Effect of inoculation of
1. Introduction
Indonesia is a tropical country in Southeast Asia region, located between the Asia and Australia continents. In most parts of Indonesia, climate variation and high of rainfall causes intensive leaching, soil becomes low content of alkaline and the pH tend to acidic. Indonesia has acid dry land area approximately 102.8 million hectares, but only 55.8 million hectares are suitable for agricultural [1]. The arid lands in Indonesia which are generally formed from mineral soil are acidic (pH 4.6 to 5.5) and poor of nutrients. One effort to increase the soil fertility and plant productivity on acid dry land with planting legumes, such as soybean. Inoculation of root nodule bacteria on soybean plant could enhance soybean quality and its productivity [2 & 3]. Some varieties of acid tolerant soybean, such as Tanggamus, Sibayak, Seulawah, Ratai, and Nanti are issued by the Research Institute for Legumes plants and Tuber Crops Indonesia could grow at acidic soil with pH 4.5-5.0 and produced soybean up to 2000 Kg/hectares on the right growing conditions [4]. Soybeans generally grow in soil at pH 5.5-6.0 while the optimum pH is 6.8. Below pH 4.7 soybean production will decline. It is related to the chemical properties of acid soil, that is high levels of aluminium, high P fixation, iron and manganese concentration increases to the toxic level, sensitive to erosion, and poor biotic status under a low pH conditions [5]. Soybean production could be increase by symbiosis with root nodule bacteria. The effectiveness of symbiotic bacteria in legume root nodules is strongly influenced by the soil conditions. Keyser and Munns [5] suggested that aluminum (Al) with a high concentration (50 µM) is one of the stress factors that can inhibit the growth and prolong the lag phase of root nodulating bacteria. Richardson
2. Acid aluminium tolerant Bradyrhizobium japonicum
Some strains of root nodulating bacteria tolerant to acid soil conditions have been reported [8]. The bacteria has ability to fix atmospheric nitrogen (N2) and and convert into ammonium (NH3) [9].
Root nodulating bacteria can be distinguished from other bacteria by growing it on media yeast extract mannitol agar (YMA) consist of 10 g/L mannitol, 0.5 g/L K2HPO4, 0.2 g/L MgSO4.7H2O, 0.2 g/L NaCl, and 0.5 g/L yeast extract and containing 0.0025% congo red. Root nodulating bacteria can not absorb congo red or less, and the colony is colorless or pale white [2]. Bradyrhizobia growing on agar media are classified into three types based on the appearance of colonies, such as: small dry (SD), large mucoid (LM), large watery (LW), and dimorphism [13]. Colony of SD type is round, convex, translucent, and diameter of <1 mm. The LM type is circular, convex, slimy, relatively translucent, and diameter> 1 mm. The LW type is irregular shapes, flat, watery, translucent, and diameter> 1 mm. Dimorphism type strain is called to strain with a mixture of SD and LM type. Colony type can be used to predict tolerant or sensitive strain to acid-Al condition. A small dry colony type strain is more sensitive to acid-Al compare to large one and wet type colony [8]. BJ 11 is the slow-growing colony, circular shape, convex elevation, slimy, translucent, and diameter of colony > 1 mm, it is categorized large mucoid. Other root nodulating bacteria has fast growing and acid producing is classified as genus
3. Symbiotic effectiveness
Effective strains of
Symbiotic effectiveness is the relative ability of an association between legume and root nodulating bacteria. Effective nodule consist of leghemoglobin, that is an iron-containing red protein binding with O2 that controls the partial pressure of O2 (pO2) in the nodule [15]. When pO2 was below or above normal condition (0.21 atm), it could decrease the activity of N2 fixation. Leghemoglobin is induced by the interaction between
Effective nodule tends to be large size, reddish, and able to fix nitrogen gas from air. In addition, the effective root nodules have a limited number and distribution, usually found on the main root and secondary first root [14]. Ineffective nodules tend to be small, numerous, greenish white (pale), unable to fix nitrogen from air and spread the root system [14].
Symbiotic effectiveness of acid-tolerant soybean with acid-Al tolerant
The effectiveness of symbiosis can be observed in several ways viz. the determination of plant dry weight, total N content, and nitrogenase activity [2]. Dry weight of the plant is still considered relevant for evaluating the effectiveness of symbiotic root nodulating bacteria with soybean plants, because plant dry weight significantly correlated with total N content [14]. Plant dry weight is usually correlated with the dry weight of root nodules. Upper plant dry weight is used as a parameter to evaluate the binding of N, because as much as 70% of the fixing N is transported to the upper plant [14].
The symbiotic interaction between soybean and root nodulating bacteria played an important role in increasing the plant growth of soybean plant. Effectiveness of a root nodulating bacteria in fixing nitrogen is affected by the compatibility between bacteria and the soybean plant. Mubarik
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BJ 11 (wt) | 17 | 0.0397 | 71.4 | 12.54 | 0.8447 | 16.88 | 155.37 | 144.55 | ||
USDA 110 | 12 | 0.0241 | 63.0 | 12.21 | 0.6164 | 13.63 | 114.92 | 100.00 | ||
Control N | 0 | 0 | 46.0 | 0 | 0.5509 | 13.58 | 100.00 | 96.26 | ||
Control NO | 0 | 0 | 37.3 | 0 | 0.4561 | 6.91 | 83.88 | 77.55 |
The source of energy for nitrogen fixation in bacteroids depends on host photosynthate which is transported through the membrane simbiosome in the form intermediate product of the tricarboxylic acid cycle (Krebs cycle) such as succinic acid, fumaric and malic acid which is a electron donor to produce ATP and reduce N2. Pyruvic acid is the reductant that involved directly as an electron donor in the nitrogenase system [15]. The N2 binding reaction that occurs in bacteroids as follows:
Complex of nitrogenase reduces the triple bond of N2 into ammonia molecules. Nitrogenase enzyme activity can be measured by the acetylene reduction technique. Acetylene (C2H2) can be used as an alternative substrat to N2. Reduction of N2 and acetylene by nitrogenase as follows:
The comparison between the substrate N2 reduction by C2H2 is 3:1, and according to calculation [20] the total amount of N fixed by plants (µg) = μmol C2H4 x 28.
While the C2H2 reduction can provide a useful tool for detecting N2-fixing activity in both legumes and non-legumes plants, the method is unsuitable for measuring N2 fixation at field scales. There are some suitability of methods for quantifying N2 fixation for crop legumes, such as measurement of N difference, relative ureide method, 15N natural abundance, and 15N isotope dilution [21]. But none of the methods for assessing N2 fixation is perfect. Some additional informations are needed to support the N2 fixation data, such as assessment of nodulation, growth analysis, rooting patterns of N2 fixing and companion non N2-fixing plants, determination of mineral N soil, and soil analysis [21].
4. Greenhouse experiments of symbiotic between acid aluminium tolerant B. japonicum and soybean on acid soils
Situmorang
Inoculation of BJ 11(19) isolate increased number of seeds and pods higher than the other treatments [22]. Acid tolerant soybean such as Slamet generally has weight 12.5 g of 100 seeds [24]. BJ 11 (19) showed the highest 13.5 g of 100 seeds. Pods that were already formed then were filled with photosynthate to form seeds. Numbers of seeds are effected by the number and size of pods. Higher number of pods also produce higher numbers of seeds [25].
Further experiments are done in acid soil plots (pH 4.5). Totally 12 plot experiments, each plot measured 1 m x 2 m x 0.2 m filled with 45 kg of acid soils (pH 4.5) and 10% (w / w) peat or rice husk as innoculant carrier. Each plot planting with soybean sprouts each with a spacing of 20 x 40 cm2. Amount of inoculant (about 1.0 x 108 cells/ml) in peat-carrier is applied to each plot. Every hole on plot planted with 5 seedling soybeans and to be reduced to 3 plants at 30 days after planting. Each plot is separated by a distance of 1 m from other plot. Results of plot experiment showed that the effectiveness of symbiotic BJ 11 (19) with soybean is significantly had higher value on the plant height, dry weight of upper crop, root nodules, nodule number, nitrogenase activity, and weight of 100 seeds. Treatment of compost before planted soybean in acid soils could produce better crops and increase producing of soybean seeds compare to without compost (Figure 3). The compost consists of plant residues and soil microbes that can improve acid soil structure becomes more fertile and porous.
5. Viability test of acid-aluminium B. japonicum inoculant using peat as carrier
Viability of
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BJ 11 (5) | Room | 9.8 x 107 cdef | 2.8 x 107 f | 1.3 x 108 abcdef |
10 ºC | 1.4 x 108 abcdef | 1.2 x 108 abcdef | 7.6 x 107 def | |
BJ 11 (19) | Room | 2.4 x 108 ab | 2.0 x 108 abc | 1.1 x 108 bcdef |
10 ºC | 1.8 x 108 abcd | 2.5 x 108 a | 1.8 x 108 abcd | |
BJ11 (wt) | Room | 1.6 x 108 abcde | 1.1 x 108 bcdef | 4.2 x 107 ef |
10 ºC | 1.3 x 108 abcdef | 1.1 x 108 bcdef | 1.9 x 108 abcd |
6. Field trial of application of acid-aluminium tolerant B. japonicum on soybean
There are three locations for field trials to apply of the formula acid-aluminium tolerant
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C (%) | 1.18 | 0.73 | 2.54 |
N (%) | 0.16 | 0.11 | 0.20 |
P (%) | 0.0342 | 0.0142 | 0.0749 |
Mg (%) | 0.02 | 0.01 | 0.02 |
K (%) | 0 | 0 | 0.01 |
Ca (%) | 0.23 | 0.14 | 0.67 |
C/N (%) | 7.38 | 6.64 | 12.7 |
Al-dd | 2.56 | 0.88 | 0.15 |
Capacity of cation exchange | 7.55 | 2.45 | 7.14 |
pH : aquadest | 4.61 | 5.56 | 6.18 |
pH :KCl | 4.72 | 4.76 | 4.81 |
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Jasinga-West Java | 4.0 x 105 | 0 |
Sukadana -Lampung | 5.9 x 105 | 0 |
Tambang Ulang -South Kalimantan | 7.4 x 105 | 0 |
The field trial was conducted to examine the efficiency of BJ 11 (wt) and BJ 11 (19) on the growth, nodulation and yield of soybean variety Tanggamus and Anjasmoro. Tanggamus is one of leading variety which can adapt to dry acid soil, Anjasmoro generally showed good adaptation on paddy fields.
The seeds were coated with the inoculum formula before sowing. Seeds were sown by hand in each hole and planted 3 seeds per hole at a depth of 3 cm, distance of hole 20 cm x 40 cm. Fertilizer was placed at other hole besides of seeds hole. Watering was carried regularly if no rain. Removal of weeds or grasses are done as far as possible.
Soybean seed are sown by hand in a hole at soil. There were three seeds per polybag. Soybean seeds were selected based on the same size and healthy (able to shoot). Some treatments were conducted to soybean seed as follows: 1. inoculated by
Each treatments were done at 150-200 m2 and replicated two times per treatment. Mineral fertilization 100% N treatment consisted of 100 Kg ha-1 urea + 200 Kg ha-1 TSP (trisodium phosphate) + 100 Kg ha-1 KCl. For 50% N consisted of a half dose of urea + 200 Kg ha-1 TSP + 100 Kg ha-1 KCl + compost 1000 Kg ha-1. Compost was spread out at land surface one week before seeds planting. The compost only consisted of decaying plants and decomposed by microbes. There are not found rhizobia in compost, and consist of phosphate solubilizing bacteria as much as 320 cell.ml-1. Urea used twice at one planting period viz a half dose at seeds planting and the rest at 30 days after planting (DAP) [27].
Growth parameters such as plant height at 30 days after planting (DAP), number of pods at 90 DAP, total number of seeds, total of seed weight, and weight of 100 seeds numbers of pods compare to control were determined. Growth parameters were measured from 10 plants per treatments. Data were analyzed using completely randomized design and the means at p<0.05 level of significance.
The results of field experiments showed that there were a significant effect of
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BJ 11 (19) + 1 N | 30.6 d | 1 c | 28.2 cd | 34.2 c | 1.5 c | 10.8 c | 17.5 d | 2.83 cd | 13.42 b | |
BJ 11 (19) + 1/2 N + C | 35.8 b | 0.7 cd | 31.2 bc | 41 b | 2.6 a | 16.2 b | 29.4 bc | 4.33 bc | 12.97 b | |
BJ 11 (19) | 31.9 cd | 0.3 de | 19.9 e | 37.5 bc | 1.4 c | 10 c | 18.3 d | 2.27 de | 12.60 b | |
BJ 11 (WT) + 1 N | 40.1 a | 1.7 b | 35.4 b | 46.5 a | 2.3 ab | 19.7 b | 35 b | 4.55 b | 13.21 b | |
BJ 11 (WT) + 1/2 N + C | 42 a | 2.3 a | 45.2 a | 51.9 a | 2.5 a | 31.3 a | 51.1 a | 6.29 a | 12.55 b | |
BJ 11 (WT) | 33.6 bc | 0.4 de | 21.8 de | 34.c1 | 0.1 d | 5.5 c | 7.1 e | 1.02 e | 15.06 a | |
1 N | 41.3 a | 1.6 b | 36.4 b | 38.1 bc | 1.6 bc | 16.6 b | 25,5 bcd | 3.43 bcd | 12.75 b | |
1/2 N + C | 42.2 a | 2.1 ab | 29.9 bc | 38.1 bc | 1.7 bc | 17.1 b | 29.8 bc | 4.23 bc | 13.17 b | |
Control | 26.4 e | O e | 20.5 e | 34.7 c | 1.8 bc | 10.9 c | 21.8 cd | 2.95 cd | 13.26 b | |
BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05). | ||||||||||
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Treatment | Plant height at 45 DAP (cm) | Number of branch | Number of flower | Plant height at 90 DAP (cm) | Number of branch at 90 DAP | Number of pods | Number of seed | Total of seed weight (g) | Weight of 100 seeds (g) | |
BJ 11 (19) + 1 N | 30.6 d | 1 c | 28.1 cd | 32.6 cd | 1.6 cd | 14.5 bc | 23.9 cd | 2.37 cd | 7.95 b | |
BJ 11 (19) + 1/2 N + C | 35.8 b | 0.7 cd | 31.2 bc | 33.2 cd | 1 d | 12.1 cd | 17.8 d | 1.38 de | 7.11 b | |
BJ 11 (19) | 31.9 cd | 0.3 de | 19.8 e | 36.1 bc | 1.6 cd | 21.8 a | 29.2 bc | 2.68 bcd | 8.32 b | |
BJ 11 (WT) + 1 N | 40.1 a | 1.7 b | 35.4 b | 43 a | 2.1 bc | 23.5 a | 34.9 abc | 3.72 ab | 10.74 a | |
BJ 11 (WT) + 1/2 N + C | 42.1 a | 2.4 a | 45.1 a | 42 ab | 2.8 ab | 24. 8 a | 38.6 ab | 3.42 abc | 9 ab | |
BJ 11 (WT) | 33.6 bc | 0.4 de | 21.8 de | 41.7 ab | 3.1 a | 25.9 a | 42.6 a | 4.02 a | 8.95 ab | |
1 N | 41.3 a | 1.6 b | 36.3 b | 31.7 cd | 1.3 cd | 19.5 ab | 25.1 cd | 2.32 cd | 8.66 ab | |
1/2 N + C | 42.2 a | 2.1 ab | 29.9 bc | 29.4 cd | 0.9 d | 10.6 cd | 15.3 de | 1.39 de | 9.42 ab | |
Control | 26.4 e | O e | 20.5 e | 27.4 d | 0.1 e | 5.5 d | 5 e | 0.42 e | 8.44 ab |
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BJ 11 (19) + 1 N | 38.3 de | 66.8 a | 33.1 a | 3 ab | 3.3 ab | 54.8 a | 109.2 a | 17.3 ab | 14.7 abc |
BJ 11 (19) + 1/2 N + C | 38.3 de | 61.5 b | 28 bc | 2.7 abc | 3.1 ab | 60.2 a | 71.5 c | 10.4 cd | 13.5 bc |
BJ 11 (19) | 41.6 bc | 52.8 c | 26.4 cd | 2.2 c | 3 ab | 41.2 b | 123.1 a | 18.7 a | 13.4 bc |
BJ 11 (WT) + 1 N | 53 a | 68.9 a | 22.6 de | 2.5 abc | 2.3 d | 34.7 bc | 81 bc | 14.1 bc | 15 abc |
BJ 11 (WT) + 1/2 N + C | 44. 3 b | 71.2 a | 29 abc | 2 c | 3 ab | 58.2 a | 75.9 c | 12.3 cd | 15.3 ab |
BJ 11 (WT) | 40.9 cd | 69.9 a | 32 ab | 3.3 a | 3.6 a | 67.7 a | 101.1 ab | 17.5 ab | 13.3 c |
1 N | 53.4 a | 50.8 cd | 21.6 ef | 3 ab | 2.9 bc | 27.9 cd | 46.3 d | 6 e | 16.1 a |
1/2 N + C | 35.8 e | 47.6 d | 21.2 ef | 2.3 bc | 3.1 ab | 27.2 cd | 63.1 cd | 9.2 de | 13.3 c |
Control | 41 cd | 45.8 d | 17.3 f | 3.2 a | 2.4 cd | 15.6 d | 40.6 d | 5 e | 14.6 abc |
BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05). | |||||||||
Tanggamus | |||||||||
Treatment | Plant height at 30 DAP (cm) | Plant height at 90 DAP (cm) | Number of leaf at 90 DAP | Number of branch at 30 DAP | Number of branch at 90 DAP | Number of pod at 90 DAP | Number of seed | Total of seed weight (g) | Weight of 100 seeds (g) |
BJ 11 (19) + 1 N | 28.6 d | 62.5 a | 32.9 a | 1.2 ab | 3 ab | 37.9 abc | 49.3 bcd | 4.3 bc | 11.0 ab |
BJ 11 (19) + 1/2 N + C | 30.4 cd | 59.4 ab | 24.8 b | 1.5 a | 2.7 bc | 33.6 bcd | 56.2 bc | 4.9 bc | 10.4 b |
BJ 11 (19) | 51.1 c | 50.5 c | 18.5 c | 1.5 a | 2.2 c | 21.8 e | 47.1 cd | 4.8 bc | 10.3 b |
BJ 11 (WT) + 1 N | 33.8 ab | 57.3 b | 31.8 a | 1.7 a | 3.2 ab | 41.1 ab | 74.9 a | 6.9 a | 11.4 ab |
BJ 11 (WT) + 1/2 N + C | 35 a | 59 ab | 32.6 a | 1.7 a | 2.7 bc | 45.1 a | 64.9 ab | 6.8 a | 10.9 ab |
BJ 11 (WT) | 33.9 ab | 55.4 b | 25 b | 0.6 bc | 2.6 bc | 31.6 cd | 64.4 ab | 5.8 ab | 12.1 a |
1 N | 34.9 a | 45.8 c | 23.3 bc | 1.9 a | 2.5 bc | 21.7 e | 36.7 d | 3.5 c | 10.3 b |
1/2 N + C | 32.4 bc | 45.7 c | 21.1 bc | 1.7 a | 2.7 bc | 19.8 e | 38.8 d | 4.1 c | 11.4 ab |
Control | 28.8 d | 48.9 c | 22.7 bc | 0.5 c | 3.5 a | 26.3 de | 50.3 bcd | 4.4 bc | 10.6 b |
BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05). |
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BJ 11 (19) + 1 N | 40.9 b | 61.5 ab | 1.5 ab | 2.4 a | 54.9 a | 60.6 ab | 7.6 bc | 12.1 ab |
BJ 11 (19) + 1/2 N + C | 46.7 a | 66.2 a | 1.9 a | 2.9 a | 60.2 a | 64.6 ab | 10.3 a | 13.6 ab |
BJ 11 (19) | 22.4 d | 67.8 a | 0 c | 0 b | 41.2 b | 13.4 e | 1 e | 10.5 ab |
BJ 11 (WT) + 1 N | 46.7 a | 50.2 bc | 1.4 ab | 2.4 a | 34.8 bc | 73.9 a | 9.5 ab | 16.1 a |
BJ 11 (WT) + 1/2 N + C | 40.6 b | 44.4 c | 1 b | 2.4 a | 58.2 a | 66.2 ab | 7.2 bc | 11.2 ab |
BJ 11 (WT) | 25.3 d | 64.7 a | 0.2 c | 0.2 b | 67.7 a | 35.2 d | 3.1 de | 8.8 b |
1 N | 43.2 ab | 68.4 a | 1.5 ab | 3.0 a | 27.9 cd | 53.8 bc | 7 c | 11.7 ab |
1/2 N + C | 40.9 b | 64.3 a | 1.6 ab | 2.1 a | 27.2 cd | 57.4 bc | 6.4 c | 10.1 ab |
Control | 33.6 c | 55.3 abc | 1 b | 0.9 b | 15.6 d | 42.9 cd | 4.1 d | 9.6 b |
BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05). | ||||||||
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BJ 11 (19) + 1 N | 32.4 bc | 61 a | 3.5 ab | 4.3 ab | 62.3 a | 131.6 a | 10.7 b | 7.4 a |
BJ 11 (19) + 1/2 N + C | 35.4 b | 60.3 a | 3.7 ab | 4.5 a | 61.4 a | 127 a | 10.2 b | 7.3 a |
BJ 11 (19) | 25.1 d | 34.5 f | 0.5 d | 1.2 c | 18.4 b | 33.1 c | 2.2 c | 6.8 a |
BJ 11 (WT) + 1 N | 31.1 c | 51 bcd | 3.1 b | 4.7 a | 51.6 a | 128.5 a | 9.7 b | 7.8 a |
BJ 11 (WT) + 1/2 N + C | 36.1 a | 57 ab | 3.9 ab | 4.3 ab | 58.3 a | 130.1 a | 10.1 b | 7.4 a |
BJ 11 (WT) | 26.3 d | 42.1 e | 1.2 cd | 1.8 c | 21.3 b | 43.3 bc | 3 c | 7.1 a |
1 N | 33 abc | 53.6 bc | 4.4 a | 4.8 a | 69.1 a | 145.3 a | 16.1 | 7.3 a |
1/2 N + C | 32.3 bc | 59.4 a | 3.1 b | 4.3 ab | 60.4 a | 129 a | 10.6 b | 7.9 a |
Control | 29.7 c | 47.2 d | 1.6 c | 3.1 b | 35.2 b | 73.1 b | 5.5 bc | 7.8 a |
BJ 11 = BJ 11 inoculant formula; N = 100 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl); ½ N = 50 Kg.Ha-1 urea + 200 Kg. Ha-1 TSP and 100 Kg.Ha-1 KCl; C = compost. Control = without fertilizer (NPK) and inoculants. Numbers on the same column followed by the same letter were not significantly different based on Duncan Multiple Range Test (α = 0.05). |
7. Conclusion
Effectiveness symbiotic between soybean and acid-toleran aluminium root nodule bacteria, such as
Acknowledgments
This project was supported by Integrated Excelence Research, The Ministry of Research and Technology, Republic of Indonesia in 1996 to TI Sunatmo and Incentive Programs for Applied Research, The Ministry of Research and Technology, Republic of Indonesia in 2007-2009 to NR Mubarik. We thank our colleaguaes and students for support in the project.
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