Abstract
Shepherdia ×utahensis ‘Torrey’ (hybrid buffaloberry) is an actinorhizal plant that can form symbiotic nodules with the actinobacterial genus Frankia. However, little research has been conducted to investigate the presence of Frankia in their nodules and the effects on plant growth. In this study, plants were grown in a Metro-Mix® 820 substrate and inoculated with soils collected from Mohave County, AZ, or in a low organic-matter substrate inoculated with soils from North Logan, UT. The presence of Frankia was quantified using PolF/PolR primers to amplify their nitrogenase (nifH) gene sequences. In the Metro-Mix 820 substrate, plants irrigated with nitrogen (N)-free Hoagland’s solution at pH 6.5 formed nodules at week 12 after experiment initiation, whereas those receiving the same solution with 2 mm ammonium nitrate (NH4NO3) appeared healthy, but no nodules formed. In the low organic-matter substrate, nodules formed in 5 weeks when plants were irrigated with N-free Hoagland’s solution at pH 7.5. Four 300-bp fragments of query sequences (SU1, SU2, SU3, and SU4) were obtained from nodules. When compared with nifH gene sequences reported in the literature using the Basic Local Alignment Search Tool (BLAST), more than 90% similarity to the nifH of Frankia spp. was obtained. The Frankia strains in the nodules shared nifH sequences similar to those of the same host-specific group of Shepherdia. Furthermore, Frankia strains with similar nifH genes have been reported in nodules of Shepherdia argentea (silver buffaloberry). Additionally, Frankia strains belonging to cluster 3 infective strains consisting of Elaeagnaceae and Rhamnaceae infective Frankia showed high similarity to the query sequences. This research demonstrates that nodulation of S. ×utahensis is inhibited at 2 mm NH4NO3. Apart from N, nodule formation may be associated with the substrate type and pH of the nutrient solution. Based on nifH gene sequence amplification, Frankia strains in the root nodules may have the potential to fix atmospheric nitrogen (N2). These Frankia strains have signature gene sequence characteristics of Elaeagnaceae-infective Frankia, suggesting that S. ×utahensis shares Frankia strains similar to its parents.
Actinorhizal plants are able to fix atmospheric nitrogen (N2) through symbiosis with Frankia, a genus of actinobacteria, and have great potential for sustainable landscaping (Kratsch and Graves, 2004). Plant growth and development may be improved when the symbiotic association is established, as reported by previous studies (Laws and Graves, 2005; Schwencke and Caru, 2001). However, excessive N may inhibit nodulation of actinorhizal plants. For instance, nodule formation of Alnus maritima (seaside alder) was prevented by either 2.7 g⋅L−1 of 15N–3.9P–10K controlled-release fertilizer (CRF) or 4 mm NH4NO3 (Beddes and Kratsch, 2010; Laws and Graves, 2005). Therefore, to successfully induce symbiotic nodules in nursery production, it is important to examine the nodulation of inoculated actinorhizal plants irrigated with nutrient solutions with different N levels.
Frankia in symbiotic nodules are highly diverse (Schwencke and Caru, 2001). Phylogenetic analyses have been conducted to investigate the diversity of Frankia strains in nodules of inoculated actinorhizal plants (Jeong and Myrold, 2001; Myrold and Huss-Danell, 1994). Recently, researchers used comparative sequence analyses (e.g., glnII, nifH, recA, and 16S rRNA) to investigate the phylogeny of Frankia strains in nodules (Pawlowski and Bergman, 2007). The phylogenetic research of Frankia using nitrogen fixation (nif) gene, 16s rRNA, or other genes is recognized by most researchers, who agree that all infective Frankia strains can be classified in one of three groups (Benson et al., 2004; Normand et al., 1996; Schwencke and Caru, 2001). According to the review study by Benson et al. (2004), group 1 contains Frankia strains from nodules of plants in Betulaceae, Casuarinaceae, and Myricaceae; group 2 contains Frankia strains from nodules of plants in Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus of Rhamnaceae; and group 3 contains effective Frankia strains that can fix N2 from nodules of plants in Elaeagnaceae, Myricaceae, Rhamnaceae, and Gymnostoma of Casuarinaceae and noneffective strains from Betulaceae, Rosaceae, and genera in Casuarinaceae, except Gymnostoma, and genera in Rhamnaceae, except Ceanothus. Comparative sequence analyses are also important to identify other microorganisms in the nodules of actinorhizal plants. According to Huss-Danell (1997), actinorhizal plants formed nodules without Frankia because a fungus, Penicillium nodositatum, can induce nodules that are incapable of fixing N2.
Shepherdia argentea and S. rotundifolia (roundleaf buffaloberry) are native actinorhizal plants in the U.S. Intermountain West (Mee et al., 2003). Shepherdia argentea can tolerate a wide range of soil conditions (Sriladda et al., 2016), whereas S. rotundifolia has strong drought tolerance. Although S. rotundifolia is more aesthetically appealing compared with S. argentea (Mee et al., 2003), S. rotundifolia has high mortality in nursery conditions (Sriladda et al., 2016). Shepherdia ×utahensis is an interspecific hybrid of S. argentea and S. rotundifolia that tolerates wet and disturbed soil and drought stress (Sriladda et al., 2016). It has high potential for low-water landscaping. The genetic, morphological, and physiological traits of this plant have been studied by Sriladda et al. (2016). As an actinorhizal species, it is able to establish symbiotic associations with Frankia. However, previous studies associated with actinorhizal plant nodulation have focused on A. maritima in the eastern United States (Beddes and Kratsch, 2010; Laws and Graves, 2005), and few studies have investigated the nodulation of Shepherdia. Because of different soil chemical and physical properties in the U.S. Intermountain West (Heaton and Koenig, 2010; Sriladda et al., 2014), characteristics of Shepherdia nodulation might be different from Alnus. Frankia strains in the nodules of S. ×utahensis might be similar to those in nodules of its parents, S. argentea and S. rotundifolia.
The objectives of this study were to 1) evaluate plant growth and nodulation of S. ×utahensis in conditions that mimic nursery environments and natural habitats of S. rotundifolia; and 2) investigate the diversity of Frankia strains in nodules using comparative sequence analyses of polymerase chain reaction (PCR)-amplified nifH gene fragments.
Materials and Methods
Nodulation of S. ×utahensis
Expt. 1.
On 22 Mar. 2019, terminal cuttings (≈10 cm) of S. ×utahensis were collected from the Utah Agricultural Experiment Station's (UAES) Greenville Research Farm (North Logan, UT) (41.765741, −111.813175). Leaves at the bottom of the cuttings were removed, leaving two to three pairs of leaves at the top. Cuttings were dipped in 8000 mg⋅L−1 indole-3-butyric acid (Hormodin 3; OHP, Mainland, PA) and stuck in a soilless substrate containing 80% perlite (Hess Perlite, Malad City, ID) and 20% peatmoss (Canadian sphagnum peatmoss; Sun Gro Horticulture, Agawam, MA). Cuttings were kept on a mist bench with temperatures set at 22 °C in the UAES Research Greenhouse (Logan, UT). On 15 May 2019, nodule-free rooted cuttings were transplanted to 3.8-L injection-molded, polypropylene containers (PC1D-4; Nursery Supplies, Orange, CA) filled with Metro-Mix 820 substrate (Sun Gro Horticulture), which is primarily peatmoss. Plants were irrigated with deionized water before the experiment. All plants were grown in the UAES Research Greenhouse with temperatures set at 25/22 °C (day/night). A heated silicon chip pyranometer (SP-230; Apogee Instruments, Logan, UT) mounted to a weather station at the UAES Greenville Research Farm, ≈1000 m away from the greenhouse, was used to record light intensities. The daily light integral inside the greenhouse, calculated using a light transmission rate of 68%, was 32.6 ± 9.8 mol⋅m−2⋅d−1 (mean ± sd) from 17 June to 4 Sept. 2019.
On 17 June 2019, a factorial treatment combination was created with (F+) or without (F−) Frankia inoculation and with (N+) or without (N−) N application. A total of 84 uniform plants were used, with 22 plants in each of the F+N+ and F+N− groups and 20 plants in each of the F−N+ and F−N− groups. The plants with inoculation were topdressed with 30 mL of soil collected from the root zone of a wild S. rotundifolia plant in Mohave County, AZ (36.881550, −112.895690), with symbiotic nodules observed in the rhizosphere (Fig. 1); the plants without inoculation did not receive the soil topdressing treatment. The plants with N treatment were irrigated with quarter-strength N-free Hoagland’s solution (Hoagland and Arnon, 1950) with 2 mm NH4NO3 at pH 6.5; those without N treatment were irrigated with the same solution without NH4NO3.
Root nodules observed in the soil sample collected from the root zone of a wild Shepherdia rotundifolia at Mohave County, AZ (36.881550, −112.895690) (A), and nodules formed on the roots of Shepherdia ×utahensis ‘Torrey’ during Expts. 1 (B) and 2 (C).
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Root nodules observed in the soil sample collected from the root zone of a wild Shepherdia rotundifolia at Mohave County, AZ (36.881550, −112.895690) (A), and nodules formed on the roots of Shepherdia ×utahensis ‘Torrey’ during Expts. 1 (B) and 2 (C).
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Root nodules observed in the soil sample collected from the root zone of a wild Shepherdia rotundifolia at Mohave County, AZ (36.881550, −112.895690) (A), and nodules formed on the roots of Shepherdia ×utahensis ‘Torrey’ during Expts. 1 (B) and 2 (C).
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Ten plants in each treatment were randomly selected and destructively harvested on 29 July 2019 (7 weeks after experiment initiation; first harvest), and the remaining plants were harvested on 4 Sept. 2019 (12 weeks after experiment initiation; second harvest). Plant heights from the surface of the substrate to the highest terminal bud and number of shoots (>5 cm) were recorded at the initiation of the experiment and on both harvest dates. A soil plant analysis development (SPAD) 502 meter (Minolta Camera, Osaka, Japan) was used to measure the relative chlorophyll content (SPAD reading) of each plant at both harvest dates, and the average values of five randomly selected mature leaves per plant from the canopy were recorded. The leaf area of each plant was recorded using a leaf area meter (LI-3000; LI-COR Biosciences, Lincoln, NE) at both harvest dates. Shoots were dried in an oven at 80 °C for 3 d, and shoot dry weight (DW) was recorded. Roots were harvested, washed with deionized water, and checked for nodulation. The NO3-N concentration of leachate was recorded using a NO3-N meter (LAQUA Twin; Horiba, Kyoto, Japan) on 27 July and 2 Sept. 2019. The pH of the leachate solution was recorded only on 2 Sept. 2019, using a pH meter (LAQUA Twin; Horiba).
Expt. 2.
On 6 Aug. 2019, 60 nodule-free plants propagated using the aforementioned method were transplanted to 656-mL cone-tainers (D40H; Stuewe and Sons, Tangent, OR) filled with perlite (Hess Perlite, Malad City, ID) and sorted into four blocks. After plants were transplanted, 30 mL of field soil collected from the rhizosphere of a nodulated S. ×utahensis plant at the UAES Greenville Research Farm was used to inoculate plants. Plants were irrigated with 250 mL quarter-strength N-free Hoagland’s solution at pH 7.5 every other day. The experiment was initiated on 6 Aug. 2019, and it ended on 18 Nov. 2019. One plant per block was randomly selected and harvested weekly to study nodulation. At harvest, the number of nodules was counted and the diameter (mm) and fresh weight (mg) of each nodule were measured. All plants were also grown in the UAES Research Greenhouse, and the daily light integral from 6 Aug. to 18 Nov. 2019 was 23.9 ± 10.6 mol⋅m−2⋅d−1 (mean ± sd; calculated as described). Supplemental light was provided using 1000-W high-pressure sodium lamps (Hydrofarm, Petaluma, CA) from 0600 to 2200 hr. Lamps were turned on at an average intensity of 130.4 ± 18.0 μmol⋅m−2⋅s−1 (mean ± sd) at plant canopy level when greenhouse light intensity was less than 544 μmol⋅m−2⋅s−1.
Phylogenetic analyses of Frankia strains
DNA extraction.
DNA was extracted from root nodules using a DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany). The quantity and quality of DNA were analyzed using a spectrophotometer (Thermo NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA).
Amplification of nifH gene.
Polymerase chain reaction (PCR) amplification of nifH gene was conducted following the method of Gtari et al. (2007). In brief, primers PolF (5′ TGC GAY CCS AAR GCB GAC TC 3′) and PolR (5′ATS GCC ATC ATY TCR CCG GA 3′) (Poly et al., 2001) were used to amplify nifH gene of Frankia strains in nodules in a reaction volume of 20 μL containing 10 μL Master Mix (Thermo Fisher Scientific), 1 μL of each primer, 1 μL DNA template, and 7 μL distilled water. The PCR was performed in a thermocycler (Eppendorf Mastercycler; Eppendorf, Hamburg, Germany) under the following conditions: primary denaturation at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, and a 5-min extension at 72 °C for the last cycle. PCR products were analyzed by electrophoresis on 1% agarose gel in a TAE buffer after staining with ethidium bromide at 0.5 μg⋅mL−1.
Sequence analyses.
The amplified nifH gene fragments were purified using a Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific GeneJET; Thermo Fisher Scientific) according to the manufacturer’s instructions. The purified amplicons were sequenced at the Utah State University’s Center for Integrated BioSystems (Logan, UT) using an ABI PRISM 3730 DNA Analyzer with an ABI BigDye terminator (Applied Biosystems, Foster City, CA).
Phylogenetic analyses.
SeqMan Pro (DNASTAR, Madison, WI) was used to check the mapped reads of sequences manually. Nucleotide sequences were aligned using the ClustalW algorithm (Thompson et al., 1994) and manually trimmed off primers using molecular evolutionary genetics analysis (MEGA) (Kumar et al., 2018). The generated sequences were analyzed and compared using BLAST (Altschul et al., 1990).
Experimental design and statistical analyses.
Expt. 1 had a completely randomized design with a factorial treatment combination of two factors with 20 blocks, whereas Expt. 2 had a randomized complete block design with 4 blocks. A two-way analysis of variance (ANOVA) procedure was used to test the effects of N treatment and Frankia inoculation on growth and nodulation during Expt. 1, whereas a one-way ANOVA procedure was used to test the effect of time on nodulation during Expt. 2. A χ2 test was conducted using the PROC FREQ procedure in SAS Studio (Version 3.8; SAS Institute, Cary, NC) to test the effects of N or Frankia inoculation on the nodulation of S. ×utahensis. Mean separation among treatments was adjusted using the Tukey-Kramer method for multiplicity at α = 0.05 during Expt. 1. During Expt. 2, regression analyses of time, nodule number, diameter, and fresh weight were conducted. Except for the χ2 test, all statistical analyses were performed using PROC Mixed procedures in SAS Studio.
Results
Nodulation of S. ×utahensis
During Expt. 1, S. ×utahensis plants irrigated with quarter-strength N-free Hoagland’s solution with 2 mm NH4NO3 had a greater NO3-N concentration in the leachate solution at both harvest dates (Fig. 2) than those without NH4NO3. At the time of termination of the experiment, the pH values of the leachate solution were 6.2 ± 0.1 (mean ± se) and 5.8 ± 0.1 (mean ± se) for plants irrigated with quarter-strength N-free Hoagland’s solution with or without 2 mm NH4NO3, respectively. No nodules were observed at week 7, but nodules were found on plants in the F+N− and F−N− groups at week 12 (Table 1, Fig. 1). Eight of 12 plants in the F+N− group formed nodules, whereas 4 of 10 plants in the F−N− group had nodules (Tables 2 and 3). According to the χ2 test, nodulation was affected by 2 mm NH4NO3 in quarter-strength N-free nutrient solution (χ2 = 19.8; df = 1; P < 0.0001). However, although nodulation was greater for those in the F+N− group than those in the F-N- group, inoculation did not affect nodulation (χ2 = 0.98; df = 1; P = 0.32).
The nitrate–nitrogen (NO3-N) concentration of leachate solution collected after Shepherdia ×utahensis ‘Torrey’ was irrigated during Expt. 1. Four treatments were created with a factorial design with (F+) or without (F−) Frankia inoculation and irrigation with quarter-strength nitrogen-free Hoagland’s solution with (N+) or without (N−) 2 mm ammonium nitrate at pH 6.5. Plants were harvested at 7 weeks (first harvest) and 12 weeks (second harvest) after experiment initiation. The error bars represent the se of five samples. The same lowercase letters above column bars within harvest dates denote no significance among treatments according to Tukey-Kramer method for multiplicity at α = 0.05.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
The nitrate–nitrogen (NO3-N) concentration of leachate solution collected after Shepherdia ×utahensis ‘Torrey’ was irrigated during Expt. 1. Four treatments were created with a factorial design with (F+) or without (F−) Frankia inoculation and irrigation with quarter-strength nitrogen-free Hoagland’s solution with (N+) or without (N−) 2 mm ammonium nitrate at pH 6.5. Plants were harvested at 7 weeks (first harvest) and 12 weeks (second harvest) after experiment initiation. The error bars represent the se of five samples. The same lowercase letters above column bars within harvest dates denote no significance among treatments according to Tukey-Kramer method for multiplicity at α = 0.05.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
The nitrate–nitrogen (NO3-N) concentration of leachate solution collected after Shepherdia ×utahensis ‘Torrey’ was irrigated during Expt. 1. Four treatments were created with a factorial design with (F+) or without (F−) Frankia inoculation and irrigation with quarter-strength nitrogen-free Hoagland’s solution with (N+) or without (N−) 2 mm ammonium nitrate at pH 6.5. Plants were harvested at 7 weeks (first harvest) and 12 weeks (second harvest) after experiment initiation. The error bars represent the se of five samples. The same lowercase letters above column bars within harvest dates denote no significance among treatments according to Tukey-Kramer method for multiplicity at α = 0.05.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Plant height, relative chlorophyll content [soil plant analysis development (SPAD) reading], number of shoots, leaf area, shoot dry weight (DW), nodulation rate, number of nodules per plant, and nodule DW of Shepherdia ×utahensis ‘Torrey’. Four treatments were created with a factorial design with (F+) or without (F−) Frankia inoculation and irrigated with quarter-strength nitrogen-free Hoagland’s solution with (N+) or without (N−) 2 mm ammonium nitrate at pH 6.5.z
Chi-square test of the nodulation of Shepherdia ×utahensis ‘Torrey’ irrigated with Hoagland’s solution with (N+) or without (N−) 2 mm ammonium nitrate (NH4NO3) at the time of the second harvest. The overall chi-square statistic (χ2) is the sum of all of cell χ2 with their degree of freedom (df) and P value.
Chi-square test of the nodulation of Shepherdia ×utahensis ‘Torrey’ with (F+) or without (F−) Frankia inoculation as measured at the time of the second harvest. The overall chi-square statistic (χ2) is the sum of all of cell χ2 with their degree of freedom (df) and P value.
At both harvest dates, the plant height, SPAD reading, number of shoots, leaf area, and shoot DW of plants irrigated with quarter-strength N-free Hoagland’s solution with 2 mm NH4NO3 increased compared with those treated with the same solution but without NH4NO3 (Table 1). Furthermore, the number of shoots of plants in the F+N+ group increased compared with that in the F−N+ group at the first harvest, whereas the height and shoot DW of plants in the F+N+ group increased compared with that in the F−N+ group at the second harvest.
During Expt. 2, nodules were first observed at week 5 after the experiment was initiated (Figs. 1 and 3). Positive correlations were observed between weeks and number of nodules (P < 0.0001), nodule diameter (P < 0.0001), and fresh weight (P < 0.0001) of the largest nodule (Fig. 3).
Regression analyses of the number of nodules (A), diameter of the largest nodule (B), and fresh weight of the largest nodule (C) of inoculated Shepherdia ×utahensis ‘Torrey’ plants grown in pure perlite, a low organic-matter substrate, irrigated with quarter-strength nitrogen-free Hoagland’s solution at pH 7.5 during Expt. 2. Four plants were randomly chosen and harvested weekly. Nodules were found at week 5 after experiment initiation.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Regression analyses of the number of nodules (A), diameter of the largest nodule (B), and fresh weight of the largest nodule (C) of inoculated Shepherdia ×utahensis ‘Torrey’ plants grown in pure perlite, a low organic-matter substrate, irrigated with quarter-strength nitrogen-free Hoagland’s solution at pH 7.5 during Expt. 2. Four plants were randomly chosen and harvested weekly. Nodules were found at week 5 after experiment initiation.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Regression analyses of the number of nodules (A), diameter of the largest nodule (B), and fresh weight of the largest nodule (C) of inoculated Shepherdia ×utahensis ‘Torrey’ plants grown in pure perlite, a low organic-matter substrate, irrigated with quarter-strength nitrogen-free Hoagland’s solution at pH 7.5 during Expt. 2. Four plants were randomly chosen and harvested weekly. Nodules were found at week 5 after experiment initiation.
Citation: HortScience horts 56, 7; 10.21273/HORTSCI15726-21
Phylogenetic analyses of Frankia strains
Four query sequences (≈300-bp fragments) were obtained from PCR reactions, including a sequence from the nodule induced with soils from Mohave County, AZ (SU1), and three sequences from three nodules induced with soils from North Logan, UT (SU2, SU3, and SU4).
When compared with sequences reported in the literature using BLAST, SU1 had 98% similarity with the nifH gene of Frankia strain NRRLB-16306 (accession number JF273735.1] (Table 4). High similarity (98%) was also observed between SU1 and the nifH gene of Frankia strains G2 (accession number HM026367.1), Cg70.1 (accession number HM026362.1), R43(2009) (accession number FJ477447.1), and CeSI5 (accession number FJ477443.1). Additionally, more than 97% similarity with the nifH gene was found between the Frankia strains in our study and uncultured Frankia clone T2P1-7 (accession number LT840168.1) (data not shown).
List of 10 Frankia species that have the highest nifH gene similarity with each of four query sequences (SU1, SU2, SU3, and SU4) obtained from nodules of Shepherdia ×utahensis ‘Torrey’.
The nifH gene sequences SU2 and SU3 had 92% and 99% similarity, respectively, with the nifH gene of Frankia strain BMG5.12 (accession number AJ545031.1), FMc5 (accession number KP342119.1), FMc4 (accession number KP342118.1), FMc3 (accession number KP342117.1), FMc2 (accession number KP342116.1), and FMc1 (accession number KP342115.1), whereas nifH gene of Frankia strain BMG5.15 (accession number JF273726.1), BMG5.1 (accession number AJ545034.1), and BMG5.2 (accession number AJ545032.1) had 91% and 98% similarity with SU2 and SU3, respectively. The nifH gene of the uncultured Frankia clone T2P1-7 (accession number LT840168.1) had 90% and 99% similarity with the SU2 and SU3, respectively (data not shown). The SU4 shared 97% similarity with the nifH gene of Frankia strain EUN1f (accession number HM026364.1), G2 (accession number HM026367.1), Cg70.1 (accession number HM026362.1), CeSI5 (accession number FJ477443.1), and NRRLB-16306 (accession number JF273735.1).
Discussion
Nodulation of S. ×utahensis
Inoculation is a preferred practice in nursery production to induce symbiotic nodules on actinorhizal plants (Schwencke and Caru, 2001). Soils collected from the rhizosphere of wild actinorhizal plants have been used for inducing nodules in previous studies (Beddes and Kratsch, 2010; Jeong and Myrold, 2001; Laws and Graves, 2005). However, increasing N levels decreased the nodule number of inoculated plants (Laws and Graves, 2005). Nodulation of Purshia mexicana (mexican cliffrose) and Purshia tridentata (antelope bitterbrush) was inhibited at 6 mm NH4NO3 during a greenhouse study (Righetti et al., 1986). For Ceanothus species, no nodules formed on Ceanothus griseus (Carmel ceanothus) at 2.68 mm NH4NO3 (Thomas and Berry, 1989). Nodulation of Alnus glutinosa (black alder) was inhibited with 2 mm potassium nitrate (Huss-Danell et al., 1982), whereas that of A. maritima was not completely inhibited until 4 mm NH4NO3 was used (Laws and Graves, 2005). It appears that S. ×utahensis may be more sensitive to environmental N levels than A. maritima, C. griseus, P. mexicana, and P. tridentata in terms of nodulation, but less sensitive than A. glutinosa; however, a direct comparison is needed to study N sensitivity of actinorhizal plants. Because this plant is sensitive to environmental N content, the N concentration in irrigation solutions needs to be monitored to maximize nodule formation when producing nodulated plants.
Research is needed to determine the optimal N level or fertilizer level to effectively produce nodulated plants. Increased N levels decrease the nodule number, but they improve plant growth (Laws and Graves, 2005). During our study, plant quality without N treatment was poor, although nodulation was present. Laws and Graves (2005) also reported that A. maritima without N treatment had the greatest nodule number but showed irregular shoot shape and yellowing leaves. Consequently, proper fertilizer application is important for producing nodulated plants with acceptable visual quality and minimal N leaching. A study conducted in Oct. 2019 suggested that 2.1 g⋅L−1 of 15N–3.9N–10K CRF was an appropriate application rate to produce nodulated S. ×utahensis (Chen et al., 2020).
Interestingly, nodulation was observed 7 weeks earlier during Expt. 2 than during Expt. 1, which may be the result of the differences in substrates and the pH of nutrient solutions. The commercial growing substrate contains peatmoss, which is a material containing more than 80% organic matter (Brady, 1990). Organic matter in commercially used growing substrates increases the water-holding capacity (Hudson, 1994) and decreases pH (McCauley et al., 2017). However, soils in the Intermountain West are different from the commercial growing substrate and contain low organic matter because of the desert climate and lack of plant coverage (Heaton and Koenig, 2010). Sriladda et al. (2014) reported that soil organic matter in the habitat of wild S. rotundifolia in the Intermountain West was between 0.7% and 8.7%. Growing native plants in conditions mimicking native habitats improves their growth. For example, according to Beddes and Kratsch (2009), seed germination of S. rotundifolia is optimized in a low organic-matter substrate. Therefore, although peatmoss is commonly used in nursery production, a substrate containing low organic matter may be better for inducing nodulation of S. ×utahensis plants.
The pH of the irrigation solution might also explain the discrepancy in nodule formation during Expt. 1 and Expt. 2. Nodulation of actinorhizal plants is associated with substrate pH (Huss-Danell, 1997). The pH of the growing substrate was affected by the irrigation solution pH (Bailey et al., 2000). The optimal pH for nodulation of Alnus glutinosa and Alnus incana (grey alder) was 5.5, whereas pH less than 4.5 inhibited nodule formation (Berry and Torrey, 1985). However, for Alnus rubra (red alder), more nodules were found when plants were grown in a substrate with pH 4.5 than with pH 5.6 or 7.2 (Crannell et al., 1994). Shepherdia argentea and S. rotundifolia, parents of the S. ×utahensis, are native to the Intermountain West regions with alkaline soil. Shepherdia rotundifolia thrives in soil pH ranging from 6.5 to 7.9 (Sriladda et al., 2014), whereas S. argentea has been found in soil with pH between 7.0 and 8.0 (Mee et al., 2003). Consequently, an alkaline environment may be better for the nodulation of S. ×utahensis because nodules formed 7 weeks earlier during Expt. 2 (irrigation solution with pH 7.5) than during Expt. 1 (irrigation solution with pH 6.5); however, a symbiotic association may be established in a slightly acid soil.
Nodule formation also occurred in the rhizosphere of plants in the F−N− group. Previous publications found infective Frankia strains persist in locations outside the habitat of host plants, such as the rhizosphere of nonhost plant stands or places where host plants have disappeared over time (Benecke, 1969; Smolander and Sundman, 1987; Wollum et al., 1968). Jeong and Myrold (2001) reported that Ceanothus integerrimus (deer brush), Ceanothus sanguineus (redstem ceanothus), and Ceanothus velutinus (snowbrush ceanothus) formed nodules when inoculated with soils collected from a site dominated by Pseudotsuga menziesii (Douglas fir) for more than 100 years. Additionally, Wollum et al. (1968) reported that soils collected from a 300-year-old conifer stand contained Ceanothus-infective Frankia strains. Casuarina, which is native to Australia, formed nodules when first grown in Florida (Benson and Silvester, 1993). During our study, no difference in nodulation was observed between the inoculated and uninoculated plants. This suggests that infective Frankia strains might exist in the nonsterilized commercial substrate used in our study because limited airflow occurred in the greenhouse and inoculated plants were kept apart from uninoculated plants to prevent cross-contamination.
Phylogenetic analysis
The presence of the nifH gene has been considered an indicator of potential N2 fixation (Young, 1992). In our study, nifH amplifications were obtained from Frankia strains in the nodules of S. ×utahensis, suggesting these Frankia are effective strains. However, their N2-fixing capacity is unclear because Benson et al. (2004) revealed that effective Frankia strains in nodules might have poor N2-fixing ability. Therefore, further studies are needed to investigate the possible N2 fixation abilities of the Frankia strains using an acetylene reduction assay or 15N-labeling techniques (Huss-Danell, 1997; Laws and Graves, 2005).
Comparative sequence analyses using nifH genes revealed that nifH sequences of Frankia strains obtained during our study were consistent with the results of previous research. Nouioui et al. (2011) conducted comparative sequence analyses of 38 Frankia strains using glnII, gyrB, and nifH genes and classified all Frankia strains into four clusters: infective Frankia strains in Betulaceae, Casuarinaceae, and Myricaceae in cluster 1; uncultured Frankia strains in Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus in Rhamnaceae in cluster 2; infective Frankia strains in Elaeagnaceae and Rhamnaceae in cluster 3; and noninfective and/or non-N2-fixing Frankia strains in cluster 4. These results are consistent with those of Normand et al. (1996). Frankia strains CeSI5, FMc5, NRRLB-16306, and R43(2009), all of which shared a highly similar nifH gene with the four sequences (SU1, SU2, SU3, and SU4) obtained during our study, were all classified in cluster 3 (Nouioui et al., 2011; Welsh et al., 2009; Wilcox and Cowan, 2016). In addition, Frankia strains BMG5.12 and EUN1f were in cluster 3 and isolated from root nodules of Elaeagnus angustifolia (Russian olive) and Elaeagnus umbellata (autumn olive), respectively (Gtari et al., 2007; Jamann et al., 1993). Frankia strain BMG5.12 has a nifH gene similar to sequences SU2 and SU3, whereas Frankia strain EUN1f was similar to sequences SU1 and SU4. These results suggested that Frankia strains in the nodules of S. ×utahensis were similar to those in nodules of plants in Elaeagnaceae (Normand et al., 1996; Nouioui et al., 2011; Valdés et al., 2005).
Frankia strains with nifH gene sequences highly similar to those in our study have been observed in the nodules of S. argentea, one of the parents of S. ×utahensis. Tekaya et al. (2018) obtained uncultured Frankia clone T2P1–7 from the nodules of S. argentea. Frankia strain Cc1.17 had nifH gene sequences highly similar to those from the symbiotic nodules of S. argentea (Mirza et al., 2009). In another study, 93% of sequences obtained from the nodules of S. argentea shared 98.3% similarity with strain EUN1f, whereas the remaining 7% of sequences showed 99.6% similarity to strain BMG5.12 (Tekaya et al., 2018). The nifH gene sequences of these strains were similar to those obtained during our study. Therefore, Frankia strains inhabiting the nodules of S. ×utahensis are similar to those in the nodules of S. argentea.
The BLAST results also revealed that Frankia strains in our study had nifH gene sequences highly similar to symbionts found in four genera (Casuarina, Colletia, Elaeagnus, and Morella) (Table 4), and all four of these genera were not in the same host-specific group (HSG) (Benson and Silvester, 1993). Because Casuarina-infective Frankia strains belong to HSG 2, and because Elaeagnaceae-infective Frankia strains belong to either HSG 3 or HSG 4 (Huss-Danell, 1997), cross-boundary infectivity might exist in Frankia strains in the nodules of S. ×utahensis. The cross-boundary infectivity of Frankia strains has been reviewed by Huss-Danell (1997). Frankia strains in the nodules of S. ×utahensis might infect diverse plants to form symbiotic nodules.
Conclusions
This study showed that nodulation of S. ×utahensis was restricted by 2 mm NH4NO3. Nodules formed earlier when plants were grown in a low organic-matter substrate and irrigated with quarter-strength N-free Hoagland’s solution at pH 7.5. These results suggest that nodulation was improved when plants were grown in conditions similar to their native, low-fertility, arid habitat. A preliminary genetic analysis of Frankia in root nodules was conducted during this study. Four nifH sequences were obtained from nodules of S ×utahensis. Results of phylogenetic analyses support that Frankia strains in nodules have high similarity to those in Elaeagnaceae nodules and suggest that they potentially have N2-fixing ability. Frankia strains obtained during our study are similar to those observed in S. argentea nodules. Further research is needed to study the phylogenetic characteristics and N2-fixing ability of Frankia strains in the nodules of S. ×utahensis. Further comparative sequence analyses involving several genes (e.g., glnII, gyrB, and 16S rDNA) are needed to clarify the phylogeny of the Frankia spp. in S. ×utahensis nodules.
Literature Cited
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. 1990 Basic local alignment search tool J. Mol. Biol. 215 403 410
Brady, N.C. 1990 The nature and properties of soils 10th ed Prentice Hall Englewood Cliffs, NJ
Bailey, D.A., Nelson, P.V. & Fonteno, W.C. 2000 Substrates pH and water quality North Carolina State University Raleigh, NC 6 Mar. 2021. <http://www.nurserycropscience.info/water/source-water-quality/other-references/substrateph-and-water-quality.pdf>
Beddes, T. & Kratsch, H.A. 2009 Seed germination of roundleaf buffaloberry (Shepherdia rotundifolia) and silver buffaloberry (Shepherdia argentea) in three substrates J. Environ. Hort. 27 129 133
Beddes, T. & Kratsch, H.A. 2010 Nodulation of seaside alder topdressed with controlled-release fertilizer HortTechnology 20 740 745 doi: 10.21273/HORTTECH.20.4.740
Benecke, U. 1969 Symbionts of alder nodules in New Zealand Plant Soil 30 145 149
Benson, D. & Silvester, W.B. 1993 Biology of Frankia strains, actinomycete symbiont of actinorhizal plants Microbiol. Rev. 57 293 319
Benson, D.R., Vanden Heuvel, B.D. & Potter, D. 2004 Actinorhizal symbioses: Diversity and biogeography 99 128 Gillings, M. & Holmes, A. Plant microbiology BIOS Scientific Publishers Oxford, UK
Berry, A.M. & Torrey, J.G. 1985 Seed germination, seedling inoculation and establishment of Alnus spp. in containers in greenhouse trials Plant Soil 87 161 173
Chen, J., Kratsch, H., Norton, J., Sun, Y. & Rupp, L. 2020 Nodulation and plant growth of Shepherdia ×utahensis ‘Torrey’ topdressed with controlled-release fertilizer HortScience 55 1956 1962 doi: 10.21273/HORTSCI15260-20
Crannell, W.K., Tanaka, Y. & Myrold, D.D. 1994 Calcium and pH interaction on root nodulation of nursery-grown red alder (Alnus rubra Bong.) seedlings by Frankia Soil Biol. Biochem. 26 607 614 doi: 10.1016/0038-0717(94)90249-6
Gtari, M., Brusetti, L., Hassen, A., Mora, D., Daffonchio, D. & Boudabous, A. 2007 Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis Soil Biol. Biochem. 39 372 377 doi: 10.1016/j.soilbio.2006.07.005
Heaton, K. & Koenig, R. 2010 Solutions to soil problems, V. Low organic matter Utah State University Logan, UT 14 May 2020. <https://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=1957&context=extension_curall>
Hoagland, D.R. & Arnon, D.I. 1950 The water culture method for growing plants without soil California (Berkeley) Agriculture Experiment Station Circ. 347
Hudson, B.D. 1994 Soil organic matter and available water capacity J. Soil Water Conserv. 49 189 194
Huss-Danell, K. 1997 Actinorhizal symbioses and their N2 fixation New Phytol. 136 375 405 doi: 10.1046/j.1469-8137.1997.00755.x
Huss-Danell, K., Sellstedt, A., Flower-Ellis, A. & Sjöström, M. 1982 Ammonium effects on function and structure of nitrogen-fi xing root nodules of Alnus incana (L.) Moench Planta 156 332 340
Jamann, S., Fernandez, M.P. & Normand, P. 1993 Typing method for N2-fixing bacteria based on PCR-RFLP application to the characterization of Frankia strains Mol. Ecol. 2 17 26 doi: 10.1111/j.1365-294X.1993.tb00095.x
Jeong, S.C. & Myrold, D.D. 2001 Population size and diversity of Frankia in soils of Ceanothus velutinus and Douglas-fir stands Soil Biol. Biochem. 33 931 941 doi: 10.1016/S0038-0717(00)00241-8
Kratsch, H.A. & Graves, W.R. 2004 Nitrogen fixation as a stress-avoidance strategy among actinorhizal (non-legume) trees and shrubs J. Crop Improv. 10 281 304 doi: 10.1300/J411v10n01_12
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018 MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms Mol. Biol. Evol. 35 1547 1549 doi: 10.1093/molbev/msy096
Laws, M.T. & Graves, W.R. 2005 Nitrogen inhibits nodulation and reversibly suppresses nitrogen fixation in nodules of Alnus maritima J. Amer. Soc. Hort. Sci. 130 496 499 doi: 10.21273/JASHS.130.4.496
Mee, W., Barnes, J., Kjelgren, R., Sutton, R., Cerny, T. & Johnson, C. 2003 Water wise: Native plants for intermountain landscapes Utah State University Press Logan, UT
Mirza, B.S., Welsh, A., Rasul, G., Rieder, J.P., Paschke, M.W. & Hahn, D. 2009 Variation in Frankia populations of the Elaeagnus host infection group in nodules of six host plant species after inoculation with soil Microb. Ecol. 58 384 393 doi: 10.1007/s00248-009-9513-0
Myrold, D.D. & Huss-Danell, K. 1994 Population dynamics of Alnus-infective Frankia in a forest soil with and without host trees Soil Biol. Biochem. 26 533 540 doi: 10.1016/0038-0717(94)90239-9
McCauley, A., Jones, C. & Olson-Rutz, K. 2017 Soil pH and organic matter Montana State University Extension Bozeman, MT 2 Mar. 2021. <https://landresources.montana.edu/nm/documents/NM8.pdf>
Normand, P., Orso, S., Cournoyer, B., Jeannin, P., Chapelon, C., Dawson, J., Evtushenko, L. & Misra, A.K. 1996 Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae Intl. J. Syst. Bacteriol. 46 1 9 doi: 10.1099/00207713-46-1-1
Nouioui, N., Ghodhbane-Gtari, F., Beauchemin, N.J., Tisa, L.S. & Gtari, M. 2011 Phylogeny of members of the Frankia genus based on gyrB, nifH and glnII sequences Antonie van Leeuwenhoek 100 579 587 doi: 10.1007/s10482-011-9613-y
Pawlowski, K. & Bergman, B. 2007 Plant symbioses with Frankia and cyanobacteria 165 178 Bothe, H., Ferguson, S.J. & Newton, W.E. Biology of the nitrogen cycle Elsevier Amsterdam, Netherlands
Poly, F., Ranjard, L., Nazaret, S., Gourbiere, F. & Monrozier, L.J. 2001 Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties Appl. Environ. Microbiol. 67 2255 2262 doi: 10.1128/AEM.67.5.2255-2262.2001
Righetti, T.L., Chard, C.H. & Backhaus, R.A. 1986 Soil and environmental factors related to nodulation in Cowania and Purshia Plant Soil 91 147 160
Schwencke, J. & Caru, M. 2001 Advances in actinorhizal symbiosis: Host plant-Frankia interactions, biology, and applications in arid land reclamation. A Review Arid Land Res. Manage. 15 285 327
Smolander, A. & Sundman, V. 1987 Frankia in acid soils of forests devoid of actinorhizal plants Physiol. Plant. 70 297 303 doi: 10.1080/153249801753127615
Sriladda, C., Kratsch, H.A., Larson, S.R., Monaco, T.A., Shen, F. & Kjelgren, R.K. 2016 Interspecific hybrid of xeric Shepherdia rotundifolia and riparian Shepherdia argentea: Description, and traits suitable for low-water urban landscapes HortScience 51 822 828 doi: 10.21273/HORTSCI.51.7.822
Sriladda, C., Kjelgren, R., Kratsch, H., Monaco, T., Larson, S. & Shen, F. 2014 Ecological adaptation of the endemic Shepherdia rotundifolia to conditions in its Colorado Plateau range West. N. Amer. Nat. 74 79 91
Tekaya, S.B., Guerra, T., Rodrigue, D., Dawson, J.O. & Hahn, D. 2018 Frankia diversity in host plant root nodules is independent of abundance or relative diversity of Frankia populations in corresponding rhizosphere soils Appl. Environ. Microbiol. 84 1 17 doi: 10.1128/AEM.02248-17
Thomas, K.A. & Berry, A.M. 1989 Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth of Ceanothus griseus var. horizontalis Plant Soil 118 181 187
Thompson, J.D., Higgins, D.G. & Gibson, T.J. 1994 CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res. 22 4673 4680 doi: 10.1093/nar/22.22.4673
Valdés, M., Pérez, N.O. & Estrada-de, L.S.P. 2005 Non-Frankia actinomycetes isolated from surface-sterilized roots of Casuarina equisetifolia fix nitrogen Appl. Environ. Microbiol. 71 460 466 doi: 10.1128/AEM.71.1.460-466.2005
Welsh, A., Mirza, B.S., Rieder, J.P., Paschke, M.W. & Hahn, D. 2009 Diversity of Frankia in root nodules of Morella pensylvanica grown in soils from five continents Syst. Appl. Microbiol. 32 201 210 doi: 10.1016/j.syapm.2009.01.002
Wilcox, D.A. & Cowan, D.A. 2016 Diversity of Frankia in root nodules of six Morella sp. from the Cape flora of South Africa Plant Soil 406 375 388
Wollum, A.G., Youngberg, C.T. & Chichester, F.W. 1968 Relation of previous timber stand age to nodulation of Ceanothus velutinus For. Sci. 14 114 118 doi: 10.1093/forestscience/14.2.114
Young, J.P.W. 1992 Phylogenetic classification of nitrogen-fixing organisms 43 86 Stacey, G., Burris, R.H. & Evans, H.J. Biological nitrogen fixation Chapman and Hall New York, NY
