated by a strong relationship between the root by maintaining internal Na+ concentration in the mycelial colonization and height, biomass, and carbohydrate content network, thuspreventingitsaccumulationandtranslocation and multivariate analysis through PCA projection. Based on into the photosynthetic tissues [38, 39]. Salinity may also these results, the tolerance of L. leucocephala was improved induce the accumulation of glomalin, a typical heat shock to some degree, while also opening up the possibilities to protein produced by AMF in the soil environment [40]. further study in acquiring a deeper understanding of other However, the distribution of this protein was more likely to possible mechanisms facilitated through an AMF symbiosis improve the soil integrity to maximize mineral and nutrient with the plants. absorption for the host. It would be interesting for further study to evaluate these parameters in salt-stressed Data Availability L. leucocephala. Tissue dehydration under salt stress may be prevented by the accumulation of osmolytes such as proline, (e data are available upon request provided by the cor- sugars, organic acids, amino acids, and trehalose [41]. (ese responding author’s laboratory record at the Department of molecules serve as osmoprotectants to stabilize protein and Forestry, Faculty of Forestry, Universitas Sumatera Utara, membrane integrity and scavenging of outgoing ROS in- Indonesia. cidence [42]. Here, the leaf proline content of salt-stressed L. leucocephala was quantified and showed that there was no Conflicts of Interest significant interaction between salinity levels and AMF treatments. (e reports on the accumulation of proline have (e authors declare that there are no conflicts of interest been inconsistent between mycorrhizal and nonmycorrhizal regarding the publication of this work. plants [18]. However, it is generally accepted that under salt stress the accumulation of proline as a stress marker by Acknowledgments plants is stable due to the presence of AMF involving other possible mitigation mechanisms [43]. Accumulation of (e authors would like to express their highest gratitude to carbohydrates or soluble sugars by plants is also considered the head of laboratory and fellow researchers in the Labo- as another salt-induced mechanism in adjusting the osmotic ratory of Forest Biotechnology, Research Center for Bio- balance and as carbon storage [44]. Here, the sugar content resources and Biotechnology (PPHSB), IPB University, in L. leucocephala leaves was considerably similar in control Bogor, for facilitating the instrumentation and the labora- plants; however, there was a significant interaction between tory facilities. salinity levels and AMF treatments with the highest sugar contentinthe150mMNaClcomparedtocontrolplants and References then decreasing hereafter. (e relationship between root colonization and carbohydrate content was strongly cor- [1] C. L. Garcia, S. Dattamudi, S. Chanda, and K. 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Improved Salt Tolerance of Lamtoro (Leucaena leucocephala) through the Application of Indigenous Mycorrhiza

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Copyright © 2021 Delvian Delvian and Adrian Hartanto. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi International Journal of Forestry Research Volume 2021, Article ID 8100480, 11 pages https://doi.org/10.1155/2021/8100480 Research Article Improved Salt Tolerance of Lamtoro (Leucaena leucocephala) through the Application of Indigenous Mycorrhiza 1 2 Delvian Delvian and Adrian Hartanto Department of Forestry, Faculty of Forestry, Universitas Sumatera Utara, Medan, North Sumatra 20155, Indonesia Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, North Sumatra 20155, Indonesia Correspondence should be addressed to Delvian Delvian; delvian@usu.ac.id Received 17 May 2021; Accepted 24 July 2021; Published 31 July 2021 Academic Editor: Monika Markovic´ Copyright © 2021 Delvian Delvian and Adrian Hartanto. (is is an open access article distributed under the Creative Commons AttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkis properly cited. 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 stressthroughthestructuralandphysiologicaladjustment.(isstudyaimedtodeterminearelationshipbetweensalinity 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. (ese 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. to thrive in the environment by decreasing plant height, 1. Introduction lowering photosynthetic capacity and nutrient absorption Conversion of agriculture land into human settlement and with death penalty [5]. Some strategies have been employed industry has led to the decrease territory of arable lands. to maximize the use of saline soils, one of which through the Upcoming climate change with consequences on the rise of application of soil-borne microorganisms, such as bio- sea level, sea water intrusion, and high evaporation was amelioration by arbuscular mycorrhizal fungi (AMF) [6, 7]. regarded as a major environmental issue which also posed Lamtoro (Leucaena leucocephala (Lam.) de Wit) from some challenges in the cultivation of economical crops [1]. family Fabaceae is a pioneer legume species from Central (e consequence of this land conversion has directed America known for its notable nitrogen fixation capacity farmers to utilize marginal lands such as saline soils [2]. and fast-growing woody plant. (e species has been utilized Saline soils are characterized by their high salt content as biofertilizer or green leaf manuring to improve soil fer- (NaCl, Na CO , Na SO ) with electric conductivity >4dS/ tility, as forage for animal feed, and a prominent source of 2 3 2 4 m≈40mM NaCl which deter the optimum growth and high quality timber [8–10]. In Indonesia, the species has development of many horticultural crops around the world been cultivated since 1800 with some of its cultivars being [3]. Saline soils in Indonesia cover an area of 27,4 million ha recognizedasimportanttimberproducersduetoitsvaluable with potential being utilized for cultivation of salt tolerant traits [11, 12]. (erefore, the species is well-adapted towards crops[4].However,excessivesaltionswilllimitnativeplants abioticstress in marginallands whilebeing proveduseful for 2 International Journal of Forestry Research afforestation and landscaping [13]. Studies have also 2.2. Plant and Soil Treatments. Seeds of L. leucocephala, revealed the significant contribution of AMF to the im- provided by the Research Institute of Haurbentes (Bogor, proved growth performance of L. leucocephala seedlings Indonesia), were disinfected with 1% (v/v) NaOCl solution under stress condition especially in heavy metal contami- for 20min, washed three times with distilled water, and nated sites [14–16]. soaked in sterile water for 24h. After that,the seedswere put Plants maintain their growth and development under on a seed tray covered with river sand and rapidly germi- salinity stress through biological adjustment to thrive in the nated in a greenhouse. (e seedlings were exposed to harsh environments. (e adjustment may vary such as ac- sunlight for 12h every day, and the water was given cumulation of compatible solutes, production of ROS- conditionally. Once the seedlings had grown to 10cm and scavenging enzymes, induction of phytohormones, and ion- produced two leaves, they were put into a plastic cup homeostasis balancing [17]. However, the performance may (93.4mm ×65.82mm, 5 holes) filled with sterilized zeolite belimitedduetotherapidlychangingenvironmentandhigh (particle size �1mm)+AMF inoculum in a ratio of 1:1 influx of salt ions which demand a more stable adjustment (w/w) or in a ratio of 2:1:1 for AMF consortium (Glomus sp1+ Glomus sp2) and maintained in a greenhouse for from the plant [18]. Symbiotic establishment between AMF and plant species was initiated 400 million years ago and salinity treatment. considered as stable mutualism which form inter-regulation and enhancement on the survivability of terrestrial plants 2.3. Experimental Design. (e experiment was arranged in a [19]. (e salt resistance and growth improvement conferred Randomized Complete Block Design (RCBD) with two by AMF may be different across species and strain origins. factors (Figure 1): AMF inoculation (Glomus sp1, Glomus Claroideoglomus etunicatum colonizing the roots of rice sp2, and Glomus sp1+ Glomus sp2) and salt stress (0, 150, plant (Oryza sativa L.) showed enhancements in terms of 300, and 450mM) and five replications for each, totaling photosynthetic productivity and stomatal conductance 4 ×4 ×5 �80 pots. (e 60 seedlings inside the plastic cups under salinity stress [20]. Another strain of C. etunicatum were transplanted in contact with the top layer of colonizing the roots of a halophytic C grass species 15cm ×14cm pots (no hole) filled with saline waters (150, Aeluropus littoralis has been reported to increase the shoot 300, and 450mM) and grown in a greenhouse. Pots without and root biomass of its host following other adaptive traits AMF inoculum were prepared as nonmycorrhizal controls. such as improved stomatal conductance, synthesis of (e seedlings were watered with distilled water every day. compatible solutes, and balanced ion uptakes [21]. Another Supplementation of Hyponex (N, P, K �25%, 5%, 20%) examplefromlegumespeciesalfalfa(Medicago sativaL.),the ® was given once in two weeks with a concentration of 2g/L. coinoculation of AMF and rhizobia improved the yield (e treatments were maintained for 1 month. through increases in mycorrhizal colonization, rhizobia nodulation, root Ca content, and shoot proline content [22]. Based on our understanding, the information of AMF 2.4. AMF Colonization. After 1 month of saline treatment, colonization in L. leucocephala under saline conditions is the seedlings were harvested, and the fresh roots were still limited. (e present study then investigates the role of collected. (e roots were washed, cut into 1cm segments, two indigenous AMF following their biological outputs in and fixed with 10% KOH at 90 C until being colorless. (e maintaining the normal growth of L. leucocephala under root segments were fixed with 10% HCl and stained with salinity stress by examining physiological adjustment by 0.05% trypan blue at 90 C for 15min [24]. (e AM colo- the plant. (is study will also discriminate which fungal nization rate was determined by the gridline intersection species is the better option as an AM fungal inoculant or method [25]. Data were recorded as the proportion of root AMF consortium for improving salt tolerance in length colonized. L. leucocephala. 2.5. Plant Height and Total Biomass. Measurementof growth 2. Materials and Methods response under saline treatment was expressed as the av- erage growth in plant height over one month, determined 2.1. AMF Identification and Inoculum Preparation. Two from the beginning and at the end of a 1-month saline indigenous AMF isolates, Glomus sp1 and Glomus sp2, were treatment. (e seedlings were dried in an oven at 60 C until isolated in a previous study using Pueraria javanica as host constant weight (g) to obtain the total biomass of through trap cultures and single spore isolation. Spore L. leucocephala. suspension was prepared for molecular identification. Ge- nomic DNA extraction and nested polymerase chain reac- tion (PCR) were performed commercially by Macrogen, Inc. 2.6. Total Protein Content. Fresh leaves from each treatment (Singapore). Molecular identification of AMF was based on were sampled and washed with running tap water. One g of the amplified nuclear rDNA fragments using a pair of leaves was crushed and diluted with 20mL of phosphate SSUmAf/LSUmAr and SSUmCf/LSUmBr primers [23]. buffer saline (pH 7.4). (e solution was centrifuged at Crude inoculum was prepared in sterilized zeolite filled with 10,000rpm for 10min and then supernatants were collected. colonized root segments of P. javanica maintained in dry Five mL of Quick Start Bradford Protein Assay (Bio-Rad, state. (e AMF inoculum was then preserved for further US) containing Coomassie Brilliant Blue G-250 was mixed experiment. with 0.1mL of sample solution [26]. Samples were incubated International Journal of Forestry Research 3 AMF AMF AMF Nonmycorrhizal Treatment 1 Treatment 2 Consortium (T0) (T1) (T2) (T3) AMF = Arbuscular mycorrhizal fungi 0 mM NaCl S0 = (–) NaCl (S0) T0 = (–) AMF T1 = Glomussp1 T2 = Glomussp2 150 mM NaCl T3 = Glomussp1 + Glomussp2 (S1) 300 mM NaCl (S2) 450 mM NaCl (S3) Figure1:Experimentallayout(4 ×4factorial)forthegreenhouseexperiment,includingsalinityandAMFtreatments.Eachtreatmenthad5 replications. at room temperature for 5min and then read at A . Es- Glomus sp1 was identified as Glomus manihotis while timation of leaf protein (mg/g) content was compared to the Glomus sp2 was identified as Glomus etunicatum compared standard solution using bovine serum albumin (BSA). to the DNA sequence of Archaeospora trappei as an out- group (Figure 2). 2.7. Total Carbohydrate Content. Total carbohydrate con- taining polysaccharide and free sugars was estimated from 3.2. Effect of AMF Inoculation on Height and Biomass of the leaf sample solution as prepared previously using L. leucocephala under Salt Stress. Results obtained after 1 anthrone method [27]. (e solution was acid-hydrolyzed month of growing L. leucocephala in greenhouse showed using HCN and added with anthrone reagent. Samples were that the increasing NaCl concentration caused significant read at A and estimated for their carbohydrate content reduction to plant height of L. leucocephala seedlings from (mg/g) with the standard solution using glucose. 21.5% (150mM) to44.6% (450mM) compared to controls at 0mM (Figure 3). (e biomass of L. leucocephala also ex- perienced significant reduction from 20.96% (150mM) to 2.8. Proline Content. Leaf proline content was estimated 61.35% (450mM) compared to controls at 0mM (Figure 4). from the leaf sample solution as prepared previously using In the presence of AMF, the height and biomass of the method described by Monneveux and Nemmar [28]. L. leucocephala seedlings were higher and significant com- Samples were read at A and estimated for their proline pared to controls at all levels of salinity stress. (e appli- content (µmol/g) with the standard solution using proline. cation of AMF consortium (G. manihotis+ G. etunicatum) was observed to significantly alleviate the salt stress better 2.9. Data Analysis. (e data were analyzed using a two- than the application of single AMF species even matching factor analysis of variance (ANOVA) obtained from salinity the biomass in control plants at 150mM and 300mM level and AMF treatment at the α level of 5%, followed by a treatments. In addition, the AMF consortium also promoted pairwisecomparisonwith TukeytestusingMinitabver.16.0. the best growth performance (height, biomass) of GraphicalimagesweregeneratedusingGraphPadPrismver. L. leucocephala seedlings in this study. 8.0.2. 3.3. Effect of AMF Inoculation on the Total Protein, Carbo- 3. Results hydrate, and Proline Contents of L. leucocephala Leaves under 3.1. Identification of Indigenous AMF. Two samples of in- Salt Stress. (e total protein, total carbohydrate, and total digenous AMF were sent for molecular identification. DNA and proline contents in L. leucocephala fresh leaves were sequencing result showed the sequence of amplified region quantified after 1 month of salt stress and the results showed (partial 18S (SSU), 5.8S (ITS), and partial 28S (LSU)) rDNA asignificantincrease inAMF-inoculated plants comparedto of two Glomus isolates, each with a size of 1,796bp for control plants at all concentrations (Figures 5 and 6). (e Glomus sp1 and 1,764bp for Glomus sp2. Based on the interaction between salinity and AMF treatment was not bioinformatic analysis and phylogenetic construction significant for total protein and proline content indicating among members of Glomeromycota, it was revealed that that the salinity did not affect the AMF performance in 4 International Journal of Forestry Research 97 Glomus sp1 Glomus manihotis (Y17648.3) Glomus fasciculatum (Y17640.2) Glomus intraradices (X58725.1) Glomus microcarpum (LC379062.1) Glomus geosporum (Y17643.1) Glomus fragilistratum (AJ276085.2) Glomus mosseae (U96139.1) Glomus coronatum (AJ276086.2) Glomus luteum (AJ276089.3) Glomus lamellosum (AJ276087.2) Glomus etunicatum (AJ852598.1) Glomus sp2 Archaeospora trappei (Y17634.3) 0.010 Figure 2: Phylogenetic tree of partial 18S (SSU), 5.8S (ITS), and partial 28S (LSU) rDNA sequences of 14 Glomeromycota representatives. Neighbor-joining (NJ) analysis including Archaeospora trappei as outgroup. Bootstrap values are given for each branch (BV>50). Scale bar indicates the number of substitutions per site. ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF gh fg i hi 40 c ef 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 3: Effects of AMF inoculation on the height growth of L. leucocephala seedlings at different NaCl stress. AMF: arbuscular my- ∗ ∗∗ corrhizal 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. elevating or lowering the total protein and proline contents G. etunicatum did not play an important role in the accu- in L. leucocephala. Meanwhile, the total carbohydrate mulation of the proteins and sugars in L. leucocephala leaves content in L. leucocephala leaves was significant in both compared to control plants. treatment and its interactions between salinity and AMF treatment (Figure 7). (e total protein and carbohydrate 3.4. Effect of Salt Stress on the Root Colonization of contents in control plants were observed for its maximum value at 150mM NaCl, although insignificant for total L. leucocephala. Root colonization of L. leucocephala by carbohydrate parameter. (e highest proline content in AMF isolates was observed microscopically after 1 month of control plants was observed at 300mM and insignificant saltstress.Incontrolplants,nocolonizationwasobservedby among the group. (e inoculation of AMF consortium other indigenous AMF due to the application of sterilized increased the total protein, total carbohydrate, and proline zeolites as growth medium. (e microscopic images showed content of L. leucocephala seedlings which almost was that each AMF treatment was successful as indicated by the matched by the single inoculation of G. manihotis. Based on presence of hyphal structures in all samples with some these results, it was revealed that the inoculation of vesicles andfewarbuscules (Figure8).Soil salinity decreased Plant height (cm) International Journal of Forestry Research 5 2.0 ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF 1.5 bb cd 1.0 d d ef ef fg gh hi 0.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 4: Effects of AMF inoculation on the biomass 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. 20.0 ∗∗ ∗∗ ns P P P Salt AMF Salt×AMF 17.5 15.0 a a ab ab 12.5 bc bc cd cd cd cde de def def 10.0 ef 7.5 5.0 2.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium 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. therootcolonizationof L. leucocephalainalltreatments.(e among parameters was priorly analyzed using Pearson’s AMF colonization decreased by 30%, 29%, and 32% (in the correlation coefficient test (Table 1). (e height of G. manihotis, G. etunicatum, and AMF consortium, re- L. leucocephala was negatively correlated with the biomass spectively) in the 450mM treatments compared to control which may indicate the limit of growing the seedlings in plants (0mM NaCl) (Figure 9). (ere was a significant greenhouse. (e carbohydrate content in L. leucocephala difference between salinity and AMF treatments. (e ap- leaves was negatively correlated with the plant height and positivelycorrelatedwiththebiomass.(eprolinecontentwas plication of single AMF isolate, G. manihotis, almost matched the root colonization by the AMF consortium in negatively correlated with the biomass indicating a stress the 150, 300, and 450mM treatments while the colonization responsetothesaltstress.RootcolonizationbyAMFshoweda by G. etunicatum was recorded the lowest in our study. 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 3.5. PCA Analysis. Multivariate analysis of all datasets was proline content in L. leucocephala leaves was less likely cor- meant to depict the physiological response patterns of relatedwiththerootcolonizationbyAMFwhichgaveusaclue L. leucocephalatosaltstressandAMFinoculation.Correlation on other tolerance mechanisms under salt stress. All Total protein (mg/g) Total biomass (g) 6 International Journal of Forestry Research 5.0 ∗∗ ∗∗ ns P P P Salt AMF Salt×AMF 4.5 4.0 abcd 3.5 abcd abc bcde bdef efgh cdef defg 3.0 fghi fghij ijk ghijk hijk jk 2.5 2.0 1.5 1.0 0.5 0.0 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 6: Effects of AMF inoculation on the proline 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. ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF bc 20 c ef de h def a c 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 7: Effects of AMF inoculation on the total carbohydrate 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 rep- 4. Discussion resentative dimensions. (e distance between treatments in the score plot of PCA showed the similarity of tested pa- Salinity stress is a global limiting factor that reduces the rameters. (e PCA components, PC1 and PC2, accounted for growth and viability of valuable plants cultivated in the agricultural fields [29]. Excessive salt ions in soil environ- 57%and25%ofthevariance,respectively,yetwerecategorized as optimum in depicting the correlation among parameters ment impose serious physiological stressors to the plants with the biological consequences like reduced photosyn- (PC >70%). Based on the plot, it can be seen that control 1+2 plants and AMF-inoculated plants were separated in different thesis capacity, cellular dehydration, and nutrient defi- quadrants showing distinct results of individual parameters ciencies [7, 18, 30]. (e effect of AMF inoculation to (Figure 10). (e growth response of L. leucocephala seedlings L. leucocephala plants was studied through an experiment of also tends to be grouped together under type of AMF treat- different NaCl concentrations (0, 150, 300, and 450mM) for ment rather than at different salt levels. (e application of 1 month under greenhouse. Inoculation of AMF into either single AMF species or consortium could enhance the L. leucocephala seedlings has been reported previously and studied mostly under heavy metal toxicity experiments. salttoleranceof L. leucocephalaseedlings,especiallyat150and 300mM NaCl. AMF species such as Glomus aggregatum, Glomus Total carbohydrate (mg/g) Proline content (µmol/g) International Journal of Forestry Research 7 ∗∗ ∗∗ ∗∗ P P P Salt AMF Salt×AMF bc de def f ef h h h h 0 150 300 450 Salinity level (mM) (–) AMF Glomus etunicatum Glomus manihotis Consortium Figure 8: Examples of AMF structures as indicators of L. leucocephala colonization. A. Dissected root of L. leucocephala observed at 4x showing (a) external hyphae of AMF; B. Internal root tissue of L. leucocephala stained with trypan blue showing (a) hyphae and (b) vesicle observed at 400x; C. Root cell containing an (c) arbuscule observed at 1000x. (a) b c (b) (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. etunicatum (syn. Claroideoglomus etunicatum), Acaulospora one of the indigenous AMF was considered unique since the longula, and Acaulospora scrobiculata have been imple- species was known to be abundant in cassava [33, 34]. In mented successfully as bioameliorants in planta [15, 31, 32]. addition, there is still no recent record on the application of Ourfindingon G. manihotis(syn. Rhizophagus manihotis)as G. etunicatum, especially G. manihotis on L. leucocephala Root colonization (%) 8 International Journal of Forestry Research Table 1: Pearson’s correlation matrix (r) of all morphological and physiological parameters. Height Biomass Protein Carbohydrate Proline Colonization Height Biomass −0.820 Protein −0.461 0.329 Carbohydrate −0.672 0.595 0.431 Proline 0.135 −0.492 0.277 0.157 Colonization 0.762 0.595 0.431 0.815 0.157 S2 S2 2 S2 S2 S2 S2 S3 S3 S3 S3 S2 S2 S2 S3 S1 S3 S1 S2 S3 S3 S1 S1 S1 S2 S3 S3 S1 S3 S1 S1 S2 S3 S1 S1 S0 S1 –1 S0 S0 S0 S0 S1 S0 S0 S0 S0 S0 S0 –2 S0 –3 –4 –3 –2 –1 0 1 2 3 PC1 (57%) Control Glomus etunicatum Glomus manihotis Consortium Figure 10: PCA plots of the two principal components of L. leucocephala, S0: control/0mM NaCl, S1:150mM NaCl, S2: 300mM NaCl, S3: 450mM NaCl; PC1: principal component 1, PC2: principal component 2. undersalinity stress. Hence, our resultsmay be beneficial for control plants (0mM NaCl) in this study. Application of future investigation and possible formulation as a product. AMF is effective to support the vegetative growth of plants (e AMF colonization rate in L. leucocephala roots was through improved root absorption especially phosphorus considered as stable under severe salt stress, although the (P) under abiotic stress which helps to maintain the highest percentage of colonization only reached >30% for structural integrity or biomass of the plants [36]. Mea- G. manihotis and AMF consortium in the 450mM NaCl surement of total soluble protein in NaCl-affected treatment. Salt stress may inhibit the mycelial growth and L. leucocephala seedlings was meant to estimate the stability formation of vesicles in the mycelial network due to the of dissolved membrane and pigment proteins. In this study, the interaction between salinity levels and AMF inoculation toxicity of excessive sodium ions [35]. A significant decline in root colonization also indicated that the protective effect was not significant which means that there are other factors affecting the result. A study on a tall perennial cane species, by preinoculation of AMF was less potent at higher salinity levels and diminished hereafter. In this study, the excessive Arundo donax, showed that the leaf protein content was salt ions reduced the root colonization capacity by AMF in lower in salt-stressed plants than control plants and the the 150 and 300mM NaCl. (e early response of interaction was more likely by nutrition than AMF treat- L. leucocephala towards salt stress may be regulated in the ment. In addition, the chlorophyll content was not signif- form of delayed growth. Reduced cell growth of icant in the moderate salt stress as supplied by adequate C L. leucocephala manifested in the form of shorter plant and and P nutrition [37]. Another possible mechanism as low biomass under salinity stress is normal but the appli- revealed from the correlation test between root colonization cation of AMF also proved to promote growth better than and leaf protein content is that AMF may also stabilize the PC2 (25%) International Journal of Forestry Research 9 Na translocation from the root system to the shoot system as indicated by a strong relationship between the root by maintaining internal Na+ concentration in the mycelial colonization and height, biomass, and carbohydrate content network, thuspreventingitsaccumulationandtranslocation and multivariate analysis through PCA projection. Based on into the photosynthetic tissues [38, 39]. Salinity may also these results, the tolerance of L. leucocephala was improved induce the accumulation of glomalin, a typical heat shock to some degree, while also opening up the possibilities to protein produced by AMF in the soil environment [40]. further study in acquiring a deeper understanding of other However, the distribution of this protein was more likely to possible mechanisms facilitated through an AMF symbiosis improve the soil integrity to maximize mineral and nutrient with the plants. absorption for the host. It would be interesting for further study to evaluate these parameters in salt-stressed Data Availability L. leucocephala. Tissue dehydration under salt stress may be prevented by the accumulation of osmolytes such as proline, (e data are available upon request provided by the cor- sugars, organic acids, amino acids, and trehalose [41]. (ese responding author’s laboratory record at the Department of molecules serve as osmoprotectants to stabilize protein and Forestry, Faculty of Forestry, Universitas Sumatera Utara, membrane integrity and scavenging of outgoing ROS in- Indonesia. cidence [42]. Here, the leaf proline content of salt-stressed L. leucocephala was quantified and showed that there was no Conflicts of Interest significant interaction between salinity levels and AMF treatments. (e reports on the accumulation of proline have (e authors declare that there are no conflicts of interest been inconsistent between mycorrhizal and nonmycorrhizal regarding the publication of this work. plants [18]. However, it is generally accepted that under salt stress the accumulation of proline as a stress marker by Acknowledgments plants is stable due to the presence of AMF involving other possible mitigation mechanisms [43]. Accumulation of (e authors would like to express their highest gratitude to carbohydrates or soluble sugars by plants is also considered the head of laboratory and fellow researchers in the Labo- as another salt-induced mechanism in adjusting the osmotic ratory of Forest Biotechnology, Research Center for Bio- balance and as carbon storage [44]. Here, the sugar content resources and Biotechnology (PPHSB), IPB University, in L. leucocephala leaves was considerably similar in control Bogor, for facilitating the instrumentation and the labora- plants; however, there was a significant interaction between tory facilities. salinity levels and AMF treatments with the highest sugar contentinthe150mMNaClcomparedtocontrolplants and References then decreasing hereafter. (e relationship between root colonization and carbohydrate content was strongly cor- [1] C. L. Garcia, S. Dattamudi, S. Chanda, and K. 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International Journal of Forestry ResearchHindawi Publishing Corporation

Published: Jul 31, 2021

References