Differentiating Indigenous Soybean Bradyrhizobium and Rhizobium spp. of Indian soils; Indian Journal of Microbiology; Parveen. G. Ansari and D. L. N. Rao; Indian Institute of Soil Science, Bhopal, India;

Supplementary material-1: Origin and Characteristics of Soybean Rhizobia

Strain
code / NCBI Acc. no. / Origin / Colony Size
(mm) / EPS / Colony on CRYEMA / Growth Rate / Acid/alkali production / Growth in
Hoffers
alkaline
broth / Reduction
of BCP
in GPA / Litmus
milk
test
1 / R3 / JQ514071 / Anooppur / 8 / +++ / Watery translucent / F / Acid / No / No / Acid
2 / R4 / JQ514072 / Betul / 6 / +++ / Watery translucent / F / Acid / Yes / No / Acid
3 / R5 / JQ514073 / Betul / 4 / ++ / Watery translucent / F / Acid / Yes / No / Acid
4 / R8 / JQ514074 / Chhindwara / 5 / +++ / Watery translucent / F / Acid / Yes / Yes / Acid
5 / R9 / JQ514075 / Dewas / 5 / ++ / Watery translucent / F / Acid / Yes / Yes / Acid
6 / R10 / JQ514076 / Dhar / 5 / ++ / Watery translucent / F / Alkali / Yes / Yes / Acid
7 / R11 / JQ514077 / Dhar / 6 / ++ / Watery translucent / F / Acid / Yes / No / Acid
8 / R12 / JQ514078 / Harda / 5 / ++ / Watery translucent / F / Acid / Yes / No / Acid
9 / R14 / JQ514079 / Narsinghpur / 4 / ++ / Watery translucent / F / Acid / Yes / No / Acid
10 / R18 / - / Seoni / 5 / +++ / Watery translucent / F / Acid / No / No / Alkali
11 / R20 / - / Vidisha / 5 / +++ / Watery translucent / F / Alkali / Yes / No / Acid
12 / R22 / JQ665272 / Indore 3c / 3 / + / Water translucent / F / Acid / Yes / No / Acid
13 / R23 / - / Indore 5a / 2 / + / Watery translucent / F / Acid / Yes / Yes / Acid
14 / R24 / - / Indore 7.1 / 3 / + / Watery translucent / F / Acid / Yes / Yes / Acid
15 / R25 / - / Indore 7a / 5 / + / Milky / F / Acid / Yes / Yes / Acid
16 / R1 / - / Bhopal / 10 / - / Pink / M / Acid / Yes / No / Acid
17 / R6 / - / Chhattarpur / 2 / + / Pink / M / Alkali / Yes / Yes / Acid
18 / R7 / - / Chhattarpur / 3 / - / Pink with
yellow center / M / Acid / Yes / No / Acid
19 / R13 / - / Indore / 5 / - / Pink / M / Acid / Yes / No / Alkali
20 / R21 / - / Indore 1a / 2 / + / Pink / M / Alkali / Yes / No / Acid
21 / R2 / - / Geelakhedi / 2 / + / White opaque / S / Acid / No / No / Acid
22 / R15 / - / Rajgarh / 1 / + / White opaque / S / Acid / No / No / Alkali
23 / R16 / JQ514080 / Rajgarh / 1 / + / White opaque / S / Alkali / No / No / Alkali
24 / R17 / - / Rajgarh / 1 / ++ / White opaque / S / Alkali / No / No / Alkali
25 / R19 / JQ514081 / Tikamgarh / 4 / + / White opaque / S / Acid / Yes / Yes / Acid
26 / R26 / - / Indore 9b / + / White opaque / S / Alkali / Yes / Yes / Acid
27 / R27 / - / Bradyrhizobium japonicum
USDA-110 / 5 / + / Water translucent / S / Acid / Yes / Yes / Acid
28 / R28 / - / B. japonicum
USDA-122 / 2 / + / White opaque / S / Acid / Yes / No / Acid
29 / R29 / - / B. elkanii
USDA-31 / 5 / + / Water translucent / S / Acid / Yes / Yes / Acid
30 / R30 / - / Liphatech
isolate / 1 / + / White opaque / S / Alkali / Yes / No / Alkali
31 / R31 / - / Liphatech
isolate / 1 / + / White opaque / S / Alkali / Yes / No / Alkali
32 / R32 / JQ665273 / Liphatech-reisolate / 1 / + / White opaque / S / Alkali / Yes / No / Alkali
33 / R33 / JQ665274 / Vidisha / 1 / + / White opaque / S / Alkali / No / No / Alkali
34 / R34 / JQ514083 / Vidisha / 1 / + / White opaque / S / Alkali / Yes / No / Acid
35 / R35 / - / Vidisha / 1 / + / White opaque / S / Alkali / Yes / No / Alkali
36 / R36 / - / Vidisha / 1 / + / White opaque / S / Alkali / No / No / Alkali
37 / R50 / JQ514084 / Parbhani / 2 / + / White opaque / S / Alkali / No / No / Acid
38 / R51 / JQ514085 / Pantnagar / 2 / + / White opaque / S / Alkali / No / No / Acid
39 / R52 / - / Palampur / 2 / + / White opaque / S / Alkali / Yes / No / Alkali
40 / R53 / - / Ludhiana / 2 / + / White opaque / S / Alkali / Yes / No / Alkali

*S=slow grower M=medium grower F=Fast grower; CRYEMA= Congo-red yeast extract mannitol agar; EPS=Extra-polysaccharide; BCP= Bromocresol purple; GPA= Glucose peptone agar; all the strains were negative for ketolactose test.Note: The soybean isolates (R1-R2, R32-R36) are from IISS culture collection- R-3 to R-20 from the AICRP-BNF survey collection at JNKVV, Jabalpur; R21-26 from Directorate of Soybean Research, Indore; R30-31 were isolated from a commercial liquid inoculant;R 32 is a re-isolate from commercial inoculant after plant passage, R50-53 are soil isolates.

Characteristics of the soil sampling sites.

Zone / Location / Soil Type / pH / EC
(mS/cm) / Organic C
(%)
Central India / 16 sites in
Madhya Pradesh / Vertisol / 6.7 - 7.90
(7.4) / 0.10-0.85
(0.26) / 0.39-1.04
(0.62)
Western India / Parbhani / Vertisol / 7.7 / 0.87 / 1.19
North India / Palampur / Alfisol / 5.1 / 0.44 / 1.16
Pantnagar / Mollisol / 8.1 / 0.48 / 0.38
Ludhiana / Inceptisol / 8.0 / 0.61 / 0.39

* values in row 1 are range and mean (in parenthesis)

Nodulation testing

The isolates were tested for nodulating ability on soybean (var. JS 335); seeds were surface sterilized, pre-germinated and transplanted into paper cups containing sterile sand; 3 seedlings per cup, 5 replicate cups per strain were kept in a plant screen house. The rhizobial strains were grown in yeast extract mannitol broth for 3 days (fast growing strains), 5 days (medium) and 7 days (slow growers). One ml of the culture broth of each strain (containing ~ 106 cells ml-1)was added to each plant in the pot at the base of the seedling. In controls, 1 ml of uninoculated sterile YEM broth was added to each plant. The plants were irrigated on alternate days with boiled tap water and weekly with four times diluted modified Arnon and Hoagland’s solution (N-free). Sterile water was added to compensate for evapo-transpiration losses. The plants were uprooted after 55 days and those strains that formed pink healthy nodules at the base of tap root were considered as effective. The total number of nodules per plant were counted and dried to record their mass along with shoot biomass. On an average the slow, medium and fast growing strains showed 18, 12 and 16 nodules per plant, with mean nodule dry mass of 15, 21 and 27 mg per plant respectively. The dry weight of the plants inoculated with the slow, medium and fast growing strains was 1.20, 1.28 and 1.05 g per plant respectively at 55 days.