Skip to content
BY 4.0 license Open Access Published by De Gruyter Open Access February 3, 2021

The nutmeg seedlings growth under pot culture with biofertilizers inoculation

Reginawanti Hindersah, Agusthinus Marthin Kalay, Henry Kesaulya and Cucu Suherman
From the journal Open Agriculture


Nutmeg is important for national and community revenue mainly in Maluku Province where nutmeg seedlings are grown in low-fertility soil without fertilizer. A greenhouse experiment was performed to evaluate the response of nutmeg seedlings following the application of two different biofertilizer concortia. The experimental design was completely randomized block design, which tested the combination treatments of two rates and the application methods of biofertilizer concortium. The rates of “bacillus biofertilizer” was 0.15 and 0.3%, while the rates of “mixed biofertilizer” was 0.5 and 1.0%. Both biofertilizer were inoculated by foliar spray and soil application. The results verified that at 24 weeks after inoculation, biofertilizers increased the seedling growth traits which included plant height, shoot dry weight, leaf surface area, root number, and root dry weight over the control. Soil application by 1% of “mixed biofertilizer” consists of nitrogen-fixing bacteria and phosphate-solubilizing microbes resulted in better seedlings performance. However, the highest plant height was demonstrated by seedlings treated with 0.3% “bacillus biofertilizer” composed of phosphate solubilizing Bacillus. Biofertilizer inoculation also enhanced soil microbes and leaf surface area but did not change the root-to-shoot ratio of the seedlings. The results showed that biofertilizer inoculation improves the growth of nutmeg seedlings.

1 Introduction

Indonesia is the world’s second largest exporter of nutmeg (Myristica fragrans Houtt). The native of nutmeg is Maluku Islands including Ambon where nutmeg is cultivated for generations with other crops in agroforestry system known as Dusung (Matinahoru 2014; Leatemia et al. 2017). In addition to nutmeg, communities grow perennial and annual food crops such as fruit, spices, horticultural, and medicinal plants in the Dusung (Rehatta and Raharjo 2014). Nutmeg productivity in Ambon Island is low, 0.39–0.77 t ha−1 (Leatemia et al. 2017) even though the potency of nutmeg production in Ambon island is predicted up to 3 t ha−1 (Basir et al. 2018).

The obstacles to increase nutmeg production include the unavailability of good-quality seedlings in the nursery. The local farmers believe that growing nutmeg naturally from seed is the best way to have best and long-term plant productivity compared to vegetative-propagated seedlings. In Maluku, nutmeg seedlings were produced mainly from mature seeds with soil as the only growth media without fertilizer. Mature fruits were obtained from mother trees with varied leaf, fruit, seed, and mace characteristics (Hetharie et al. 2015), so that the performance of the seedling was diverse. The constraint of tree seedling production in tropical soil is low nitrogen (N) and phosphorus (P) contents that have to be overcome by chemical fertilizer. In most islands of Maluku, the chemical fertilizer price is high and occasionally not available in the market.

Biofertilizer is an alternative technology to substitute chemical fertilizer and can be used in nutmeg seedling production. The active ingredients of biofertilizers are commonly N-fixing bacteria (NFB) and P-solubilizing microbes (PSMs) isolated from the soil. Both bacterial groups play an important role in nutrient cycling in soil. The nitrogenase of NFB catalyzes the fixation of N2 to available NH3. Organic acid excretion is a main mechanism of PSMs to change the solubility of unavailable P to soluble phosphate. They assist nutrient acquisition and increase the availability of N and P for root uptake (Rubio et al. 2013; Sharma et al. 2017).

Biofertilizer usually contains phytohormones indole acetic acid (IAA), gibberellins (GAs), and cytokinins (CKs) excreted by certain beneficial microbes. Phytohormones production by NFB Arthrobacter, Azotobacter, Azospirillum, Pseudomonas, and Bacillus have been documented (Fibach-Paldi et al. 2012; Yu et al. 2012; Rubio et al. 2013; Li et al. 2017). Phosphate-solubilizer Bacillus, Pseudomonas, Aspergillus, and Penicillium were also reported to secrete the same phytohormones (Araújo et al. 2005; Mittal et al. 2008; Sharma et al. 2017). Certain soil microbes also produced siderophore and exopolysaccharide (EPS) to improve the uptake of essential metals (Emtiazi et al. 2004, Ahmad et al. 2008). Microbial EPS has been documented to enhance soil aggregation and improve nutrient uptake (Alami et al. 2000; Costa et al. 2018).

Biofertilizer application in tree and forest nursery have not only improved nutrient uptake and plant growth but also induced plant tolerance to abiotic stress (Asif et al. 2018). In addition to nutrients’ supply by the soil and fertilizer, woody plants require phytohormones to induce their growth (Aloni 2007; Yuan et al. 2019). Actually, the composition of chemical fertilizer consist of only macro- and micronutrients, so that biofertilizer application is a way to supply phytohormones to the tree. Nowadays nutmeg is considered as the medicinal plants that have been studied broadly. Microbial inoculation is beneficial for the growth, nutrient uptake as well as active substance of medicinal plants (Solaiman and Anawar 2015). Moreover, biofertilizers are ecofriendly and cost-effective inputs for the farmers since they are renewable by using appropriate technology. Though the biofertilizer has a positive effect on plant growth, sole application of biofertilizer might not be effective to provide all nutrients needed by nutmeg seedling. Organic matter and reduced dose of chemical fertilizer application are still needed.

Research in biofertilizer inoculation for nutmeg seedlings is very limited. Inoculating nutmeg seedlings by beneficial microbes give plant roots the access to form the root–microbe partnership which will be effective to promote plant growth and development once the seedlings grow in the fields. Nair and Chandra (2001) stated that Azospirillum and Azotobacter inoculation is beneficial for increasing nutmeg seedling growth. However, information concerning the biofertilizer rates and application method of NFB and PSB co-inoculation for nutmeg are not yet available. Therefore, the objective of this pot experiment was to evaluate the response of nutmeg seedlings, mainly the growth of shoots and roots to different rates and the application methods of two kinds of liquid biofertilizer that contain NFB and PSM as well as phytohormones.

2 Material and method

2.1 Experimental site

The study was conducted in the greenhouse of Faculty of Agriculture, Pattimura University, Ambon city, Maluku Province, Indonesia, in July 2018–January 2019. The research location is in the tropics at an altitude of 10 m above sea level (asl) with average day temperature of 24–30oC and humidity of 75–80%. First-transplanted seedlings (10 weeks old) of nutmeg var Banda were prepared from mother seeds by the Nutmeg Nursery Community of Lilibooi Village, Leihitu District, Maluku Tengah Regency, Maluku. The Lilibooi Village is located 2 m asl with the climate similar to the Ambon city.

2.2 Biofertilizer

Two kinds of liquid biofertilizer consortia used in this trial were bacillus biofertilizer (BB) and mixed biofertilizer (MB). First biofertilizer contained three Bacillus species prepared by the Plant Physiology Laboratory, Faculty of Agriculture, Pattimura University. The second one was formulated by using NFB and PSM microbes belonging to Soil Biology Laboratory, Faculty of Agriculture, Universitas Padjadjaran. In the preparation of both biofertilizer formulations, all microbes (Table 1) of equal volume were mixed and the total bacillus and fungal densities were counted. The density of Bacillus spp. in BB was at least 107 colony-forming unit (CFU) per 1 mL, while bacterial and fungal populations in MB were at least 107 and 105 CFU mL−1, respectively. The activity of N fixation and P dissolution as well as phytohormones production of fertilizers are depicted in Table 1. Based on the in vitro test, all PSMs produced organic acid, Bacillus produced siderophore and Azotobacter chroococcum produced EPS in significant amounts.

Table 1

Nitrogen-fixing and phosphate-solubilizing capacity and phytohormone production of Bacillus and mixed biofertilizers

Microbes Acetylene reduction (nmol g−1 h−1) Phosphate solubilizing (mg L−1) Phythormones (mg L−1)
Bacillus biofertilizer a
Bacillus subtilis SWI16b 12.4 IAA 5.6; GAs 6.3
Bacillus mojavensis JCEN3 13.2 IAA 0.4; GAs 5.7
Bacillus subtilis HPC21 12.3 IAA 0.3; GAs 4.2
Mixed biofertilizer
Azotobacter chroococcum 74.1 IAA 1.08; CKs 0.5, GAs 0.3
Azotobacter vinelandii 142.1 IAA 1.3; CKs 4.6
Azopsirillum sp. 219.9 IAA 12.2
Acinetobacter sp. 96 IAA 10.6
Pseudomonas cepacia 2.69 IAA 7.8; CKs 5.1; GAs 10.6
Penicillium sp. 4.15 IAA 10.1; CKs 2.9; GAs 10.9

IAA: indole acetic acid, CKs: cytokinins, GAs: gibberellins.

  1. a

    Source: Kesaulya et al. (2017).

2.3 Experimental design

The greenhouse experiment was carried out in completely randomized block design with nine treatments and three replications. The treatments were the combination of two rates and two application methods of each biofertilizers. The application rates of BB were 0.15 and 0.3% while that of MB was 0.5 and 1%. Both biofertilizers were inoculated to nutmeg seedlings by foliar spray and soil application. The control treatment received no biofertilizer.

2.4 Experimental establishment

Inceptisols collected from Lilibooi area was silty clay loam and had pH of 4.7. The soil contained 1.27% organic carbon, 0.1% total N, 14.13 mg kg−1 total P2O5, 22.19 mg kg−1 total K2O, 4.14 mg kg−1 soluble P, and cation exchange capacity of 10.62 cmol kg−1. Soil was taken from the top soil (20 cm depth) of Dusung agroforestry in Lilibooi Village and cleaned of the plant debris. The growth medium was prepared by mixing the soil evenly with chicken manure at a volume ratio of 3:1. The chicken manure had pH of 6.7, 28% water content, C/N ratio of 14.2, 1.1% N, 2.7% P, and 0.9% K. As much as 5 kg of growth media was put into a 20 × 30 cm (width × height), black polyethylene bag and incubated for 1 week prior to transplanting of the 10-week-old seedlings.

Each biofertilizers were diluted according to the treatment rates with ground water shortly before inoculation. For each concentration and application method, 20 mL of biofertilizer was inoculated every 3 weeks from 1 week to 20 weeks after transplanting. A total of 3 g of compound fertilizer (NPK 15-15-15) was applied to individual plants including control plants at 4, 12 and 20 weeks after transplanting. All seedlings were maintained in the greenhouse for 24 weeks. During the experiment, there was no diseases or pests attack and therefore no pesticides were applied. Seedlings were irrigated with groundwater 2–3 times a week depending on the weather.

2.5 Plant growth evaluation and statistical analysis

Plant height, shoot dry weights, leaf surface area (leaf area [LA]), secondary (lateral) root number, root weight, leaf number, and stem diameter were measured at 24 weeks after transplanting. Plant height was measured from the base of the stem to the shoot apex. Leaf surface area was measured by using “easy leaf area,” an automated digital image analysis (Easlon and Bloom 2014). Total root length was calculated by summing the length of each roots of individual plant. The dry weight of shoots and roots was weighed after heating the biomass at 60oC for 72 h. Secondary root number was counted based on roots extending laterally from the primary root; while the leaf number included all leaves that perfectly open. Stem diameter was measured at 10 cm from the stem base. Root-to-shoot ratio (R/S) was calculated based on the root and shoot dry weights of 24-week transplants.

For individual polybag, soil samples for bacterial counting were taken up from the soil 5 cm away from the roots. Soil samples were collected at the depth of 10 cm and evenly mixed. Soil samples were transferred into sealed plastic bag and stored at 4°C prior to microbiological analysis. Those soil samples have also been used for soil acidity analysis. Population of NFB and PSB were enumerated at 24 weeks after transplanting by serial dilution plate method (Suliasih and Widawati 2005). Nitrogen-free Ashby’s mannitol and Pikovskaya media were used to count NFB and PSB, respectively.

All data were subjected to analysis of variance (ANOVA) and then the least significant difference (LSD) test (P < 0.05) was done. All statistical analyses have been done by using Minitab 18.

  1. Ethical approval: The conducted research is not related to either human or animal use.

3 Results

3.1 Shoot growth traits

Biofertilizer treatments significantly enhanced plant height and dry weight at 24 weeks after transplanting. Seedlings inoculated with 0.3% BB by soil application and 1% MB by foliar as well as soil application demonstrated higher plant height over the control and other treatments (Figure 1a). Seedling that received 1% MB through soil application produced the highest dry weight (up to 9.90 g) but statistically did not significantly differ with 0.3% BB and 1% MB by foliar spray as well as 0.5% MB by soil application (Figure 1b).

Figure 1 
                  Effect of biofertilizer on plant height (a) and shoot dry weight (b) of nutmeg seedling at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Figure 1

Effect of biofertilizer on plant height (a) and shoot dry weight (b) of nutmeg seedling at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Biofertilizer treatments have no effect on stem diameter and leaf number (Table 2). We found no great variation in stem diameter between treatments, and the average diameter was 0.54 cm. The leaf number was 14.7–20.0, which depended on the treatment. The control seedling had the lowest leaf number due to leaf fall. The seedlings that received 1% MB by foliar dressing showed higher leaf number but not significantly different from other treatments. Based on LSD test (p < 0.05), biofertilizer-treated seedlings showed appreciable increase in LA during 24 weeks in the greenhouse (Table 3). Generally, LA of all inoculated seedlings was 25.4–43.8% higher than the control. Irrespective of the statistical analysis, the highest LA (57.8 cm2) was seen in the seedling treated with 1% MB by soil application.

Table 2

Effect on biofertilizer on leaf number and leaf surface area of nutmeg seedlings at 24 weeks after transplanting in the greenhouse

Treatments Stem diameter (cm) Leaf number Leaf surface area (cm2)
A: Control 0.53 ± 0.020 14.7 ± 1.28 40.2b ± 2.29
B: 0.15% BB, foliar spray 0.56 ± 0.006 18.8 ± 3.30 51.5a ± 7.05
C: 0.15% BB, soil application 0.57 ± 0.050 13.2 ± 1.89 50.4a ± 5.00
D: 0.3% BB, foliar spray 0.56 ± 0.041 18.2 ± 5.42 55.0a ± 3.33
E: 0.3% BB, soil application 0.53 ± 0.040 17.3 ± 1.15 50.7a ± 4.84
F: 0.5% MB, foliar spray 0.53 ± 0.032 18.7 ± 0.57 52.2a ± 6.32
G: 0.5% MB, soil application 0.53 ± 0.061 17.5 ± 2.29 55.2a ± 4.15
H: 1% MB, foliar spray 0.53 ± 0.036 20.0 ± 0.86 51.4a ± 4.51
I: 1% MB, soil application 0.55 ± 0.142 18.2 ± 2.25 57.8a ± 1.91

Means followed by the same letter in a column are not significantly different at p < 0.05 according to the least significant different test. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Table 3

Effect of biofertilizer on root-to-shoot ratio of nutmeg seedlings at 24 weeks after transplanting in the greenhouse

Treatments Root-to-shoot ratio
A: Control 0.38 ± 0.06
B: 0.15% BB, foliar spray 0.39 ± 0.08
C: 0.15% BB, soil application 0.41 ± 0.03
D: 0.3% BB, foliar spray 0.31 ± 0.03
E: 0.3% BB, soil application 0.45 ± 0.09
F: 0.5% MB, foliar spray 0.40 ± 0.06
G: 0.5% MB, soil application 0.35 ± 0.03
H: 1% MB, foliar spray 0.38 ± 0.12
I: 1% MB, soil application 0.37 ± 0.10

A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

3.2 Root traits

Based on ANOVA, no significant difference was found among treatment for root length but did for root dry weight at 24 weeks after transplanting. The total root length of all treated seedlings was 21.42–24.88 cm, which was not significantly different with the control (Figure 2a). However, biofertilizer treatment had a potency to increase the total root length up to 9.1% in average compared to the control. Based on the LSD test (p < 0.05), root dry weight in seedling with 1% MB through either foliar or soil dressing was significantly higher compared with the control and other treatments (Figure 2b). Both treatments gain dry root weight of 62 and 60%, respectively, over the control.

Figure 2 
                  Effect of biofertilizer on total root length (a) and root dry weight (b) of nutmeg seedlings at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Figure 2

Effect of biofertilizer on total root length (a) and root dry weight (b) of nutmeg seedlings at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Fertilizer treatment was significant at p < 0.05 for root number. Based on LSD test (p < 0.05), seedlings treated with 1% MB by soil dressing had higher root number over the control and other treatments (Figure 3). Arrosing 1% MB around the stem produced 16.8 lateral roots which is 71.4% higher than the control.

Figure 3 
                  Effect of biofertilizer on root number of nutmeg seedlings at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

Figure 3

Effect of biofertilizer on root number of nutmeg seedlings at 24 weeks after transplanting in the greenhouse. A: control, B: 0.15% BB foliar spray, C: 0.15% BB soil application, D: 0.3% BB foliar spray, E: 0.3% BB soil application, F: 0.5% MB foliar spray, G: 0.5% MB soil application, H: 1% MB foliar spray, I: 1% MB soil application. BB: bacillus biofertilizer, MB: mixed biofertilizer.

3.3 Root-to-shoot ratio

Biofertilizer treatments did not change the R/S ratio based on LSD test (p < 0.05), suggesting that both biofertilizer and their application methods had no effect on R/S (Table 3). The average R/S ratio of nutmeg seedlings at 24 weeks after transplanting was 0.38–0.45. We found that 0.3% BB by soil application had a potency to increase the R/S ratio of seedling compared to the other treatments.

3.4 Bacterial population and soil acidity

Biofertilizers significantly increased NFB and PSB count in soil around the seedling roots (Table 4). Before experiment, the untreated soil contained 103 CFU g−1 of NFB and 106 CFU g−1 of PSB. The increase in NFB was clearly demonstrated by MB treatment but the higher population of PSB was shown by BB treatments. In general, the higher increase in both bacterial population was caused by higher concentration of biofertilizer by soil application.

Table 4

Effect of biofertilizer treatments on nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and pH in soil at 24 weeks after transplanting in the greenhouse

Treatments Microbial population Soil acidity
NFB (106 CFU g−1) PSB (108 CFU g−1)
A: Control 0.05d ± 0.24 0.73g ± 0.09 5.29 ± 0.11
B: 0.15% BB, foliar spray 0.19c ± 0.24 1.22f ± 0.05 5.41 ± 0.13
C: 0.15% BB, soil application 0.22c ± 0.09 3.36bc ± 0.05 5.38 ± 0.11
D: 0.3% BB, foliar spray 0.30c ± 0.33 2.57cd ± 0.02 5.38 ± 0.04
E: 0.3% BB, soil application 0.29c ± 0.24 5.83a ± 0.03 5.33 ± 0.14
F: 0.5% MB, foliar spray 1.33b ± 0.17 1.84ef ± 0.22 5.35 ± 0.16
G: 0.5% MB, soil application 2.55b ± 0.07 1.98de ± 0.03 5.27 ± 0.10
H: 1% MB, foliar spray 2.56b ± 0.04 2.81c ± 0.01 5.40 ± 0.04
I: 1% MB, soil application 4.65a ± 0.06 4.27ab ± 005 5.26 ± 0.18

Note: Means followed by the same letter in a column are not significantly different at p < 0.05 according to least significant different test. BB: bacillus biofertilizer, MB: mixed biofertilizer. NFB: nitrogen-fixing bacteria, PSB: phosphate-solubilizing bacteria.

The acidity of soil taken form Lilibooi Village before the experiment was 4.7, which is categorized as strongly acid soil (Table 4). At the end of experiment, the range of pH in soil was raised to 5.27–5.40.

4 Discussion

Biofertilizer increased the plant height, shoot dry weight, LA, root number, and root dry weight over the control. Stem diameter, leaf number, root length, and R/S ratio remained unchanged after inoculation. The highest plant height demonstrated by seedling received 0.3% BB by soil application, but the value was not significantly differ with the said trait of 1% MB treatments. Seedling inoculation with 1% MB by soil application significantly produced highest shoot dry weight, LA, root number, and root dry weight. In addition, lower rate of MB also significantly increased the plant height, shoot dry weight, and LA over the control and BB. The results agree with the increased in plant height and shoot and root dry weight of walnut seedling after treating with NFB Arthrobacter and PSB Pseudomonas chlororaphis (Yu et al. 2012).

The consistency of MB to affect plant growth traits over BB could be related to the microbial composition in biofertilizer formulation. The soil in this trial contained low total nitrogen and available P. Nitrogen content in soil was as low as 0.1% and may induce nitrogenase activity of NFB since nitrogenase is inhibited by high nitrogen available such as NH4Cl (Yin et al. 2015). This repression is in accordance with the sensitivity of nif genes in NFB to fixed nitrogen (Yan et al. 2010; Poza-Carrión et al. 2014).

Low content of soluble P2O5 (4.14 mg kg−1) in Inceptisols used in this experiment induced the PSMs to produce phosphatase for organic P mineralization, and organic acid for solubilizing inorganic P which is unavailable in lower soil pH (Sharma et al. 2013; Kalayu 2019). All PSMs in both biofertilizer produce organic acid and based on in vitro assay were able to produce available P. This biological properties are agree with the metabolism of another PSM; F Agrobacterium spp., and Bacillus circulans (Babalola and Glick 2012), and Aspergillus and Penicillium fungi (Saxena 2013).

Since the soil was low in N and available P, nitrogen and P contents in nursery growth media might be increased for root uptake after inoculation. Both nutrients are essential for perennial plant growth seedling. Vegetative growth needs a lot of N for amino acid synthesis to build the cell and metabolism (de Oliveira Ferreira et al. 2016). Hence, amino acid (N) portion in leaves improved the efficiency of photosynthesis (Perchlik and Tegeder 2018). The role of P in vegetative growth is in biosynthesis of ATP for energy transfer as well as proteins and carbon. Under P deficiency circumstances, ATP production is limited and then CO2 fixation through phytosynthesis is reduced (Carstensen et al. 2018).

The records concerning fertilization on nutmeg seedling included in Indonesia was very limited although the Indonesian Spice and Medicinal Research Institute recommended the application of 4 g NPK fertilizer (15:15:15) for individual nutmeg seedlings. The N-fixer and P-solubilizer microbes can be beneficial to plant growth mainly in N- and P-deficient soil. In walnut seedling, the N-fixing Arthrobacter inoculated together with P-solubilizing P. chlororaphis resulted in maximum content of available N and P in soils (Yu et al. 2012). Moderately elevated N content in shoots and roots and P content in roots of mangrove seedlings following mixed inoculation of NFB and PSB have also been reported (Xiong et al. 2016).

Plant growth inoculated with high rates of biofertilizer showed more improvement relative to the lower rates. The improvement was caused by an increase in NFB and PSB population around the root seedlings. Before experiments, the soil contained 103 CFU g−1 of NFB and 106 CFU g−1 of PSB. Despite the presence of NFB and PSB in preplant soil, the effectivity and competence of indigenous beneficial microbes might be lower than that of biofertilizer. Increase in NFB and PSB population in growth media at the end of experiment (Table 4) could have an impact on the ability of both microbial groups to provide N and P for root uptake and then plant growth. Microbial inoculation along with seedling establishment provides a niche in the rhizosphere for microbial growth although competitive and synergic effect with indigenous microbes may have taken place (Trabelsi and Mhamdi 2013). The higher growth of nutmeg seedling with higher rate of biofertilizer application might be attributed to the synergic effect between native and introduced microbes. This positive interaction may increase N fixation, P solubilization, and phytohormone production. Synergetic effects between PSB and NFB have been demonstrated to enhance soil ammonium, inorganic N, and available P, which then resulted in performance improvement of the 60-day-old Cyclocarya paliurus seedling (Wang et al. 2019).

The soil acidity was very strongly acid (pH 4.7) before experiment and became less acid at the end of the experiment. The acid soil was less supportive to NFB growth which is optimum at neutral soil although some Azotobacter and Azospirillum isolates are able to tolerate the acid condition (Singh 2011; Verma et al. 2011). Low soil pH is more suitable for the growth of PSMs Bacillus and Penicillium (Koni et al. 2017). The better effect of MB on the growth of nutmeg seedlings may be due to the adaptability of NFB and Penicillium in acid soil (pH 5.29–5.40) of this experiment.

Seedling inoculation with high rate of MB by soil application enhanced plant growth (plant height, shoot dry weight, and root number) and microbial population compared to foliar spray. This is due to the increase in NFB and PSB population in soil after the trial (Table 4). Soil application of liquid biofertilizer allows microbes to colonize the rhizosphere faster over foliar spray. Once the biofertilizer inoculated to the plant, the desired beneficial microbes proliferate and colonize the rhizosphere where they metabolize the root exudates and affect plant growth (Huang et al. 2014) and then interact with plant roots to enhance plant growth. Biofertilizer application by foliar spray allow beneficial microbes to colonize the phyllosphere. Compared to the microbes in the rhizosphere, phyllosphere microbes will face more abiotic stress such as nutrient and water limitations as well as solar radiation (Thapa et al. 2017; Truchado et al. 2017).

Irrespective of the biofertilizer dose and type, we found that LA of seedling treated with biofertilizer enhanced about 31.9% over control. Further, the biomass and the growth of nutmeg seedling that received both the biofertilizers in the field might be better. The results are in line with the findings of Rashid et al. (2018) who demonstrated that Azospirillum combined with PSMs and P-mobilizing microbes enhanced LA of physic nut (Jatropha curcas L.) seedlings.

The results showed that biofertilizer treatments had not change the R/S ratio. We found the average R/S ratio of nutmeg seedling at the end of trial was about 0.38 due to higher proportion of shoots over roots (Figures 1a and 2b). Plants with high proportion of shoots adsorb more light photon and synthesize more photosynthate and hence greater shoot biomass, but limited water and nutrient uptake by the seedling roots of 17 eudicot species (Mašková and Herben 2018). Nonetheless soil treatments in the field can lead to better tree growth since R/S ratio is not strongly relevant to the imbalance partitioning of the resource between shoots and roots (Rogers et al. 2019).

Researchers have concluded that inoculation of soil beneficial microbes in estate plantation plays a significant role in plant establishment and ensures the robustness of the seedlings (Nair and Chandra 2001; Taryo-Adiwiganda et al. 2006; Xiong et al. 2016). Moreover, biofertilizer application enables to decrease the rate of chemical fertilizer (Çakmakç et al. 2012) and increase the yield (Rohman et al. 2019). The results of the experiment verified that biofertilizers are the promising nutrient input for nutmeg seedling production.

5 Conclusion

Some shoot and root traits of nutmeg seedlings treated with biofertilizer types and their method of application showed better performance compared to the control. In general, nutmeg seedling inoculation with 1% MB by soil application produced the highest shoot dry weight, LA, root number, and root dry weight. Dressing the soil with MB was preferred than BB to increase plant growth although both treatments had not change the R/S ratio at 24 weeks. Soil application of 0.3% BB also resulted in the highest plant height. The biofertilizers enhanced leaf surface area but the R/S ratio remained unchanged following inoculation of both biofertilizers. The R/S ratio was lower than 1, so that growth of nutmeg seedlings aerial part in the greenhouse was higher than roots. For better quality of local variety nutmeg seedling, we recommend to treat nutmeg seeds with microbial preparations before planting in the substrate.


We thank the Community of Nutmeg Nursery Center in Lilibooi Village, Leihitu District, Maluku Tengah Regency, and Regional Planning and Development Agency of Maluku Province.

  1. Funding information: The research was funded by Maluku Corner Centre of Excellence of Universitas Padjadjaran year 2018 (contract number: 542/UN6.WR3/TU/2018).

  2. Authors’ contributions: Reginawanti Hindersah wrote the proposal and manuscript, controlled the quality of the mixed biofertilizer, and supervised the experiment; Agusthinus Marthin Kalay did the experiment, traits’ measurement, and statistical analysis as well as wrote the materials and methods section; Henry Kesaulya controlled the quality of bacillus biofertilizer and did the soil and microbial analyses; and Cucu Suherman wrote the manuscript.

  3. Conflict of interest: Authors declare no conflict of interest.

  4. Data availability statement: The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


[1] Ahmad F, Ahmad I, Khan MS. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res. 2008;163(2):173–81.10.1016/j.micres.2006.04.001Search in Google Scholar

[2] Alami Y, Achouak W, Marol C, Heulin T. Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots. Appl Environ Microbiol. 2000;66(8):3393–8.10.1128/AEM.66.8.3393-3398.2000Search in Google Scholar

[3] Aloni R. Phytohormonal mechanisms that control wood quality formation in young and mature trees. In: Entwistle K, Harris P, Walker J, (Eds.). The compromised wood workshop. The Wood Technology Research Centre, University of Canterbury, Christchurch, New Zealand; 2007.Search in Google Scholar

[4] Araújo FF, Henning AA, Hungria M. Phytohormones and antibiotics produced by Bacillus subtilis and their effects on seed pathogenic fungi and on soybean root development. World J Microbio Biotechnol. 2005;21:1639–45.10.1007/s11274-005-3621-xSearch in Google Scholar

[5] Asif M, Mughal AH, Bisma R, Mehdi Z, Saima S, Ajaz M, et al. Application of different strains of biofertilizers for raising quality forest nursery. Int J Curr Microbiol App Sci. 2018;7(10):3680–6.10.20546/ijcmas.2018.710.425Search in Google Scholar

[6] Babalola OO, Glick BR. The use of microbial inoculants in African agriculture: current practice and future prospects. J Food Agric Environ. 2012;10(3–4):540–9.Search in Google Scholar

[7] Basir A, Neswati R, Baja S, Lopulisa C. Climate index for estimating nutmeg plant suitability under tropical rainforests in Maluku Province, Indonesia. SSRG – IJAES. 2018;5(6):23–30.10.14445/23942568/IJAES-V5I6P105Search in Google Scholar

[8] Çakmakç R, Erturk Y, Dönmez F, Şekban R. Tea growth and yield in relation to mixed cultures of N2-fixing and phosphate solubilizing bacteria. Conference paper presented in 23rd İnternational Scientific-Experts Congress on Agriculture and Food Industry at İzmir, Türkiye; 2012. in Google Scholar

[9] Carstensen A, Herdean A, Schmidt SB, Sharma A, Spetea C, Pribil M, et al. The impacts of phosphorus deficiency on the photosynthetic electron transport chain. Plant Physiol. 2018;177(1):271–84.10.1104/pp.17.01624Search in Google Scholar

[10] Costa OYA, Raaijmakers JM, Kuramae EE. Microbial extracellular polymeric substances: ecological function and impact on soil aggregation. Front Microbiol. 2018;9:1636.10.3389/fmicb.2018.01636Search in Google Scholar

[11] Easlon HM, Bloom AJ. Easy leaf area: automated digital image analysis for rapid and accurate measurement of leaf area. Appl Plant Sci. 2014;2(7):1400033.10.3732/apps.1400033Search in Google Scholar

[12] Emtiazi G, Ethemadifar Z, Habibi MH. Production of extracellular polymer in Azotobacter and biosorption of metal by exopolymer. Afr J Biotech. 2004;3(6):330–3.10.5897/AJB2004.000-2060Search in Google Scholar

[13] Fibach-Paldi S, Burdman S, Okon Y. Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense. FEMS Microbiol Lett. 2012;326(2):99–108.10.1111/j.1574-6968.2011.02407.xSearch in Google Scholar

[14] Hetharie H, Raharjo S, Rahado K, Hehanussa ML. Morphological diversity of nutmeg mother trees and seedlings in Lilibooi Village, Ambon Island. Proceeding of the 1st International Seminar of Basic Science, Faculty of Mathematics and Natural Sciences, Pattimura University, Ambon; 2015.Search in Google Scholar

[15] Huang XF, Chaparro JM, Reardon KF, Zhang RF, Shen QR, Vivanco JM. Rhizosphere interactions: root exudates, microbes, and microbial communities. Botany. 2014;92(4):267–75.10.1139/cjb-2013-0225Search in Google Scholar

[16] Kalayu G. Phosphate solubilizing microorganisms: promising approach as biofertilizers. Int J Agron. 2019;2019:4917256.10.1155/2019/4917256Search in Google Scholar

[17] Kesaulya H, Hasinu JV, Tuhumury GNC. Potential of Bacillus spp. produces siderophore in suppressig the wilt diseases of banana plants. IOP Conf Ser Earth Environ Sci. 2017;102:012016.10.1088/1755-1315/102/1/012016Search in Google Scholar

[18] Koni TNI, Hanim RC, Zuprizal Z. Effect of pH and temperature on Bacillus subtilis FNCC 0059 oxalate decarboxylase activity. Pak J Biol Sci. 2017;20(9):436–41.10.3923/pjbs.2017.436.441Search in Google Scholar

[19] Leatemia JA, Rehatta H, Uluputty MR, Mahulete AS. Productivity of nutmeg (Myristica spp.) in agroforestry system (Dusung) in Ambon Island. In: Yamamoto S, Leatemia JA, (Eds.). Future collaboration on island studies between Pattimura University and Kagoshima University. Occasional Papers Kagoshima University Research Center for the Pacific Islands, vol. 58; 2017. p. 69–76.Search in Google Scholar

[20] Li H-B, Singh RK, Singh P, Song Q-Q, Xing Y-X, Yang L-T, et al. Genetic diversity of nitrogen-fixing and plant growth promoting Pseudomonas species isolated from sugarcane rhizosphere. Front Microbiol. 2017;8:1268.10.3389/fmicb.2017.01268Search in Google Scholar

[21] Mašková T, Herben T. Root: shoot ratio in developing seedlings: how seedlings change their allocation in response to seed mass and ambient nutrient supply. Ecol Evol. 2018;8:7143–50.10.1002/ece3.4238Search in Google Scholar

[22] Matinahoru JM. A review on dusun as an indigenous agroforestry system practiced in small islands. Occasional papers Kagoshima University Research Center for the Pacific Islands, vol. 54; 2014. p. 53–60.Search in Google Scholar

[23] Mittal V, Singh O, Nayyar H, Kaur J, Tewari R. Stimulatory effect of phosphate-solubilizing fungal strains (Aspergillus awamori and Penicillium citrinum) on the yield of chickpea (Cicer arietinum L. cv. GPF2). Soil Biol Biochem. 2008;40(3):718–27.10.1016/j.soilbio.2007.10.008Search in Google Scholar

[24] Nair SK, Chandra N. Effect of biofertilizer application on growth of nutmeg (Myristica fragrans Houtt.) seedlings. J Trop Agric. 2001;39:65–6.Search in Google Scholar

[25] de Oliveira Ferreira EV, Novais RF, Dubay GR, Pereira GL, Araujo WL, Jackson RB. Nitrogen supply affects root and shoot amino acid composition in eucalyptus clones. AJCS. 2016;10(3):280–90.Search in Google Scholar

[26] Perchlik M, Tegeder M. Leaf amino acid supply affects photosynthetic and plant nitrogen use efficiency under nitrogen stress. Plant Physiol. 2018;178:174–88.10.1104/pp.18.00597Search in Google Scholar

[27] Poza-Carrión C, Jiménez-Vicente E, Navarro-Rodríguez M, Echavarri-Erasun C, Rubio LM. Kinetics of nif gene expression in a nitrogen-fixing bacterium. J Bacteriol. 2014;296(3):595–603.10.1128/JB.00942-13Search in Google Scholar

[28] Rashid M, Rasool M, Lal SB. Application of bio-fertilizers for quality seedling production of (Jatropha curcas L.). Int J Chem Stud. 2018;6(2):1729–32.Search in Google Scholar

[29] Rehatta H, Raharjo SHT. Common local fruits in dusun system in Leitimur, Ambon Island (abstract only). In: Yamamoto S, Raharjo SHT, (Eds.). New horizon of island studies in the asia pacific region. Occasional Papers Kagoshima University Research Center for the Pacific Islands; 2014. p. 54.Search in Google Scholar

[30] Rogers ER, Zalesny Jr, RS, Hallett RA, Headlee WL, Wiese AH. Relationships among root–shoot ratio, early growth, and health of hybrid poplar and willow clones grown in different landfill soils. Forests. 2019;10:49.10.3390/f10010049Search in Google Scholar

[31] Rohman F, Wachjar A, Santosa E, Abdoellah S. Humic acid and biofertilizer applications enhanced pod and cocoa bean production during the dry season at kaliwining plantation, Jember, East Java, Indonesia. J Trop Crop Sci. 2019;6(3):153–63.10.29244/jtcs.6.03.153-163Search in Google Scholar

[32] Rubio EJ, Montecchia MS, Tosi M, Cassán FD, Perticari A, Correa OS. Genotypic characterization of Azotobacteria isolated from Argentinean soils and plant-growth-promoting traits of selected strains with prospects for biofertilizer production. Sci World J. 2013;519603.10.1155/2013/519603Search in Google Scholar

[33] Saxena J. Phosphate solubilization by a few fungal strains belonging to the genera Aspergillus and Penicillium. Afr J Microbiol Res. 2013;7(41):4862–9.10.5897/AJMR2013.5991Search in Google Scholar

[34] Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus. 2013;2:587.10.1186/2193-1801-2-587Search in Google Scholar

[35] Sharma P, Verma PP, Kaur M. Phytohormones production and phosphate solubilization capacities of fluorescent Pseudomonas sp. isolated from Shimla dist. of Himachal Pradesh. IJCMAS. 2017;6(8):2447–54.10.20546/ijcmas.2017.609.301Search in Google Scholar

[36] Singh S. Selection of effective Azotobacter isolates for tomato (Lycopersicon esculentum Mill.). M.SC. (AG.) Thesis. College of Agriculture Indira Gandhi Krishi Vishwavidyalaya, Raipur India; 2011.Search in Google Scholar

[37] Solaiman ZM, Anawar HM. Rhizosphere microbes interactions in medicinal plants. In: Egamberdieva D, Shrivastava S, Varma A, (Eds.). Plant-growth-promoting rhizobacteria (PGPR) and medicinal plants, vol 42. Soil Biol; 2015. p. 19–41.10.1007/978-3-319-13401-7_2Search in Google Scholar

[38] Suliasih S, Widawati S. Isolation and identification of phosphate solubilizing and nitrogen fixing bacteria from soil in Wamena biological garden, Jayawijaya, Papua. Biodiversitas. 2005;6(5):175–7.10.13057/biodiv/d060307Search in Google Scholar

[39] Taryo-Adiwiganda Y, Tarigan B, Purba B. Effect of bio fertilizer on mature oil palm in north Sumatra and Riau. Indones J Agric Sci. 2006;7(1):20–6.10.21082/ijas.v7n1.2006.p20-26Search in Google Scholar

[40] Thapa T, Prasanna R, Ranjan K, Velmourougane K, Ramakrishna B. Nutrients and host attributes modulate the abundance and functional traits of phyllosphere microbiome in rice. Microbiol Res. 2017;204:55–64.10.1016/j.micres.2017.07.007Search in Google Scholar

[41] Trabelsi D, Mhamdi R. Microbial inoculants and their impact on soil microbial communities: a review. Biomed Res Int. 2013;863240.10.1155/2013/863240Search in Google Scholar

[42] Truchado P, Gil MI, Reboleiro P, Rodelas B, Allende A. Impact of solar radiation exposure on phyllosphere bacterial community of red-pigmented baby leaf lettuce. Food Microbiol. 2017;66:77–85.10.1016/ in Google Scholar

[43] Verma R, Chourasia SK, Jha MN. Population dynamics and identification of efficient strains of Azospirillum in maize ecosystems of Bihar (India). 3 Biotech. 2011;1(4):247–53.10.1007/s13205-011-0031-7Search in Google Scholar

[44] Wang Z, Chen Z, Fu X. Integrated effects of co-inoculation with phosphate-solubilizing bacteria and N2-Fixing bacteria on microbial population and soil amendment under C deficiency. Int J Env Res Public Health. 2019;16:2442.10.3390/ijerph16132442Search in Google Scholar

[45] Xiong Y, Zhang X, He X, Li M, Liao B. Species-specific effects of P-solubilizing and N2-fixing bacteria on seedling growth of three salt-tolerant trees. Russ J Ecol. 2016;47:259–65.10.1134/S1067413616030164Search in Google Scholar

[46] Yan Y, Ping S, Peng J, Han Y, Li L, Yang J, et al. Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri. BMC Genomics. 2010;11(11):A1501.10.1186/1471-2164-11-11Search in Google Scholar

[47] Yin TT, Pin UL, Ghazali AHA. Influence of external nitrogen on nitrogenase enzyme activity and auxin production in Herbaspirillum seropedicae (Z78). Trop Life Sci Res. 2015;6(1):101–10.Search in Google Scholar

[48] Yu X, Liu X, Zhu T-H, Liu G-H, Mao C. Co-inoculation with phosphate-solubilizing and nitrogen-fixing bacteria on solubilization of rock phosphate and their effect on growth promotion and nutrient uptake by walnut. Eur J Soil Biol. 2012;50:112–7.10.1016/j.ejsobi.2012.01.004Search in Google Scholar

[49] Yuan H, Zhao L, Guo W, Yu Y, Tao L, Zhang L, et al. Exogenous application of phytohormones promotes growth and regulates expression of wood formation-related genes in Populus simonii x P. nigra. Int J Mol Sci. 2019;20:792.10.3390/ijms20030792Search in Google Scholar

Received: 2020-05-30
Revised: 2020-10-02
Accepted: 2020-10-06
Published Online: 2021-02-03

© 2021 Reginawanti Hindersah et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.