African Crop Science Journal, Vol. 6. No. 2, pp. 159-169, 1998
ENVIRONMENTAL CONSTRAINTS TO NODULATION AND NITROGEN FIXATION OF PHASEOLUS VULGARIS L. IN TANZANIA. I. A SURVEY OF SOIL FERTILITY, ROOT NODULATION AND MULTI-LOCATIONAL RESPONSES TO RHIZOBIUM INOCULATION
F. Amijee and K.E. Giller
Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH
(Received 31 December, 1996; accepted 2 February, 1998)
A survey of selected fields in bean growing regions of Tanzania and a more detailed survey of bean fields in the Lushoto area of northern Tanzania were carried out to establish soil properties limiting the extent of root nodulation of Phaseolus vulgaris L. Soil samples were collected and soil pH, organic matter content, cation exchange capacity and concentrations of extractable P, K, Ca and Mg determined. Plant vigour and nodulation were recorded and indigenous populations of rhizobia nodulating Phaseolus were estimated. The majority of soils were found to have very small concentrations of extractable P and this was associated with poor nodulation and poor plant vigour. In most cases there were reasonable populations of soil rhizobia present (102-104 cells/g soil) but plants were well nodulated only when soil P concentrations were adequate. A series of trials which included treatments where rhizobial inoculants were applied demonstrated no significant responses to inoculation unless the data were combined across the 10 sites. The data confirmed the need for experiments on-farm where soil nutrient concentrations (especially P) were generally much less than those found on research stations.
Key Words: N2-fixation, phosphorus, potassium, root nodulation, soil pH
Une étude des champs sélectionnés dans les régions à haricot de la Tanzanie et une enquéte plus détaillée des champs de haricots dans la région de Lushoto, dans le nord de ce méme pays, étaient réaliseés en vue d'établir les propriétés du sol qui limitent la nodulation racinaire de Phaseolus vulgaris L. Les échantillons de sol étaient prélevés et le pH, la matière organique, la capacité d'échange cationique ainsi que les concentrations de P, K, Ca et Mg échangeables étaient determinés. La vigueur des plantes et la nodulation étaient enregistrées et les populations indigènes de rhizobium favorisant la nodulation de Phaseolus étaient estimées. La majorité des sols avaient une basse concentration de P assimilable mais qui toutefois correlait bien avec une pauvre nodulation ainsi qu'avec une pauvre vigueur des plantes. Dans la plupart des cas, il y avait des populations suffisantes de rhizobium du sol (102-104 cellules/g sol) mais les plantes étaient bien nodulées seulement quand les concentrations de P étaient adéquates. Une série d'essais comprenant des traitements inoculés au Rhizobium a failli aboutir aux réponses significatives à l'inoculation, exception faite à la combinaison des données provenant des 10 sites. Les résultats confirmaient le besoin de poursuivre les essais en champs où les concentrations des élements nutritifs du sols (spécialement P) sont généralement inférieures à celles trouvées dans les stations de recherche.
Mots Clés: La fixation de N2, phosphore, potassium, nodulation racinaire, pH du sol
Bean (Phaseolus vulgaris L.) is one of the most important grain legume crops for many countries in eastern and central Africa. It is widely grown as a subsistence crop by local farmers for whom it provides a substantial source of dietary protein and carbohydrate to supplement a diet usually based on maize and cassava (Okigbo, 1977).
In Tanzania, beans are mainly grown for local consumption and constitute one of the staple foods in many parts of the country (Karel et al., 1981); statistics indicate that an area of 470000 ha was under bean cultivation during 1988-89 (Grisley, 1991). Total production from this area was 230000 t giving an average yield of approximately 500 kg ha-1 but this figure is probably an over estimate because in many cases the same area may have been used for several croppings in a single year. The level of output has remained roughly constant over recent years, although total production has increased as a result of more land being used for bean cultivation (Grisley, 1991).
Generally, the areas in which bean is grown lie 800 m above sea level where rainfall is not limiting. In most regions the rainfall distribution is adequate to give two growing seasons; the long rains from March to June and the short rains from November to January. A major constraint for bean production in these areas is believed to be poor soil fertility (Mwandemele and Nchimbi, 1990), in particular the supply of nitrogen to support a good seed yield with a high protein content. Under such conditions the ability of Phaseolus to fix atmospheric nitrogen is likely to be an important factor determining yields.
Phaseolus vulgaris is nodulated by a range of types of fast-growing Rhizobium, including Rhizobium tropici, R. leguminosarum bv. phaseoli, R. etli and other types yet to be formally classified into species which are currently referred to as Rhizobium sp. (Phaseolus) strains (see Martinez-Romero, 1994). One of the main environmental factors which disrupts the symbiotic association between Phaseolus and Rhizobium is soil acidity (Munns, 1978; Graham, 1981). It has been suspected that the poor nodulation of bean roots commonly observed in the tropics is due in many cases to soil acidity (Franco and Day, 1980; Ssali, 1981) which sometimes causes severe problems of Al and Mn toxicity (Franco and Munns, 1982; Ssali and Nuwamanya, 1982). Anyango et al. (1995) found that the dominant types of rhizobia nodulating beans differed between an acid soil and a soil with a near-neutral pH. However, few studies have made a direct comparison between soil properties and extent of nodulation from the same field to establish whether it is pH or another soil factor that is affecting nodulation and production of beans. Certainly we are unaware of any reports on such investigations for bean growing areas of Tanzania.
The main objective for this study was first to carry out a general survey of bean fields in northern, central and southern Tanzania. Secondly, a more detailed survey was conducted in Lushoto district, an important bean growing area in the Usambara Mountains, northern Tanzania, which produced almost 25000 t of beans in 1985-86 (G. Mwaimu, District Agricultural and Development Officer, Lushoto, personal communication). For both surveys, bean fields belonging to research stations and local farmers were visited to observe plant vigour and root nodulation. Soil and plant samples were collected for chemical analyses and tests were done on the soils to estimate the indigenous populations of Phaseolus rhizobia. Our aim was to determine the variation in soil fertility and to identify soil properties which could be important for the establishment of effective nodulation under field conditions. The findings from the initial investigation also enabled us to select sites and identify treatments for subsequent field experiments in problem areas and a number of experiments were conducted to examine responses to Rhizobium inoculation.
MATERIALS AND METHODS
The general survey was carried out during the long rains (March to May 1987) and the detailed survey in Lushoto district was conducted during the short rains (October to December 1987). In the Lushoto survey, four fields in each of the thirteen villages of four different wards (Ubiri, Kwai, Gare and Lushoto) were visited. In most cases, attempts were made to meet the owner of the field to establish cropping history and note seed yields expected under normal conditions.
Plant vigour was assessed on the existing bean crop as either good (canopy height > 50cm, green foliage with > 10 pods/plant), moderate (canopy height 25-50 cm, green foliage with 5-10 pods/plant) or poor (canopy height < 25 cm, green-yellow foliage with < 5 pods/plant). Nodulation was assessed in the same field; six plants were randomly uprooted using a hand fork to observe the root system. The soil was carefully shaken off so that the extent of nodulation could be assessed as either good (> 75 nodules/root system; red/pink in colour), moderate 25-75 nodules/root system; red/pink in colour) or poor (< 25 nodules/root system, red/pink or white in colour or >25 nodules white in colour). Five soil samples per field (in a èW' pattern) were taken using a screw auger (15 cm deep). Samples from each field were bulked, air-dried, mixed and sieved (2 mm), and a sub-sample retained for analyses. Soil properties measured included the pH (1 part soil to 2.5 parts water after 2 hours), % organic matter (OM) by the method of Walkey and Black (1947), and P concentration by extraction with 0.5 M sodium bicarbonate solution at pH 8.5 (Olsen et al., 1954) and estimation colorimetrically with the ammonium molybdate-ascorbic acid method (Watanabe and Olsen, 1965). To determine exchangeable cations, the soil was leached with neutral solution of 1N ammonium acetate at pH 7 (Cahoon, 1974), and concentration of K (by flame emission spectrophotometry) and of Ca and Mg (by atomic absorption spectrophotometry) determined on the leachate using appropriate standards. The cation exchange capacity (CEC) was also measured by distilling the ammonium from the leached soil after leaching it further with potassium chloride (10% w/v). The concentrations of Zn, Mn, Cu and Fe in 0.005 M DTPA (diethylenetriamine pentacetic acid; Lindsay and Norvell, 1978) extracts were measured on selected soil samples (by atomic absorption spectro-photometry). Details for most of these methods are described by MAFF (1986).
The indigenous population of Phaseolus rhizobia was estimated in soil samples by the most probable number plant infection method (Vincent, 1970). Ten grammes of the soil sample were moistened with sterile water (10% w/v) and incubated (28°C) for 7 days. A series of 10 fold dilutions was done in sterile water and 1 ml of the suspension was used to inoculate 7 day old bean plants cv. Canadian Wonder, growing in 250 ml conical flasks containing 150 ml of nutrient solution (Hewitt, 1966) without N in 1% agar. Roots were examined for nodulation 21 days after inoculation and the number of rhizobia were estimated using tables given by Vincent (1970).
A series of ten experiments were carried out at various sites to examine a variety of factors (seed treatments, P application etc.) which all included treatments with and without inoculation using a mixed inoculum of strains. These trials included treatments examining differences in nodulation between different plant cultivars, effects of row spacing etc and are not reported in detail here as treatment effects were largely insignificant. Mean values for treatments with or without treatment with a peat inoculum containing a mixture of strains are reported.
RESULTS AND DISCUSSION
A selection of observations from representative sites throughout Tanzania (Tables 1 and 2) and a more detailed sampling from the Lushoto area (Table 3) showed that common bean was mainly grown in the more densely populated regions of Meru, Kilimanjaro and Lushoto in the north and Rukwa and Mbeya in the south (Table 1). Most of the area was cultivated by small farmers who grew bean as a sole crop or in association with maize depending upon the growing season. The seed was commonly from bush type (Types I to III) cultivars, often a mixture that was genetically diverse as indicated by their seed size and colour, which was raised by local farmers with no fertilizer or pesticide inputs. On the few large estates, bean was grown in monoculture mainly for seed production of white-seeded, èFrench Bean' varieties for the European seed markets.
TABLE 1. General observations of plant vigour and nodulation in some important bean growing regions of Tanzanla
(RS, research station; LE, large estate; FF, farmers field)
Plant vigour was good on fields managed by research stations and large estates. In sharp contrast, fields belonging to local farmers had bean plants with poor vigour (Table 1). This was usually reflected in terms of seed yield; from fields managed by research stations, yield tended to be above the national average of 500 kg ha-1, whereas it was less than 300 kg ha-1 from fields belonging to local farmers. As in earlier reports (Macartney and Watson, 1966; Souza, 1969), effective root nodulation of Phaseolus by the indigenous population of rhizobia was observed. However, it was found to be correlated with plant vigour: plants of good vigour had good nodulation and plants with poor vigour had poor nodulation (Table 1), with the exception of Karamu and Lyamungu where plant vigour was good but nodulation was moderate to poor. The intensity of nodulation does not necessarily indicate the establishment of efficient nitrogen fixation. However, observations of plants showing poor vigour indicated that availability of N and possibly P were limiting plant growth in agreement with the majority of nutrient studies on field grown Phaseolus in bean growing areas of eastern Africa (Uganda - Stephens, 1967; Tanzania - Anderson, 1974, Smithson et al., 1993; Kenya - Ssali and Keya, 1980).
The majority of soils collected from bean growing areas had pH values between 5.5 and 6.5 (Tables 2 and 3) which have been reported to be optimum for bean growth (Munns and Fox, 1976). This range of pH cannot be regarded as particularly acidic and therefore it is unlikely to be the cause of poor nodulation. Indeed, a field at Lyamungu with pH of 5.4 had bean roots with many large pink nodules, some of which were greater than 5 mm in diameter. Anderson (1974) reported that field grown beans in the Kilimanjaro area responded to lime only when soil pH was less than 5.5. A recent study (Smithson et al., 1993) showed that application of lime to bean in an experiment on a soil of pH 5.5 in the Lushoto area had no effect upon plant growth or development. Thus, although regression analyses indicated that there was a significant trend for better nodulation at higher pH across all of the sites, an alternative explanation to soil acidity must be sought for the poor nodulation of bean roots commonly observed during our investigation.
TABLE 2. Chemical analyses of soils from some important bean growing regions of Tanzania. A mean value for the analysis is presented from different number of fields at each location (with min/max values in parentheses)
TABLE 3. Chemical analyses of soils from farmers fields in the villages of Lushoto area, N. Tanzania. A mean value for the analysis is presented from four different fields at each village (with min/max values in parentheses)
The main variation in soil fertility was the difference in % organic matter (OM) and cation exchange capacity (CEC) among fields within an area (see min/max values for a batch of soil samples from different areas; Tables 2 and 3). This was probably related to the length and intensity of cropping and in some cases the variable use of manure. However, there was no conclusive evidence to relate nodulation or plant vigour to either % OM, CEC or amount of exchangeable cations in the soil (Tables 1 and 2). Some farmers in the Lushoto area apply dung collected from animals (cattle, goat and chickens) which are kept in stalls or close to the dwellings, but dung application is usually restricted to fields close to their compounds. Lower temperatures and higher rainfall, because of the variation in altitude, are probably the cause of high content of organic matter in these soils as shown elsewhere (Anderson, 1974).
The other main variation was in soil P concentration between fields managed by research stations and those by local farmers. The amount of soil P was very low in fields belonging to local farmers where plant vigour and nodulation were consistently poor (Tables 2 and 3). In fields managed by research stations or large estates, soil P concentrations were high with good plant vigour and well nodulated root systems (Tables 1 and 2). Such high soil P concentrations result from regular application of fertilizers, e.g. of 26 kg P ha-1 as triple super-phosphate are applied to fields at Lambo and Selian at the start of each cropping season. The site at TPRI was an exception as it is a pesticide research institute with no record of fertilizer application. Therefore, concentration of soil P was low and nodulation was also poor (Table 2). Local farmers may add N and P fertilizers and sometimes apply pesticides to saleable crops like tomato or potato, but traditionally, they do not add any fertilizers to their crop as it is usually grown for personal consumption and in some cases thought to play a role in maintaining or improving soil fertility. Therefore, under-standably, the majority of bean fields belonging to local farmers had very small concentrations of soil P, resulting in poor plant vigour and nodulation (Tables 2 and 3). P availability is often reduced with increasing soil acidity due to high P fixation (Sanchez and Uehara, 1980) and this is the case for some soils of eastern Africa (Le Mare, 1984). However, our data clearly show that soils were not acidic in the majority of bean fields visited (Tables 2 and 3) and, consequently, P availability should not be affected by it. The major constraint on root nodulation and plant vigour of Phaseolus in this study was most likely to be the small concentration of soil P, and not soil pH or any other soil factor measured. Although it is difficult to separate cause and effect, these results suggest that there may be an apparent correlation between soil P concentration and root nodulation and plant vigour as previously suggested (Anderson, 1964, 1974).
Chemical analyses of the soils from the detailed survey of Lushoto area (Table 3) showed that concentrations of Ca and Mg were probably adequate whereas concentrations of K were small for optimal growth of bean (i.e., < 0.5 cmol(+) kg-1, Anderson, 1973). Smithson et al.(1993) diagnosed K deficiency in bean leaves from the Usambara Mountains and found large increases in plant growth and seed yield following application of potash. Root nodulation may also be limited by K availability. Although no consistent relationships between the extent of nodulation and variation in soil K concentration were found for other bean growing areas (Tables 1 and 2), regression analyses indicated that there was a significant positive relationship between available K concentrations and nodulation. The concentra-tions of micronutrients Zn, Mn, Cu and Fe (Table 4) appeared roughly similar to those reported by Sillanpaa (1982) with the exception of Lyamungu and Karamu, where levels of Cu were relatively high due to regular application of Cu-based fungicides to coffee. Certain micronutrients are important for nodulation and nitrogen fixation, but it is difficult to determine their availability under field conditions.
TABLE 4. The coacentration of the micronutrients Zn, Mn, Cu and Fe in DTPA extracts of soils from some bean growing areas in Tanzania
The plant infection tests under laboratory conditions showed that in some soils (Table 5), Phaseolus rhizobia were present at levels of 1 - 103 cells/g soil although their effectiveness would need to be evaluated under field conditions. In other soils the indigenous population of rhizobia was much smaller (Table 5), suggesting that inoculation with rhizobia at these sites may increase nodulation and benefit plant growth. Singleton and Tavares (1986) found that inoculation increased nodulation when the indigenous rhizobial population was less than 1 - 102 cells/g soil. Later, Thies et al. (1991a, 1991b) illustrated the effect of soil rhizobial populations on the successful use of inoculants and reported that most variation in inoculation response was due to the numbers of indigenous rhizobia followed by the availability of soil N. However, our study indicates that good or poor nodulation can occur at sites with similar indigenous rhizobial populations (Tables 1, 2 and 5), e.g. the following sites had similar numbers of rhizobia but good nodulation was observed at Selian and Lambo and poor nodulation at Irente and Ubiri, agreeing with our earlier suggestion that another factor, such as soil P concentration, affected the extent of nodulation. The lack of significant responses to inoculation in individual experiments reported in Table 6 confirms that sufficient rhizobia were present at these sites and that the inoculum strains tested were not substantially better than the indigenous strains. When the response to inoculation was tested across all of the sites combined, there was a significant response in bean yield to inoculation overall due to the slightly but consistently greater yields found with inoculation, but at all the sites where the number of rhizobia was >100 cells g soil-1 this yield response was around 10% or less.
TABLE 6. Summary of results from field experiments carried out to evaluate response of Phaseolus beans to innoculation with Rhizobium.
Statistical analysis shows the combined yield increase was significant (P< 0.01 )
At some sites, it was obvious that soil moisture content was very low and the lack of water at an early stage of plant growth involving root development and nodule initiation may well have caused the poor plant vigour and poor nodulation of root systems. The other environmental factor not considered in the present study was variation in soil temperature although in these highland areas where beans are grown, soil temperatures are unlikely to be particularly high. High temperatures (> 30°C) are known to reduce nodulation (Karanja and Wood, 1988) but are unlikely to occur under the climates prevailing in these regions except in the surface 5 cm of soil.
We conclude that poor nodulation of Phaseolus root systems observed in the bean growing areas of Tanzania was not due to soil acidity. The soil chemical analyses and plant observations suggest that the extent of root nodulation and plant vigour were closely related to variation in extractable soil P concentration. The analyses further indicated that small soil P concentrations, which were simply due to the lack of P in the soil rather than strong fixation of P into unavailable forms, were important in controlling root nodulation. There was a reasonably large indigenous population of Rhizobium at many sites but nodulation was still poor. The presence of unrealistically high levels of nutrients, particularly the concentration of P, in soils managed by research stations was marked. This can be expected due to the regular application of fertilizers, nevertheless it does emphasise the need for on-farm trials where levels of soil nutrients are realistic. An accompanying paper (Giller et al., 1998) reports on-farm experiments carried out in northern Tanzania, particularly in the Lushoto area, to examine root nodulation, nitrogen fixation and seed yield in response to N and P application and Rhizobium inoculation.
This work was funded by the UK Overseas Development Administration. We are grateful to the Tanzanian Agricultural Research Organisation and to the SADCC/CIAT Regional Bean Programme for assistance during the field work. We thank Frances Harris for some of the Rhizobium population estimates during her MSc study in our group and Barry Smithson for helpful comments on the manuscript.
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