J. Life Earth Science, Vol. 1(1): July 2005 pp.

71-74

EFFECT OF INOCULATION WITH RHIZOBIUM ON NODULATION AND GROWTH OF BEAN, DOLICHOS LABLAB
Ananda Kumar Saha* and Md. Fazlul Haque
Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh Abstract
Nitrogen fixing bacteria (Rhizobium) was isolated from the healthy nodules of bean, Dolichos lablab. Pure culture of isolated bacteria was subjected to cultural, morphological, biochemical and nodulation tests for characterization. All tests showed positive results indicating that the isolated bacteria was Rhizobium. In inoculation test the highest count of nodules was on plant bacterized seed with adhesive but the lowest count was on nonbacterized seed. However, presence of few nodules on plants of nonbacterized seeds cultivated on natural soil indicating that few suitable native Rhizobium was present in that soil. Count of nodules on plants of preinoculated seeds was lower than that on plants cultivated on preinoculated soil, indicating that Rhizobium can survive well in soil than on seeds. In all cases of inoculation, nodules number was higher on plants cultivated on sterile soil mixed with Rhizobium than on natural soil. The present finding indicates that the plant growth was related to the number of nodules on roots of plant. Key word: Inoculation, Nodulation, Growth, Rhizobium

mvi msc: Dolichos lablab wmgi ^vevb z`wwZ mg~n _K bvBUvRb msebKvix evKUwiqv (Rhizobium) K c_K Kiv nqQ| c_KKZ evKUwiqvi LuvwU Pvl-Gi ewk wbYqi Rb GwUi PvlMZ, AmsvwbK, Re ivmvqwbK Ges z`wwZMZ cixv Kiv nqwQj| mKj cixv aYvZK djvdj c`kY KiQ, hvi A_ c_KKZ evKUwiqv njv Rhizobium| AYyRxe hyKiY cixvq mevP msLK z`wwZ MYbv Kiv nqwQj AvVv viv evKUwiqv hy exR _K Rbvbv Dw` wKy mewb msLK z`wwZ wQj evKUwiqv hy bq Ggb exRi Dw`| cvKwZK gvwUZ PvlKZ Ges evKUwiqv hy bq Ggb exRi Dw` wKQy msLK z`wwZi DcwwZ GUvB wb`k Ki h wKQy DchvMx vbxq Rhizobium H gvwUZ wQj| c~eAYyRxehy gvwUZ PvlKZ Dw`i Pq c~eAYyRxehy exRi Dw` z`wwZi msLv Kg wQj hv wb`k KiQ h Rhizobium exRi Pq gvwUZ fvjfve euP _vKZ cvi| mKj AYyRxe hyKiYi cvKwZK gvwUi Pq wbwRe gvwUZ PvlKZ Dw` z`wwZi msLv AwaK wQj| djvdj Aviv wb`k Ki h Dw`i ew Zvi wkKoi z`wwZi msLvi mv_ mKhy|

Introduction
Rhizobia are aerobic rod shaped, motile, gram negative, hetrotrophic, and non-spore forming bacteria. They are motile when young and have bipolar, subpoler or peritrichous flagella. Rhizobia fix atmospheric nitrogen and thus not only increase the production of inoculated crops, but also leave a fair amount of nitrogen in the soil, which benefit the subsequent crops. This successful symbiotic association requires the survival of rhizobia in sufficient number as free living bacteria in the soil ecosystem (Crozat et al., 1982). Nitrogen is the most deficient nutrient in Bangladesh soils. Urea, which is the most commonly

used nitrogenous fertilizer, has now become a costly input for most of the farmers. As such, Rhizobium inoculants may be used as a cheaper substitute for urea in the production of food legume crops (Karim, et al., 2001). The beneficial effect of rhizobial inoculates in increasing yield of leguminous crops results from the activity of its root nodule bacteria, which fix atmospheric nitrogen making it available for the plants. Inoculation types and many other factors usually affect the nodulation, nitrogen fixation and plant growth. For crop growth minimum inoculum level is necessary to obtain beneficial effects (Iswandi, 1987). Wood and Cooper (1988) showed that at increasing inoculum densities increasing numbers of Rhizobium leguminosarum

biovar trifolii survive under stress conditions in liquid medium. Postgate (1976) also mentioned that dense populations are killed to lesser extent than sparse populations. Postma et al., (1990) mentioned that lower survival percentages were found when more bacteria were inoculated. The inhibitory or stimulatory effects of soil microorganisms such as bacteria, fungi and actinomycetes on Rhizobium are known. Culture filtrates of fungi isolated from soil and those isolated from washed nodules often inhibit the growth of Rhizobia. The failure of nodulation in certain parts of Western Australian soil has been attributed to the presence of microorganisms antagonistic to Rhizobia. It is sensitive to antibiotics and other agricultural chemicals (Nutman, 1965). In the present study An attempt has been made in the present investigation to isolate Rhizobium from bean, Dolichos lablab and to study the effects of its inoculation on nodulation and growth.

Materials and Methods

A healthy, unbroken nodule was cut from the root of bean. It was surface sterilized by immersing in 0.1% w/v mercuric chloride for 2 minutes and then 95% alcohol for 2-3 minutes. Each nodule was then crushed in a small aliquot of sterile physiological saline in a sterile mortar with the help of a sterile glass rod and then milky fluid was streaked on sterile yeast extract mannitol agar (YEMA) containing congo red (1% congo red, 2.5 ml in 100 ml YEMA). The plates were incubated at 28C for five days. The typical well-isolated white, raised colonies were picked up and suspended in sterile physiological saline in a test tube and streaked on YEMA plates for purification. Pure culture of isolated bacteria was subjected to cultural, morphological, biochemical (growth on Hofers alkaline medium, growth on congo red YEMA, growth of glucose peptone agar, carbohydrate utilization) and nodulation test for characterization as described by Subba Rao (1999). For inoculation, legume seeds were disinfected with 0.2% HgCl2 (2-3 min.) followed by 6-7 washings with sterile water. Disinfected seeds (about 50) were suspended in 20-40ml thick cell suspension of

Rhizobium (1012 cells/ml) for 30 minute. Fifty disinfected seeds were also suspended in thick suspension of Rhizobium in presence of 0.5% peptone (adhesive) for 30 minutes. Another lot of 50 disinfected seeds suspended in 20-40ml sterile saline for 30 minutes were kept as control. The seeds were air dried for 30 minutes in sterile petriplates. Inoculated seeds incubated at 4C for 30 days were considered as pre-inoculated seeds. Soil sample (2 kg) was taken in polycarbonate bags and sterilized in an autoclave (121C, 1 hour) for three consecutive days. The sterile soil sample was mixed with 20-40 ml bacterial suspension (1012 cells/ml) with sterile glass rod. Similarly non-sterile natural soil sample was inoculated with Rhizobium. Inoculated soil samples were transferred separately into sterile plastic pots. Inoculated soil incubated at 4C for 30 days was considered as pre-inoculated soil. Been seeds were sown in plastic pots containing sterile, natural, freshly inoculated and pre-inoculated soil. After 45 days number of nodules per been plant was counted and recorded.

Results
On YEMA the colonies of Rhizobium were circular, cream coloured, sticky, raised and translucent structure. On YEMA containing congo red, isolate produce colourless to faint colonies indicating that the isolate was Rhizobium sp. The result of carbohydrate utilization test showed that among seven types of carbohydrates used in study Rizobium can utilize six types viz. arabinose galactose, glucose, sucrose, lactose and mannitol. Growth in Hofers alkaline medium, on Congo red medium and on glucose peptone agar indicating that it did not belong to the genus Agrobacterium. Result of nodulation test showed that no nodule was formed in non- bacterized bean plants cultivated in sterile soil in plastic pots, but remarkable number of nodules was formed in bacterized bean plant cultivated in sterile soil (plate 1). This result of nodulation test confirms that the isolate was Rhizobium sp. In inoculation experiment no nodulation on the plants of uninoculated seeds cultivated on sterile soil, indicating that sterile soil was totally free of Rhizobium.

Table 1. Number of nodules per plant on bacterized and non-bacterized bean.

Inoculation Seeds bacterized with adhesive Soil used St Nt Seeds bacterized without adhesive St Nt Non-bacterized seeds Soil bacterized by liquid concentrates Pre-inoculated seeds St Nt St Nt St Nt Pre-inoculated soil St Nt 1 No. of nodules / plant 2 3 4 5 6 15 7 11 7 0 2 10 7 3 4 10 7 12 11 12 12 0 0 8 11 4 3 12 11 17 13 10 11 0 2 14 10 5 5 8 6 12 Mean S.E.

13 11 9 13

13 13 9 0 3 9 12 0 1 13

13 11 2 7 9 8 5 9 6 9

13.33 0.92G 10.17 8 1.06G 11.17 8 0.79G 9.50 6 1.06G 0 0.00 P 1.5 1 0.43P 11.00 12 0.97G 10.17 9 0.83G 3.66 3 0.49P 5.50 5 0.89 P 9.00 9 0.82 G 8.00 7 0.73 G

on natural soil indicating that few suitable native Rhizobium was present in that soil. Number of nodules on plants of preinoculated seeds was lower than that on plants cultivated on preinoculated soil, indicating that Rhizobium can survive well in soil than on seeds. In all cases of inoculation nodules number was higher on plants cultivated on sterile soil than on natural soil. Result showed in Table 1 also indicates that the plant growth was related to the number of nodules on roots of plant.

Discussion
According to Dye (1979) and Vincent (1970) Rhizobium grows on yeast extract manitol agar (YEMA) and produce small to medium sized colonies, usually smaller than 2 mm. Rhizobium can not grow at pH 11.0 in the Hofers alkaline medium (Gaur and Sen, 1981). The rhizobial growth on glucose peptone agar medium is usually poor and causes little change in pH (Subba Rao, 1999). Result of carbohydrate utilization test support the findings of Allen and Allen (1950) and Graham and Parker (1964) who showed differences of carbohydrate utilization between fast and slow growing rhizobia. Dissacharides lactose, sucrose, and trehalose were not utilized by slow growers. Sadowsky et al., (1983) found that disaccharides were utilized only by fast growing strains. According to Gleen and Dilworth (1981) slow-growing rhizobia tend to lack both uptake-systems and catabolic enzymes for disaccharides. All these evident reveals that the isolate was a member of the fast growing rhizobia. Subba Rao (1999) reported that the number of viable rhizobia tend to diminish in pre-inoculated seeds stored indefinitely. Many leguminous seeds contain water-soluble toxic compound which adversely affect the viability of rhizobia (Millington, 1995). It may be a cause of lower viability of Rhizobium on preinoculated legume seeds than in preinoculated soil. Saha and Kapadnis (2001a) reported that there existed a significant difference in bacterial population on seeds by using different adhesives. The count of Rhizobium transformant decreased almost equally in both sterile and natural black and poita soil but the rate of decline was relatively more in natural soils than in sterile soils (Saha and Kapadnis, 2001b). It was reported that the viability of R. leguminosarum in natural soil was greatly affected by

St-Sterile, Nt-Non-sterile, G=good; P=poor

Plate-1. Nodules formed on roots of inoculated bean plant cultivated on sterile soil (BS=Seeds bacterized with adhesive; NS= Non-bacterized seeds; PS=Pre-inoculated seeds; LS=Soil bacterized by direct application of liquid concentrates) As showed in Table 1, the highest number of nodules was counted on plant bacterized with adhesive but the lowest number of nodules was on nonbacterized plant. However, presence of few nodules on plants of nonbacterized seeds cultivated

certain protozoa, fungi and bacteriophages (Subba Rao, 1999). Increasing evidence supports the view that bacteria inoculated on seeds colonize roots emerging from the germinating seed and then moves along the roots passively (Dijikstra et al., 1987). Therefore, the density of bacteria applied to seeds may determine bacterial root colonization efficacy.

Graham PH, Parker CA. 1964. Diagnostic features in the characterization of root nodule bacteria of legumes. Pl Soil 20, 383- 396. Iswandi A, Bossier P, Vandenabeele JV, Verstrate W. 1987. Influence of the inoculation density of the rhizopseudomond strain 7nsk2 on the growth and composition of root microbial community of maize and barley. Biol Fert Soil 4, 119-123. Karim MR, Islam F, Akkas Ali M, Haque F. 2001. On-farm trail with Rhizobium inoculants on lentil. Bangladesh J Agric Res 26, 93-94. Millington AJ. 1995. Deep- placement of rhizobial cultures as an aid to legume inoculation. J Aust Inst Agric Sci 21, 102-103. Nutman PS. 1965. Origin and development of root nodules. Handb Pfl Physiol 12, 1355-1379. Postgate JR. 1976. Death in macrobs and microbes. In The survival of vegetative microbes. Ed. Gray TRG, Postage JR. pp. 1-18. Cambridge University Press, Cambridge. Postma J, Hok-A-Hin, Oudo Voshaar JH. 1990. Influence of inoculum density on the growth and survival of Rhizobium leguminosarum biovor trifolii introduced into sterile and non sterile loamy sand and silt loam. FEMS Microbial Ecol 73, 49-58. Sadowsky MJ, Keyser HH, Bohlool BB. 1983. Biochemical characterization of fast growing and slow growing rhizobia that nodulate soybeans. Int J Bacteriol 33, 716-722. Saha AK, Kapadnis BP. 2001a. Effect of adhesive on population of Green Fluorescent Protein (GFP) marked Rhizobium on legume seeds and their germination. Bangladesh J Genet Biotechnol 2, 133-141. Saha AK, Kapadnis BP. 2001b. Effect of inoculum density on survival rate of Rhizobium carrying reporter gene GFP in different soils. Asian J Microbial Biotech Env Sci 3, 123-128. Subba Rao NS. 1999. Rhozobium and legume root nodulation. In Soil Microbiology. P.169. Oxford & IBH publishing Co. Pvt. Ltd. New Delhi. Vincent JM. 1970. A manual for the practical study of the root nodule bacteria, IBP Hand Book No. 15, Blackwell Scientific publications, Oxford. Wood M, Cooper JE. 1988. Acidity, aluminum and multiplication of Rhizobium trifolii: effects of initial inoculum density and growth phase. Soil Biol Biochem 1, 63-87.

Conclusion
From the above results it can be concluded that the bacteria isolated from root nodules of bean D. lablab is a species of Rhizobium. The isolate is also a fast growing rhizobial species and able to form remarkable number of healthy nodules on inoculated crops. Result of inoculation test indicates that bacterization of seeds with adhesive is the most suitable among the inoculation methods used in this experiment and inoculation is responsible for good plant growth.

Acknowledgements
The authors express their sincere gratitude to the University Grant Commission (UGC), Bangladesh for its financial support to carry out the research project.

References
Allen EK, Allen ON. 1950. Biochemical and symbiotic properties of the rhizobia. Bacterio Rev 14, 273-330. Crozat Y, Cleyet-Marel JC, Giraud JJ, Obaton M.1982. Survival rates of Rhizobium japonicum populations introduced into soils. Soil Biol Biochem 14, 401-405. Dijikstra AF, Scholten GHN, Van Veen JA. 1987. Colonization of wheat seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus subtilis. Biol Fert Soil 4, 41-46. Dye M. 1979. Functions and maintenance of Rhizobium collection. In Recent advances in biological nitrogen fixation, ed. Subba Rao NS, pp. 435-471. Oxford and IBM publishing Co. New Delhi. Gaur YD, Sen AN. 1981. Cultural and Biochemical characteristics of root nodule bacteria of chickpea [Cicer arietinum (L.)]. Zbl Bakt II Abst 136, 307-316. Gleen AR, Diworth MJ. 1981. The uptake and hydrolysis of disaccharide by fast and slow growing species of Rhizobium. Arch Microbiol 139, 233-239.