Effect of Rhizobium inoculation on the biomass production of selected green manure crops

Effect of Rhizobium inoculation on the biomass production of selected green manure crops

Ian Grange¹, Kriesing Choodee² & Amnat Bamroongkit²

1Faculty of Environment and Natural Resource Studies, Mahidol University, Salaya, Nakhon Prathom 73170. email: fridg@mahidol.ac.th

^{2Kanchanaburi Horticultural Experiment Station, Amphur Muang, Kanchanaburi 71000.

ABSTRACT
Leguminous green manure crops improve the quality of soil in many ways with these improvements often being reflected in subsequent increases in crop production. The ability of legumes to improve soil is enhanced by effective Rhizobium inoculation. The present trial found that the biomass production of pigeon pea and cowpea increased significantly when inoculated with Rhizobium, whereas velvet bean was found to be non-specific in its Rhizobium requirement and inoculation did not significantly affect biomass production.

INTRODUCTION
The role of nitrogen fixing legumes, particularly in tropical regions, is becoming increasingly important both in terms of providing essential dietary protein as well as improving the quality of the soil. It is the latter role that the present paper focuses upon and the potential benefits these can infer on field crop production systems. Leguminous green manure crops: fix nitrogen; increase soil organic matter; improve soil physical properties; increase the availability of other nutrients; reduce the risk of erosion, and; curb the incidence of pest and disease by disrupting their life cycles. All these functions are influenced by how effectively the legume has been inoculated by an appropriate Rhizobium inoculum.

Legumes often fail to nodulate when introduced to an area with no previous cropping history of that particular plant. This is due to the soil’s indigenous Rhizobium being either ineffective or insufficient in number to provide a good inoculum. Australia for example has very few, if any, native populations of Rhizobium in many of its soils (Killham, 1994). Nodules that have been effectively inoculated and are actively fixing nitrogen are pink on the inside. This pink coloration is due to leghaemoglobin and if absent, usually means nitrogen fixation is not taking place (Wild, 1988).
The main objectives of this trial were to determine how effective the indigenous Rhizobium of the clay soils of an upland cropping area in the west of Thailand were at inoculating selected legumes, as compared to introduced cultured Rhizobium, and to determine the impact of this on biomass production.

MATERIALS AND METHODS
The trial was conducted at Kanchanaburi Horticultural Experiment Station in western Thailand (13^o 58’N 99^o 27’E, 58 m above sea level). The soil belonged to the Thap Kwahng series representing 5726 ha of Kanchanaburi Province (SSCD, 1994) and is considered to be generally fertile.
Three Legume crops were selected on the basis of their availability and potential as green manure crops. These were pigeon pea (Cajanus cajan), cowpea KVC7 (Vigna sinensis) and velvet bean (Mucana spp.). For each legume crop grown in the plots, half were grown with Rhizobium inoculant, and half were grown without. Rhizobium inoculation was carried by applying a spray suspension to the seedbed. The Rhizobia used were all in a peat coating and were specific to the legumes grown, except for that of velvet bean (Rhizobium specific to Canavalia gladiata was used). The batch codes for all Rhizobium were 1509 18-6-98 (Soil Microbiology Department, DOA, Bangkok). All other treatments were the same. Seeds were sown at the end of June in the trough of the bimodal rainy season, with some irrigation initially to enable establishment.
A completely randomized experimental design was used with 3 replicates and
6 treatments. Individual plots were 2 x 3m. All seeds were sown at spacings
of 40 cm with 2 – 3 seeds per hill for pigeon pea and cowpea, and a single seed
per hill for velvet bean. All variates were analysed by one way analysis of
variance using the MSTAT-C statistical package. The biomass production was evaluated
by measuring plant heights at 20 and 60 days after sowing (DAS). At 100 DAS,
randomly sampled plants from each of the plots were carefully excavated and
the roots washed with warm water to remove soil. The root nodules were counted
and examined for effective inoculation.

RESULTS AND DISCUSSION
Pigeon pea
Sixty day old plants, inoculated with Rhizobium, were significantly taller than those plants not inoculated (Figure 1). There was no significant height difference between the treatments at 20 DAS. Pigeon pea are often described as non-specific in their Rhizobium requirements and can be effectively inoculated from the indigenous soil populations (Sukarin, 1997, Chauhan, 1993 and Skarman et al., 1988). The results in this trial however, demonstrate the benefits of inoculation for these particular soils. This increase in biomass production of the inoculated plants improves their potential as a green manure crop.

There are significantly more nodules on the inoculated plants than on non-inoculated plants (Table 1). Kumar Rao and Dart (1987) reported that nodulation continued to increase from 20 DAS until 60 – 80 DAS, followed by a decline. It was observed in the pigeon pea plants of this trial, that at 100 DAS more of the nodules on the treated plants were still actively fixing nitrogen compared with the untreated plants, where many of the nodules were already necrotic. This indicated an increased longevity of the nodules of inoculated plants with clear benefits for increased nitrogen fixing potential and production. The wide range of nodule numbers for the inoculated plants (Table 1) suggested that not all the plants had been effectively inoculated. This could perhaps have been improved upon by inoculating the seed directly before sowing.
Cowpea
Cowpea plants followed similar trends to those of pigeon pea (Figure 1). Plants inoculated with Rhizobium were significantly taller at 60 DAS, though not at 20 DAS, when compared with untreated plants. These differences occurred despite an aphid infestation which may have caused some stunted growth.
Skarman et al., (1988) recommends inoculation with a selected cowpea strain of inoculum as advantageous despite cowpea being non-specific. The present trial demonstrates this, with inoculated plants having significantly more root nodules than non-inoculated plants, many of which had no root nodules at all. The inoculated plants also had more secondary root laterals than the untreated plants. However, as with pigeon pea, the wide range of nodule numbers for the inoculated plants (Table 1) suggested that not all the plants had been effectively inoculated.
Velvet bean
Since there was no inoculum specific to velvet bean available, the Rhizobium specific to sword bean (Canavalia gladiata) was tested as a substitute. Due to the prostrate growth habit of velvet bean when lacking support, it was difficult to assess plant heights accurately at 60 DAS, and no significant differences were found between treated and untreated plants.

There was no significant difference
in the number of root nodules between treated and untreated plants suggesting
that the sword bean inoculum had no effect on velvet bean nodulation. There
were however, many nodules, though after 100 DAS with the onset of flowering,
many of these nodules were already necrotic. The few nodules that were still
active, were pink in colour for both treatments which emphasizes the non-specificity
of velvet bean and its ability to fix nitrogen in these soils without Rhizobium
inoculum.

CONCLUSION
The results suggest that indigenous soil populations of Rhizobium, specific to the pigeon pea and cowpea, are either not present or present, but in insufficient numbers to effectively inoculate the plants. If these Rhizobium are introduced to these particular soils as an inoculum, then growth of the particular legume increases significantly. This was particularly the case for pigeon pea with clear increases in biomass production. Velvet bean was found to be non-specific in its Rhizobium requirement and inoculation was not required for these soils.

REFERENCES
Chauhan, Y.S. (1993). Pigeon Pea. In; Rooting Patterns of Tropical Crops, M.Abdul Salam and P. Abdul Wahid (eds.). Tata McGraw-Hill Publishing Company Limited, New Delhi.

Killham, K. 1994. Soil Ecology. Cambridge University Press.
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Skarman P.J. Cameron, D.G. and Riveros, F. (1988). Tropical Forage Legumes (2^nd Edition). FAO Plant Production and Protection Series No 2.
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