Population Level of Bean Rhizobia in the Soil of Semi-Arid South-East Kenya and Assessment of Symbiotic Behaviour

EDXRF analysis oflocal and imported pica soil samples.

E.N.M. Njagi, A.M. Kinyua, lK. Munyua &MJ. Mangala.

19

ISSN

1029-3221

EAST AFRICAN

JOURNAL OF

SCIENCE

I

1

₂₀

_QiJ

Contents

The occurrence oflarval Contracaecum sp. (Nematoda: Heterocheilidae) in three teleostean fish species from Lake Naivasha, Kenya.

P.A. Aloo. 1

Population level of bean rhizobia in the soil of semi-arid south-east Kenya and assessment of symbiotic behaviour.

J.M. Maingi, C.A. Shisanya, N.M. Gitonga & B.Hornetz.

1

3

Linear and non-linear optical properties of amorphous Si.

J. Okumu & G.J. Morgan. 25

Computation of electronic properties of amorphous silicon (a-Si).

Y.M. Wambua, J. Okumu & T.S. Dod. 35

Electrical conductivity in doped polymers.

D.B. Bern 43

Population level of bean rhizobia in the soil of semi-arid south-east

Kenya and assessment of symbiotic behaviour

1.M. Maingi', C.A. Shisanya'", N.M. Gitonga' and B. Hornetz3

I Department of Botany, Kenyatta University, P.O. Box 43844, Nairobi, Kenya.

2Department of Geography, Kenyatta University, P.O. Box 43844, Nairobi, Kenya. 3FB VIIGeogr.- Geosciences, University of Trier, 0-54286, Trier, Germany.

*corresponding author (shisanya@avu.org)

The major objective of this study was to investigate Biological Nitrogen Fixation (BNF) in common bean in semi-arid south-east Kenya. The population of indigenous rhizobia specific to common bean was determined using the Most Probable Number (MPN) plant infection technique. The experimental design was randomised complete block design (RCBD) with four replications per treatment. The results of the MPN count indicated that the number of indigenous rhizobia resident in Kiboko soils, and specific to common bean were 2,037 - 14,850 rhizobia cells per gram of soil. Isolation carried out from the nodules indicated two different types of isolates. One has small and dry colonies (CIS), while the other one had large mucoid colonies (C2L). Presumptive and authentication tests confirmed these isolates as rhizobia.

Indigenous rhizobia were isolated from the common beans grown in Kiboko soil and tested for ability to fix nitrogen in comparison to a commercially available Rhizobium strain 446 from MIRCEN, University of Nairobi. Results showed that infectivity in common bean with Rhizobium strain 446 was good (80 nodules/plant). Bisection of the nodules showed that only 32%of them had an inner pink colour. This is an indication that a majority of the nodules were not effective in fixing nitrogen hence the need to frequently re-evaluate the homogeneity of the commercial strain 446. Greenhouse trials showed that isolate CIS was not effective in nitrogen fixation and that Rhizobium leguminosarum bv.phaseoli strain 446 and C2L from MlRCEN and

soils ofSE-Kenya, respectively, were superior in nitrogen fixation compared to CIS.

Key words: common bean; Rhizobium; semi-arid; south-east Kenya

INTRODUCTION

Common bean (Phaseolus vulgaris L.) is the most widely cultivated legume in Kenya (Shisanya,

1998). Common beans are second to maize as a subsistence crop in the country (Agwanda, 1988).

They are important in all agricultural areas of Kenya except at the coast and coastal hinterland up to about 600 m a.s.l. altitude (Acland, 1971). Despite the importance of beans as a major source of low cost protein to the majority of Kenyans, yields per hectare are still very low (Shisanya,

1996; Shisanya, 1998). One of the main factors contributing to low productivity is that the soils in most bean growing areas are low in essential nutrients like N and phosphorus (Shisanya, 1996:

Hornetz, 1997). The farmers in these areas are resource poor and cannot afford the imported and expensive chemical nitrogen fertilisers (Amara et al., 1995). This therefore calls for the need to use less expensive alternatives to improve the productivity of common beans. One such alternative is the utilization of Legume-Rhizobium symbiosis. This technology, also referred to as Biological Nitrogen Fixation (BNF) uses the legume-Rhizobium symbiosis to fix atmospheric nitrogen and make it available to the legume plant. The application of such technologies has been foundto be minimal or non-existent in semi-arid south-east Kenya (Shisanya, 1996).

14 - Maingi et al.,Bean rhizobia & symbiotic behaviour in Kenya

enumeration of specific Rhizobium in soils is also important in establishing the need for inoculation. To date the indigenous rhizobia specific to common bean has not been determined in the semi-arid areas of south-east Kenya, hence the existence of a knowledge gap. The objectives

of

• To determine the Rhizobium population in the soil of semi-arid south-east Kenya specific to common beans, and

• To investigate nitrogen fixation in common beans in Kiboko area of semi-arid south-east Kenya.

MATERIALS AND METHODS

Seeds of common beans, kathika variety (KB), were obtained from local farmers at Kiboko site in south-east Kenya. Modified Leonard jar assemblies described by Vincent (1970) were used as the .growth containers while the growth medium was nutrient free vermiculite. Nodule assessment

was carried out in the greenhouse as described by Vincent (1970). A soil sample from Kiboko (10 samples, 0 - 15 em soil depth, bulked and thoroughly mixed and sub-sampled) was used in estimating the nodulating population of rhizobia. The soil was collected prior to the onset of the rains from a field where tepary beans had been grown for four consecutive seasons. Estimation of rhizobia population was done using the Most Probable Number (MPN) plant infection technique (Beck et al., 1993). The most likely number of rhizobia specific to common bean was calculated from the MPN results (Table 1), according to the method of Vincent (1970). See also equation (1)

.MPN =(m xd)/v (1)

where:

m is the most likely number d is the lowest soil dilution, and v is the aliquot used for inoculation

Isolation of rhizobia, presumptive and authentication tests were carried out according to the methods described by Somasegaran et al. (1985). Tests carried out included: gram staining, growth of isolates on YMA, growth on YEM plus Bromothymol blue (BTB) and growth of rhizobia onYMA plus Congo red media. The plates were incubated in the dark at 28°C for 3 - 5 days. The rhizobia were routinely grown in yeast extract mannitol broth (YMA). For inoculation purposes, the rhizobia were grown in yeast extract mannitol broth (YEM). A commercial inoculum production strain Rhizobium leguminosarum biovar. phaseoli strain 446 was obtained from Microbiological Resource Centre (MIRCEN), University of Nairobi. This commercial strain was used as a standard to assess nodulation and N fixation of two other rhizobia isolates CIS and C2L obtained from common bean grown in soils obtained from Kiboko.

(1) KB-CIS:

(2)

KB-C2L:

(3) KB-446:

common bean inoculated with

Rhizobium

isolate CIS

common bean inoculated with

Rhizobium

C

S

common bean inoculated with

Rhizobium leguminosarum

biov.

phaseoli

strain 446

(4)KB-MC:

common bean uninoculated with plain mannitol broth (plants were given

--/

nutrient free solution incorporating 70 ppm ofKN03 (0

.

05

(Vincent,

1970)

(5) KB-NC:

common bean grown in nitrogen rich medium

The plants were harvested after 30 days. The plant material Wasseparated into shoots, roots

and nodules and dried to constant weight at 70

C

.

The dry weights were detenninedusing a high

precision digital Sartorius weighing balance (U6100-D2

Gottingen)

.

The experimental design was a randomised complete block design

with four

replicates per treatment

.

Data were subjected to analysis of variance

and means

separated using Duncan's multiple range test. The statistical software package used was

STATGRAPHICS.

RESULTS

The most likely number of rhizobia specific to the common bean was calculated from the

results (Table 1). See also equation 1

.

The rhizobia capable of nodulating commonbeans in the

soils ranged from 2,037 to 14,850 cells per gram of soil

.

Table 1:

MPN counts of rhizobia in one Kiboko soil

.

Soil dilution

10-1 1O-:l lO-J

10

-

4

10-'

10

-

()

1O-1S 1O-!I 1O-1U

Number of

4

4

4

4

4

2

1

1

0

0

jars with

nodulation

Presumptive tests carried out to establish the cultural characteristics of the rhizobia isolated

from common beans indicated that all the isolates were Gram negative rods. On Congo red

medium, the colonies were milky to translucent, showing very little or no absorption of the dye

.

On

growth of colonies of isolates was accompanied by a colour change of medium from

deep green to yellow indicating the production of acidic

.

substances which diffused into the

medium. Two types of isolates

of

rhizobia were observed in common bean. One type had small

dry colonies (about 2 mm in diameter).

16 - Maingi et al.,Bean rhizobia &symbiotic behaviour in Kenya

CIS. (32 nodules/plant on average) and C2L (13 nodules/plant on average) Although nodulation with strain C2L was poor, the few nodules were effective in N fixation as showed by the results of the dry weight (Table 2). Growth of the plants treated with CIS isolate was poor and chlorosis was exhibited in the leaves. The plants inoculated with phaseoli strains 446 and C2L were deep green in colour and bigger in size compared to the yellow stunted non-nodulated uninoculated controls and the poorly nodulated plants. The plant shoot dry weights are presented in Table 2. The results indicate that although treatment KB-NC plants had a higher shoot and nodule dry weight than the other treatments, there was no significant difference with treatments KB-446, KB- C2L and KB-MC, respectively (Table 2). Treatment C2L plants had higher root dry weight than the other treatments, though no significant differences with treatments KB-446, KB- NC were apparent.

Table 2: Effectiveness of rhizobia in N fixation in common bean

Treatment Shoot dry weight Root dry weight Nodule dry weight

(g plant") (g

plant ")

KB-CIS 1.36a 0.40a 4.2a

KB-C2L 1.61an _0.590 5.4ao

KB-446 1.74ao 0.50ao 5.5ao

KB-MC 1.58ao 0.45a 5.3ao

KB-NC 1.930 _{0.50ao 6.00}

Means (n=4) followed by the same letter are not significantly different by Duncan's multiple

range test at P$; 0.05 significance level

DISCUSSION

There was good nodulation in common bean in terms of nodule abundance and distribution. However, a high proportion of the nodules lacked leghaemoglobin, indicating that they were not effective in nitrogen (N) fixation. The lack of leghaemoglobin in the nodules of common beans inoculated with the commercial strain 446 is indicative of some form of change in the rhizobia used for the inoculum production. Hence, there is need for re-evaluating the homogeneity of this strain. Studies have reported that some of the indigenous rhizobia are ineffective in N fixation (Amara et al., 1995; Vincent, 1970). In this study two indigenous rhizobia isolates CIS and C2L, specific to common beans were found to exist in Kiboko soil. Of these two, C2L, though poor in nodulation (13 nodules/plant) was found to be effective in N fixation and was comparable to the commercial strain 446 (Table 2). Nodule number is frequently used as a measure of infectiveness (Beck et al., 1993. Although adequate nodulation was observed with the commercial phaseoli

strain 446, ineffective nodules exceeded the number of effective nodules, hence the possibility of inadequate N fixation (Wani et al., 1995).

1993). The MPN plant infection technique is a reliable method known since it avoids the

antagonistic effects of other micro-organisms that hamper the counting of rhizobia in the soils

(Bohlool et al., 1992; Danso, 1985; Halliday, 1984). The rhizobia population specific to common

beans was adequate for satisfactory nodulation in the soils. These numbers were above the

threshold (50 rhizobia cells per gram of soil) at which inoculation may be excluded (Theis et aI.,

1991). These results are in agreement with the findings of Nambiar et al. (1988) that. most

tropical soils have a rhizobial population of more than 100 rhizobia cells per gram of soil capable

of nodulating the legume grown in such a soil. The soils were collected during the dry season and

it is expected that during the rains, along with other soil microorganisms rhizobia numbers will

increase (Gitonga et al., 1999; Maingi et aI., 2001).

The results obtained from Gram staining and growth of isolates on YMA conformed with the

standard cultural and morphological characteristics of Rhizobium sp. described. by Somasegaran

et al. (1985). The isolates did not absorb Congo red dye at all. On bromothymol blue, the

medium changed from deep green to yellow colour indicating production of acidic substances.

Production of acidic substances is common with fast growing Rhizobium sp. The only

confirmatory test for rhizobia studies is the plant test (Vincent, 1970). Modified Leonard jar

assemblies were used for the plant tests. The method has become a standard for testing

nodulation and nitrogen fixation under greenhouse conditions (Beck et al., 1993). Plants

inoculated with isolate CIS and C2L did not nodulate well (32 and 13 nodules/plant, respectively)

compared to those inoculated with the commercial strain 446 (80 nodules/plant), However, from

the plant dry weight data (Table 2), it would appear that strain C2L is less infective but effective in N fixation in common beans.

Nitrogen fixation was estimated using plant dry weight. The method is reliable for

screening large numbers of plants for nitrogen fixation in nitrogen free medium (Brockwell et al.,

1995; Halliday, 1984). It is, however, not sensitive enough to be used in soils with high nitrogen content (Danso, 1985). In other cases, other factors besides nitrogen.do not permit the nitrogen fixed to be translated into increased dry matter yield (Danso, 1985). There was a significant

difference beween plants inoculated with CIS and the nitrogen control. In south-east Kenya, there

exist two types of colonies of bacteria CIS and C2L. CIS is not effective at N fixation compared

to C2L, which is comparable to the commercial strain 446 and to KB-MC regarding shoot dry

weight. A tentative inference from these results is that N fixation might be dependent on the

proportion of these two isolates in the soil. The response of beans to inoculation in these soils is

also likely to be dependent on the ability of the inoculant strain to compete with the indigenous

strains. There is, however, need for further research on the above two propositions. _

ACKNOWLEDGEMENTS

The work reported here is part of a larger project funded by the Volkswagen Foundation,

Hannover, Germany. The useful comments and suggestions by the two anonymous reviewers

greatly improved the quality of this paper.

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