Effect of orange peel on PGPR strain growth
To assess the relative ability of each Bv strain to grow on OP as a sole carbon source, each Bv strain was grown in a M9 minimal medium (Difco, Detroid, MI) supplemented with OP (Citrus Extracts LLC, Fort Pierce, FL 34982, USA) as a sole carbon source. The M9 minimal medium was prepared according to manufacturer’s instructions, including addition of 2 mM MgSO4 and 0.1 mM CaCl2 to a final concentration in 1X M9 base medium (Mageshwaran et al. 2014). A 10% (w/v) OP stock suspension was prepared and diluted to a final concentration of 0.5% in 1X M9 medium (pH 7.0). Overnight cultures of each PGPR strain were prepared in 10 mL of tryptic soy broth (TSB, 24 hours, 30ºC, 175 rpm). The 1X M9 medium containing 0.5% OP (M9OP) was distributed in 3 mL per culture tube while mixing. Overnight Bv cultures were subjected to centrifugation at 10,000 x g for 8 min at 25ºC to pellet cells, which were washed in 1 mL 1X M9 medium, and then pelleted again before a final resuspension in 1 mL 1X M9 medium in a sterile 1.5 mL tube. PGPR inoculum were normalized to an OD600 ~ 0.1 in 3 mL cultures of M9OP media. Once all PGPR were inoculated an OD600 was recorded, and tubes were incubated while shaking at 200 rpm at 30oC. OD600 readings were recorded every 24 hours for 72 hours. Each strain culture was replicated three times. The ∆OD600 values were calculated by the OD600 at time 72 hours subtracted from the OD600 at time 0 hours; a one-way ANOVA was performed to elucidate differences between strains using PROC GLIMMIX in SAS (SAS 9.4, SAS Institute, Cary, NC, USA) using strains as the main factor and replications as a random factor.
Greenhouse experiment to select PGPR strains
To select the most effective PGPR strains on soybean growth, inoculation with six different PGPR strains (known for their high degree of pectinolytic activity and rapid growth on OP) and orange peel were tested in a greenhouse experiment in September of 2020.
PGPR strains, soybean cultivar and growing conditions
As a substrate for the experiment, a sandy loam field soil was collected from E.V. Smith Research Center in Shorter, AL, USA and mixed with sand (1:1 proportion) to fill 15 L pots. Soil test of this mixture was performed by the Auburn University Soil Testing Laboratory resulting in a soil pH of 5.7 and soil nutrient level of 8 kg ha− 1 P, 75 kg ha− 1 K, 100 kg ha− 1 Mg, and 409 kg ha− 1 Ca. Fertilizer was applied according to the Alabama Cooperative Extension System recommendations for soybean before planting (101 kg ha− 1 of P2O5 and 45 kg ha− 1 of K2O). Six different PGPR strains, with known pectate lyase activity and rapid growth on OP (AP191, AP193, AP215, AP216, AP218, and MB315) were inoculated on soybean cultivar S49XT39 (Dyna-Gro) as a seed treatment.
PGPR spores were prepared following the methods of Hassan et al. (2019) and added to each seed at a final concentration of 1x106 spore colony forming unit (CFU) in 50 µL of sterile water. The OP suspension was prepared at a final concentration of 10 mg suspended in 200 µL per seed. Four treatments per strain were evaluated: (1) non-inoculated control (NI), (2) inoculation with orange peel suspension (OP), (3) inoculation with the PGPR strains alone (AP191, AP193, AP215, AP216, AP218, or MB315), or (4) inoculation with each PGPR strain plus orange peel (AP191 + OP, AP193 + OP, AP215 + OP, AP216 + OP, AP218 + OP, or MB315 + OP), with seven replicates in total for each treatment.
Treatments were applied on seeds at the sowing time, September 4, 2020. Five seeds were evenly placed 2.5 cm below the soil surface of each pot to ensure proper germination. Each seed received the following inoculations according with the treatment group: (NI) 250 µL of distilled water, (OP) 200 µL of orange peel powder solution and 50 µL of water, (PGPR) 50 µL of respective PGPR spores and 200 µL of distilled water, (PGPR + OP) first 200 µL of orange peel powder solution and then 50 µL of respective PGPR spores. No water was added to either treatment group for at least 24–48 hours after planting to allow the seeds to remain in contact with the PGPR and OP solution. Following this period, each pot received 500 mL of water every 2 days. After emergence (approximately one week after sowing), one seedling was kept per pot and the other ones were removed.
Pots were initially grouped by replicate and then rearranged in a randomized complete block design. The pots were rotated around the tables in the greenhouse each week, preventing any biases based on plant location and light intensity among pots. Artificial LED light (800 µmol mol− 1 PAR) was used to maintain a photoperiod of 16 h of day and 8 h of night until time of flowering, at which supplemental lights were turned off. Temperatures in the greenhouse oscillated between 18 to 30ºC during the day and 15 to 20ºC at night. Each week plants were sprayed with Tundra EC (Winfield/AgriSoulutions, Albertville, MN, USA), Talstar-Pro (FMC Inc., Philadelphia, PA, USA), and Kontos (OHP, Inc., Bluffton, SC, USA) to prevent and control insect infestation.
Physiological and growth parameters measurements
When the plants had reached the R4 growth stage (Fehr et al. 1971), SPAD values, a proxy for chlorophyll concentration, were measured using a SPAD-502 (Minolta, Tokyo, Japan). At R5, plants were harvested, and above ground plant organs were removed and separated. Total above ground biomass (g plant − 1) was calculated by separating leaves, stems, and pods, drying them at 60ºC for at least 72 h, and weighing them with an analytical balance. Before drying, total leaf area (cm2 plant− 1) was calculated by passing each trifoliated leaf through a LI-3000 Leaf Area Meter (LI-COR Biosciences, Lincoln, Nebraska, USA).
Roots were cleaned after harvesting using tap water and nodules were separated. Fresh nodules were cleaned and placed on clean white paper and then imaged with a digital camera. The pictures were analyzed for quantitative nodule characteristics using ImageJ according to Riedell et al. (2009), and nodule number and size (total cm2 plant − 1 and individual nodule cm2 plant − 1) were calculated. The imaged nodules were dried at 60ºC for at least 72 h to calculate total nodule dry weight (g plant − 1). Cleaned roots were scanned in a Winrhizo desktop scanner (Regent Instruments Inc., Sainte-Foye, Quebec, Canada) to calculate total root area (cm2 plant − 1), root volume (cm3 plant − 1), total root length (cm plant − 1), and total root average diameter (mm plant − 1). After scanning, the roots were dried at 60ºC for at least 72 h to calculate total root dry weight (g plant − 1).
Statistical analysis
A two-way ANOVA was performed for each parameter to test the effect of PGPR strain (AP191, AP193, AP215, AP216, AP218, and MB315), inoculation (NI, OP, PGPR, PGPR + OP) and their interaction. The two-way ANOVA was performed using PROC GLIMMIX in SAS (SAS 9.4, SAS Institute, Cary, NC, USA) with PGPR and inoculation as the main factors and replication as a random variable. When the main effect of PGPR and/or inoculation, or their interaction was significant, least square means post-hoc tests were performed to compare means (LSMEANS, SAS 9.4).
Field Experiment to Test the Response of Co-inoculation of PGPR plus Orange Peel Amendment with Bradyrhizobium japonicum on Soybean Cultivars
Field Experimental Design And Inoculation Treatments
During the Summer of 2021, field trials were established at E.V. Smith Research Center (EVS; Shorter, AL, USA) in a no-tillage system, with rye as winter cover crop. E.V. Smith Research Center has a Piedmont Plateau soil with a Sandy Loam soil texture with a pH of 6.3 and soil nutrient level of 36 kg ha− 1 P, 68 kg ha− 1 K, 67 kg ha− 1 Mg, and 853 kg ha− 1 Ca. Soil tests were performed two weeks before planting and fertilizer recommendations for soybean were 28 kg ha− 1 N, 112 kg ha− 1 P2O5, 190 kg ha− 1 K2O, and 22 kg ha− 1 Mg. The mean, maximum and minimum temperature during the 2021 growing season was 22.6, 32.8, 11.1ºC respectively, with a rainfall accumulation of 1,040.7 mm during the growing season. Pre- and post-emergence herbicides and pesticides were applied following the recommendations of the Alabama Cooperative Extension System.
Three commercial soybean cultivars (S49XT39, S52XT08, and AG69X0) known for their responsiveness to PGPR + OP inoculation (Pacheco da Silva et al. 2022) and a non-nodulating soybean cultivar (Lee), were evaluated. Fourteen different inoculation treatments were applied to the seeds: (1) non-inoculated (NI), (2) orange peel amendment (OP), (3) B. japonicum (Brad), (4) Brad + OP, (5) B. velezensis (Bv) AP191, (6) AP191 + OP, (7) AP191 + Brad, (8) AP191 + Brad + OP, (9) Bv strain AP193, (10) AP193 + OP, (11) AP193 + Brad, (12) AP193 + Brad + OP, (13) Cell-Tech® (B. japonicum inoculant; NexusBioAg) commercial inoculant, and (14) Vault® (B. japonicum, Bacillus amyloliquefaciens, and Bacillus subtilis; BASF) commercial inoculant.
The different PGPR spore suspensions were prepared following the methods of Hassan et al. (2019) at a final concentration of 1x109 spore CFU mL− 1. Cell-Tech® and Vault® commercial inoculant suspensions were prepared following the respective label recommendations. When included in the treatment, B. japonicum powder (Exceed® Peat for Soybean, Visjon Biologics) was applied to the seeds prior to treatment with PGPR and/or OP, according to the label recommendation.
The treatments were applied to the seeds before sowing. Each batch of untreated seed, containing 425 g of seeds, received the respective inoculant and/or PGPR suspension (2 mL for each PGPR at a 1x109 CFU mL− 1) and 18 mL of sterile water. Seeds were mixed to ensure that all the liquid was evenly spread on the seeds surface. Then 25.75 grams of OP powder was added to the seed batch. Seeds were mixed again to ensure that all the powder added was evenly distributed onto the seeds surface. Seeds were left to dry during 24 h at room temperature and later packaged for planting. Seed packages were kept in a refrigerator at 4ºC prior to planting.
The experimental design had three cultivars and a non-nodulating control, with four replications, and 14 treatments per cultivar totaling 180 plots. Plots were 20 foot long and consisted of four rows with 36 inch spacing between rows. For all the treatments, the planting density was 214,800 seed ha − 1 with a germination percentage higher than 90%. Seeds were planted with a four row Almaco Cone Planter (Almaco Inc., Nevada, IA, USA).
Physiological And Growth Measurements
Fifteen days after planting, emergence was counted twice per plot (number of seedlings/ meter) to estimate the percentage of germination. At vegetative growth stage, V2 (Fehr et al. 1971), plant vigour scores were assigned for each plot ranging from 1 to 5, where the lowest value represents significantly weaker and slower growth, and the highest value represents significantly stronger and faster growth compared to the control treatment.
When the plants had reached the R5 developmental growth stage (pod filling, Fehr et al. 1971), SPAD values were measured using a SPAD-502 in two plants per plot. Plant Height (cm) was measured at R7 in three plants per plot, from the soil surface to the apical meristem of the main stem.
At beginning of pod formation (R3, Fehr et al. 1971) aboveground biomass accumulation was measured by harvesting a 0.5-meter row. Collected plants were dried for 72 h in a forced air oven at 60ºC and later weighted on an analytical balance. The total aboveground biomass including leaves, stems, and pods was ground to pass a 1 mm screen, weighed into tin capsules, and shipped to the UC-Davis Stable Isotopes Facility (Davis, California, USA) for 15N isotope, N content, and 13C isotope analysis. Samples were analyzed using an isotope ratio mass spectrometer (IsoPrime, Elementar France, Villeurbanne) coupled to an elemental analyzer (EA3000, EuroVector, Milan, Italy).
The natural 15N isotopic ratio (δ15N) in the aboveground biomass was calculated using the formula described by (Shearer and Kohl (1986):
$${\delta }^{15}N=\frac{{R}_{sample}}{({R}_{air}-1)}*1000$$
1
where, Rsample and Rair are the isotope ratios (15N/14N) of the sample and air, respectively. The proportion of N derived from the atmosphere (%Ndfa), an estimation of the biological nitrogen fixation (BNF), was determined by the 15N natural abundance method (Shearer and Kohl 1986) following the formula:
$$\text{N}\text{d}\text{f}\text{a} \left(\%\right)=\frac{{{\delta }}^{15}{\text{N}}_{\text{r}\text{e}\text{f}} – {{\delta }}^{15}{\text{N}}_{\text{s}\text{o}\text{y}}}{{{\delta }}^{15}{\text{N}}_{\text{s}\text{o}\text{y}}-\text{B}}\times 100$$
2
where Ndfa (%) is the percentage of N coming from the atmosphere through BNF, δ15Nref is the δ15N signature of the non-fixing soybean reference (cultivar Lee) aboveground biomass, δ15Nsoy is the δ15N signature of the aboveground biomass for each treatment, and B is the δ15N value of a soybean plant growing in a N free media relying only on BNF as source of N. The B-value used in our study were obtained as the δ15N average value (-2.78‰) from previous reports for soybean sampled around R1-R2 developmental stage (Table S2).
The ratio (R) of 13C/12C was showing as 𝛿13C (‰), indicating the C isotope composition relative to Vienna Pee Dee Belemnite calcium carbonate (V-PDB):
𝛿13C = (Rsamples/Rstandard)-1
𝛿13C (‰) values were standardized to C isotope discrimination (Δ13C, ‰) data calculated as:
$${\varDelta }^{13}\text{C} \left(\text{‰}\right)=\left(\frac{{{\partial }^{13}\text{C}}_{\text{a}\text{t}\text{m}}-{\partial }^{13}{\text{C}}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}}{1+\left(\frac{{\partial }^{13}{\text{C}}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}}{1000}\right) }\right)$$
3
where 𝛿13Catm is the C isotope composition of atmospheric CO2 (-8‰; Farquhar et al. 1989), and 𝛿13Csample is the C isotope composition of the aboveground biomass sample.
Root Surface Area (cm2) and Root Volume (cm3) at the beginning of the pod developmental stage, R3 (Fehr et al. 1971), were measured by collecting two roots per plot using the shovelomic method (Seethepalli et al. 2020) and storing them in plastic bags over ice. The roots were photographed and then analyzed for root parameters using RhizoVisionExplorer (version 2.0.3) software (Seethepalli et al. 2020). At maturity, each plot was assigned with a lodging score ranging from 0 to 5, according to Table S3. The two middle rows of each plot were harvested with a two-row small plot combine (Almaco R1 Combine) and yield was re-calculated for a seed moisture content of 13%.
Statistical analysis
A two-way ANOVA was performed using PROC GLIMMIX in SAS 9.4 for each parameter to test the effect of inoculation, cultivar, and their interaction for the field experiment where inoculation and cultivar were the main factors and replication was a random variable. When the main effect of inoculation treatment and/or genotype or their interaction was significant, least square means post-hoc test was performed to compare means (LSMEANS, SAS 9.4).