**6. Inoculation techniques using compatibility between** *Rj-***genes and** *Bradyrhizobium* **strains**

In our laboratory, an experiment for testing effects of inoculation into a cylindrical soil block from just above the seed were conducted using bacterial suspension of *Bradyrhi‐ zobium japonicum* USDA110 as useful rhizobia in the past. The results showed that the oc‐ cupation ratio of USDA110 in the tap roots was as high as 70-100% and that in the lateral root was 44-77% lower than that of tap root and decreased in progression of the growth and developing of the host plant. In particular, the extreme decrease of US‐ DA110 occupation was observed in soybean variety Fukuyutakaa, which was cultivated as a recommended variety in the southwest warm region of Kyushu in Japan. It is likely that the cause of reduced occupancy in lateral roots occurred because rhizobia were ino‐ culated in the narrow range around the tap root, while lateral roots continued to grow outside of the inoculation column. If the range of inoculation were enlarged, the occu‐ pancy ratio of lateral roots probably would have increased.

In 2005, a field experiment was conducted to clarify the effect of the difference on soybean production between the inoculation of rhizobia on the seed surface compared with the plow layer [66]. Non-inoculated plot (NI), seed coat inoculation (SI) plot, and plow layer rhizobial solution inoculation (RI) plots were tested in three replications. Rhizobial concentration for inoculation was 107 cells/seed in both plots. As the result, the number of nodulation and the occupancy ratio of serotype USDA110 were highest in the SI plot, however yield (kg/10a) was higher in the RI plot versus other plots. This occurred is because the inoculum density in the SI plot was high, resulting in many nodules formed. This increased competition of photosynthetic products between the growth of soybean and nodulation, and consequently the initial growth was suppressed. For the SI plot, the effect of inoculation was expected to decline with time. In addition, since the occupancy ratio of inoculum was less than 50%, it is considered likely that the yield would increase if the seed surface inoculation were high.

Based on these results, it is important to examine the relationship between nodulation meth‐ od and the effective inoculum concentration on the seed.

Therefore, the purpose of this study was to clarify the effect of the difference in inoculation method and inoculum density of *B. japonicum* USDA110 on soybean production.

### **6.1. Materials and methods**

Soybean (*Glycine max* L. Merr.) variety Fukuyutak and *B. japonicum* USDA110 having uptake hydrogenase (Hup+ ) were used in this experiment. *B. japonicum* USDA110 was cultured in HM medium for eight days with shaking at 30˚C used for inoculation. Fukuyutaka was cul‐ tivated in the farm of Kyushu University Faculty of Agriculture, where barley was grown as the previous crop and soybean had not been grown for the past five years. Experimental plots had three replications for the treatments: non-inoculation (NI) plot, seed coat inocula‐ tion (SI) plots; 105 cells/seed (SI5) plot and 107 cells/seed (SI7) plot, and plow layer rhizobial peat-moss inoculation (PI) plots; 107 cells/seed (PI7) plot and 109 cells/seed (PI9) plot. Peatmoss inoculation was conducted using mixture of BM2 (raw materials: peat-moss, Group Berger Peat Moss Ltd., Canada) USDA110 culture inoculated into the plow layer before seed sowing to be 107 or 109 cells/seed.

Inoculum for SI plot per 100 seeds was made from 1.5 mL deionized water and 10 ml of 12% aqueous solution of gum arabic, 10 g of BM2 and 0.015 mL (SI5) or 1.5 mL (SI7) of USDA110 culture solution (1 × 109 cells/mL). Inoculum for PI plot per m2 was mixed well BM2 of 200 g spread on the plastic sheet, tap water of 40% of the maximum water holding capacity of BM2 and USDA110 culture solution of 0.25 mL (107 cells/seed) or 25.0 mL (109 cells/seed). After spraying the mixture into the row of the test plot, the rows were plowed (approximately 15 cm depth) by a tractor. The amount of USDA110 in the plow layer after inoculation was 107 or 109 cells per 1,800 cm3 (60 × 20 × 15 cm3 ) of soil occupying the root zone of one hill (three seeds sown). As a result, the inoculum density of USDA110 in the plow layer of each plot, PI9 and PI7 was estimated to be 1.7 × 103 and 1.7 × 105 cells/g DW soil. The amount of lime carbonate for adjusting to 6.5 soil pH was estimated by the buffer curve method using soil samples collected from 6 points (15 cm soil depth) in the experimental field. After applying the lime carbonate, on July 11, Mame kasei (3.0% ammonia nitrogen, 10.0% acid soluble phosphorus, 10.0% watersoluble potash) was applied at a rate of 80 kg per 10 a to all layers. Inoculation of the PI plot was done on July 18. On July 28, seeds of Fukuyutaka of all plots were sown into hills of either 20 or 60 cm apart, at a depth of approximately 2-3 cm. The seedlings were thinned to two seedlings at the trifoliate stage (V2). At V5 stage, the inter row area was cultivated and ridged. Pesticides were sprayed according to the occurrence of insect pests. Plants of one hill per each plot were sampled for roots at V6.4 and R5.7 stage, and 10 hills were harvested by cutting the stem at R8 stage (November 2 or 8).

To estimate the density of indigenous rhizobia, soils were collected from two locations in the experimental field before fertilization and the rhizobia density was measured by the most probable number (MPN) method using soybean cultivars, Orihime (non-*Rj*-genotype) and Fukuyutaka (*Rj*4-genotype). Plants sampled for roots at V6.4 and R5.7 stage [67] were divid‐ ed into shoots and roots. Roots were used for measurement of acetylene reduction activity (ARA) and counting of nodules. Counting of nodules was done after partitioning the plant into five parts the upper 3 cm of tap root, the lower part of the tap root, the lateral roots generated from the upper tap root, the lateral roots generated from the lower tap root and the superficial roots. Nodules from these samples were freeze-dried. Plant parts were airdried at 80˚C for 24-48 hrs. Also, in order to determine the effect of inoculation, the freezedried nodules were evaluated with serological tests performed using USDA110 antiserum produced previously in our laboratory. USDA110 occupancy was analyzed by the χ2 test.

After examining individual harvest yield components (effective number of pods/m<sup>2</sup> , seeds number/pods, complete seed percentage, one hundred seed weight (g), yield (g/m<sup>2</sup> ) in each plot), plant dry weight of plant part was weighed. For each growth stage, significant differ‐ ences were determined according to LSD test (Fisher) after ANOVA analysis.

### **6.2. Results and desucassion**

Based on these results, it is important to examine the relationship between nodulation meth‐

Therefore, the purpose of this study was to clarify the effect of the difference in inoculation

Soybean (*Glycine max* L. Merr.) variety Fukuyutak and *B. japonicum* USDA110 having uptake

HM medium for eight days with shaking at 30˚C used for inoculation. Fukuyutaka was cul‐ tivated in the farm of Kyushu University Faculty of Agriculture, where barley was grown as the previous crop and soybean had not been grown for the past five years. Experimental plots had three replications for the treatments: non-inoculation (NI) plot, seed coat inocula‐

moss inoculation was conducted using mixture of BM2 (raw materials: peat-moss, Group Berger Peat Moss Ltd., Canada) USDA110 culture inoculated into the plow layer before seed

Inoculum for SI plot per 100 seeds was made from 1.5 mL deionized water and 10 ml of 12% aqueous solution of gum arabic, 10 g of BM2 and 0.015 mL (SI5) or 1.5 mL (SI7) of USDA110 culture solution (1 × 109 cells/mL). Inoculum for PI plot per m2 was mixed well BM2 of 200 g spread on the plastic sheet, tap water of 40% of the maximum water holding capacity of BM2 and USDA110 culture solution of 0.25 mL (107 cells/seed) or 25.0 mL (109 cells/seed). After spraying the mixture into the row of the test plot, the rows were plowed (approximately 15 cm depth) by a tractor. The amount of USDA110 in the plow layer after inoculation was 107 or 109 cells per 1,800 cm3 (60 × 20 × 15 cm3

soil occupying the root zone of one hill (three seeds sown). As a result, the inoculum density of USDA110 in the plow layer of each plot, PI9 and PI7 was estimated to be 1.7 × 103 and 1.7 × 105 cells/g DW soil. The amount of lime carbonate for adjusting to 6.5 soil pH was estimated by the buffer curve method using soil samples collected from 6 points (15 cm soil depth) in the experimental field. After applying the lime carbonate, on July 11, Mame kasei (3.0% ammonia nitrogen, 10.0% acid soluble phosphorus, 10.0% watersoluble potash) was applied at a rate of 80 kg per 10 a to all layers. Inoculation of the PI plot was done on July 18. On July 28, seeds of Fukuyutaka of all plots were sown into hills of either 20 or 60 cm apart, at a depth of approximately 2-3 cm. The seedlings were thinned to two seedlings at the trifoliate stage (V2). At V5 stage, the inter row area was cultivated and ridged. Pesticides were sprayed according to the occurrence of insect pests. Plants of one hill per each plot were sampled for roots at V6.4 and R5.7 stage, and

To estimate the density of indigenous rhizobia, soils were collected from two locations in the experimental field before fertilization and the rhizobia density was measured by the most probable number (MPN) method using soybean cultivars, Orihime (non-*Rj*-genotype) and Fukuyutaka (*Rj*4-genotype). Plants sampled for roots at V6.4 and R5.7 stage [67] were divid‐

10 hills were harvested by cutting the stem at R8 stage (November 2 or 8).

cells/seed (PI7) plot and 109

) were used in this experiment. *B. japonicum* USDA110 was cultured in

cells/seed (SI7) plot, and plow layer rhizobial

cells/seed (PI9) plot. Peat-

) of

method and inoculum density of *B. japonicum* USDA110 on soybean production.

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

od and the effective inoculum concentration on the seed.

cells/seed (SI5) plot and 107

cells/seed.

**6.1. Materials and methods**

peat-moss inoculation (PI) plots; 107

or 109

hydrogenase (Hup+

Relationships

106

tion (SI) plots; 105

sowing to be 107

Occupation of useful rhizobium inoculated on the seed coat was known to be low because of their low competition against indigenous rhizobia. This object was to clarify the effect of in‐ oculation method and inoculum density of *B. japonicum* USDA110 on production of soybean. Five experimental plots were designed:, no inoculation (NI), seed coating inoculation with a density of 105 cells/seed (SI5) and 107 cells/seed (SI7), previous inoculation into the root zone with a density of 1.7×103 cells/g soil (PI7) and 1.7×105 cells/g soil (PI9). The PI plots were plowed after the BM2 mixture was soaked with rhizobium culture.


Mean followed by same and without letters within a column of each stage are not significantly different using LSD (*P*<0.10).


Table 11 shows the effect of inoculation methods on nodulation efficiency. The PI treatment had high efficiency for the lateral roots at the V6.4 stage, but at R5.7, the efficiency was low. For all other root positions and inoculation methods, the efficiency was low. Occupation ra‐ tio of serotype USDA110 on the total root was significantly highest for SI7 and PI9 and low‐ est in PI7 at the V6.4 stage. At the R5.7 stage, the occupation ratio in the superficial and upper lateral roots increased especially by inoculation with the *B. japonicum* USDA110, ex‐

cept the superficial roots of PI7. Also, for whole roots, the occupation ratio was significantly increased by inoculation of *B. japonicum* USDA110, except for PI7 (Table 12). However, the ARA showed a tendency to decrease with high density inoculum (Table 13). These results were complemented by seed yields of Fukuyutaka being highest in SI5 and SI7 (Table 14).




Mean followed by same and without letters within a column of each stage are not significantly different using LSD (*P*<0.10).

**Table 13.** Acetylene reduction activity (ARA: μmole g-1 DW) at each growth stage.


Mean followed by same and without letters within a column of each stage are not significantly different using LSD (P<0.10).

**Table 14.** Seed yield and yield components of each treatment.

Consequently, in SI5, SI7, and PI9 plots, occupation of serotype USDA110 was significantly high vs. other treatments. Since greater fixed nitrogen was distributed for these treatments to pods and seeds, yield (g/m2 ) significantly increased. It was thought that the density of 105 cells/seed was more effective at seed inoculation because there were no effect by increasing the inoculum above this density. And from the results of the PI9 plot, it was thought previ‐ ous inoculation using BM2 (1.7×105 cells/seed) was effective in competition with the indige‐ nous rhizobia for nodulation.

Previous work showed that to successfully compete with indigenous rhizobium, introduced inoculum must be 1000 times greater [1]. However, in Canada, standard inoculum for soy‐ bean, kidney bean, and pea were 105 cells/seed [60]. Others have reported unsuccessful nod‐ ulation at high concentrations of *B. japonicum* USDA110 [68]. In this study, we demonstrated a significant increase in yield in both SI5 and SI7 treatments with inoculation concentrations of 105 cell/seed and 1.7 x 105 cell/seed, respectively. Increased inoculum density above these levels did not increase seed yield. We concluded that it is possible to increase yield through the introduction of rhizobium species having greater nitrogen fixation rates.
