Field Crops Research, 8 (1984) 371--379 371 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
RHIZOSPHERE BACTERIAL COUNTS FOR INTERCROPPED MAIZE (ZEA MA YS L.), COWPEA (VIGNA UNGUICULA TA L.) AND 'EGUSI' MELON (COLOSYNTHIS VULGARIS L.)
T.A.T. WAHUA
Agronomy Department, University of lbadan, Ibadan (Nigeria)
(Accepted 9 January 1984)
ABSTRACT
Wahua, T.A.T., 1984. Rhizosphere bacterial counts for intercropped maize (Zea mays L.), cowpea (Vigna unguiculata L.) and 'egusi' melon (Colosynthis vulgaris L.). Field Crops Res., 8: 371--379.
The numbers of rhizosphere bacteria in sole-cropped and intercropped maize, cowpeas and melon were determined in potted plants, and in field grown plants with standard spacings and arrangements. Intercropping maize, cowpeas and melon with one another increased the rhizosphere bacterial counts for all but melon. Uptake of N and K, but not of P, by cowpeas was highly correlated with rhizosphere bacterial counts. Only the N uptake by melon correlated with rhizosphere activity and nutrient uptake. More rhizosphere bacteria were found in intra-row than in inter-row intercropping.
INTRODUCTION
Cowpea and 'egusi' melon are the most common herbaceous species intercropped with maize by traditional farmers in Nigeria. Apart from its general acceptability in the diet of most Africans, cowpea nodulates and fixes nitrogen naturally in most tropical soils, thereby greatly contributing to the improvement of soil fertility in many cropping systems. Melon, on the other hand, does not fix nitrogen but protects the softs from high solar radiation and destructive impacts of torrential rains, thus moderating soil temperature and reducing erosion. In addition, melon is being increasingly recommended for biological weed control in crop mixtures since it scarcely reduces the yields of other intercrops (Akobundu, 1981).
However, the effects of intercropping cowpeas and melon with maize or with other tropical crops on rhizosphere activities are not known. Wahua and Miller (1978) observed that tall sorghum (Sorghum bicolor) reduced the N2-fixation of intercropped soyabeans (Glycine max) while a particular dwarf sorghum variety increased it by 264%. It was suspected that allelo- chemical effects were involved in this increase, which affected both number of nodules and specific nodule activity.
Many tropical grasses and legumes are known to exude substantial amounts of organic compounds from their roots. These organic materials in the rhizosphere support large populations of micro-organisms (Patriquin and Dobereiner, 1977; Dobereiner, 1978, Sanni, 1978; Solomon, 1981). The activities of rhizosphere micro-organisms can influence nutrient uptake of associated plants (Bowen and Rovira, 1973). It is also known that dif- ferent crop species exude different types and quantities of organic materials which attract and sustain different micro-organisms. It is therefore extremely important to identify crop combinations that would increase rhizosphere activities of associated species in a mixed cropping system.
Keswani et al. (1977) reported some increase in rhizosphere bacteria of intercropped maize and soyabeans over those of sole-cropped plants. They attributed observed increases in grain yields of intercropped maize and soyabeans to increased bacterial population. However, they did not attempt to correlate the bacterial counts with either dry matter yield or nutrient uptake.
The experiment reported here was designed to: (a) investigate the effects of intercropping on the rhizosphere bacterial populations of maize, cowpeas and melon intercropped in potted softs and in the field; and (b) determine the degree of association between nutrient uptake and number of rhizosphere bacteria.
MATERIALS AND METHODS
Two experiments were conducted between March and July 1982; a potted plant experiment and a field experiment on the Teaching and Research Farm of the Agronomy Department of the University of Ibadan, Nigeria.
Pot experiment
Soil was collected from unploughed portions of a tract of land used for the field experiment. The tract was cropped to fertilized early-season maize in 1979, late-season cowpeas in 1979 and cassava (Manihot esculenta) in 1980, before it was left under natural fallow in 1981. The aquic ferrudalf soil contained 1.68% organic matter, 0.11% total N, 8.9 ppm available P (Bray's P1), 98 ppm exchangeable K, 174 ppm exchangeable Ca and had a pH of 5.9 (soft : H20, 1 : 2.5).
Forty-four 10-1 buckets, which had perforated bottoms plugged with cotton wool, were each filled with 11.5 kg of sieved soil (mesh 2 ram). The buckets were arranged, 1 m apart in four 2-m rows on metal benches in a fenced open space between two greenhouses. The soil was watered to drip- ping point and left to equilibrate overnight. The treatments, replicated eight times and randomized completely among the buckets were sole- cropped maize (vat. TZPB), sole-cropped melon (vat. Western Local), sole- cropped cowpea (vat. Ife-Brown), maize intercropped with cowpea and
373
maize intercropped with melon. Four seeds of each species were planted 2 cm deep and 6 cm apart in sole-crop buckets while two seeds of each species were planted in buckets with intercrops. Four buckets were left wi thout plants to supply rhizosphere-free soft. Initially all buckets were given 200 ml water every other day till 7 days after germination when some seedlings were carefully uprooted, leaving one of each species per bucket. This early populat ion control was necessary to ensure that only one plant of each species released exudates into the soil for the bacteria. In a later stage, watering was increased to 400 ml per bucket since evapotranspiration increased with plant age. All buckets were kept weed-free by hand-picking.
Plant sampling
Six weeks after planting, two control buckets and four buckets of each cropping treatment were randomly selected for rhizosphere activity deter- minations. Each bucket was turned over horizontally and gently shaken to loosen the soil while the plant was carefully pulled out with all roots. Any superfluous soil around the roots was shaken off and the entire plant taken to the laboratory. All hand contact with plants was above the crown region.
Laboratory operations
A quant i ty of young root material, with at tached rhizosphere soil, was cut off with sterile scissors and put into a 250-ml Kilner jar containing 95 ml sterilized distilled water and twenty-four glass beads. The shoot was then severed, weighed and dried for nutrient analysis. Each Kilner jar was tightly capped and shaken with a wrist-action shaker for 15 rain. Imme- diately after removal from the shaker, a 1-ml aliquot was transferred from the control of the rhizosphere-soil suspension into a 9-ml sterile water blank. Dilutions ranging from 10 s to 108 were obtained, using the conventional serial dilution technique. A second 1-ml aliquot of rhizosphere-soil sus- pension was placed in a tared pre-weighed aluminium dish, oven-dried at
TABLE I
Composition of agar (added to 250 ml soil extract, with pH adjusted to 7.4)
Constituents Amount (g) dissolved in 750 ml distilled water
60°C for 24 h and then reweighed. This was to obtain the dry weight of soft per ml of suspension, since colony counts are normally expressed on a per unit dry-soft weight basis.
Ten grams of rhizosphere-free soil were transferred into 90-ml sterile blanks, shaken and also diluted as stated above. From the three highest dilutions prepared, a 1-ml aliquot of freshly agitated suspension was trans- ferred to each of five sterile petri dishes into which 12 ml of soil-extract agar {Table I) prepared according to Odu and Adeoye (1970) and cooled to 42°C, was poured.
The dishes were left to solidify and then inverted for a 7-day incubation at 28°C. Three plates per sample of dilution 108 were counted, using a Quebec colony counter. At 8 weeks, another set of samples was taken and the procedures repeated.
All shoots were dried and milled to pass through a 2-mm sieve. Nitrogen was determined by the conventional micro-Kjeldahl method; P colorimetri- cally with Spectronic-20 at 400 nm after vanadomolybdate complexing; and K by flame photometer following conventional wet digestion process.
Field experiment
The tract of land described for the pot experiment was ploughed and sprayed with paraquat after one week to kill all weeds 2 days before planting on 5 May 1982. Since the pot experiment indicated that intercropping could affect bacterial populations in crop rhizospheres, it was suspected that the degree of intimacy of intercropped species determined by stand geometry, in the field, might also make a difference. The following cropping systems were tested: (a) sole maize; (b) sole cowpea; (c) sole melon; (d) maize intercropped with cowpeas between the row; (e) maize intercropped with cowpeas within the row; (f) maize intercropped with melon between the row; (g) maize intercropped with melon within the row; and (h) cowpeas intercropped with melon between the row. The cropping systems were randomized completely within each of three blocks with a space of 3 m between blocks.
Each plot was made up of five 6-m rows spaced 0.9 m apart. Each crop species, irrespective of cropping system or pattern, was planted on the same day with a spacing of 0.9 X 0.5 m. Seven days after planting, seedlings were thinned to one per hill to give a population density of 22 222 plants ha -1 for each species. Total clean weeding was done by hoeing every 2 weeks. The 3-m space between blocks was to be sampled for rhizosphere- free soft, and was therefore weeded weekly.
Seven weeks after planting, when melon had set fruit, cowpeas attained more than 50% flowering and maize was just beginning to tassel, root samples were collected. To do this a soil volume of 30 X 30 × 30 cm 3 was loosened with a shovel around each of three plants randomly selected in each plot. The plant was carefully uprooted and the shoot severed about 30 cm above
375
the crown so that as much soil as possible was shaken off without root-- finger contact.
Non-rhizosphere soil was collected from several spots along the 3-m space between blocks. All samples were transported in polythene bags to the laboratory and bacterial counts performed as described earlier.
Statistical analysis
Data from the two sample sets of the pot experiment were analysed separately. Analysis of variance, according to conventional completely randomized design, was used to test treatment effects. For the field experi- ment, a randomized complete block design was used. In each of the experi- ments, cropping system effects were compared for each crop species sepa- rately, using Duncan's Multiple Range Test (DMRT). The R : S ratio, the ratio of the number of rhizosphere bacteria (R) to the number of bacteria in non-rhizosphere soil (S), was calculated. Linear correlation coefficients between bacterial counts and N, P and K uptake by potted plants were also obtained.
RESULTS AND DISCUSSION
Effects of cropping systems on bacterial counts in the rhizospheres of intercropped maize, melon and cowpeas grown in potted soil and in the field are shown in Tables II and III, respectively. Intercropping maize and cow- peas in buckets did not significantly increase maize rhizosphere bacterial counts. With maize and melon, there seemed to be an increase in the number
TABLE II
Effects of cropping system on the number of rhizosphere bacteria in potted plants (× 10 a g-i soil)
Cropping system 6 weeks 8 weeks
Number R:S a Number R:S ratio ratio
Maize sole-cropped 191 b* 2.6 139 b 2.2 Maize intercropped with cowpea 203 ab 2.7 145 a 2.3 Maize intercropped with melon 230 a 3.1 149 a 2.4
Cowpea sole-cropped 253 a 3.4 179 a 2.8 Cowpea intercropped with maize 163 b 2.2 143 b 2.3
Melon sole-cropped 126 b 1.7 124 b 2.0 Melon intercropped with maize 153 a 2.1 139 a 2.2
*Only figures followed by different letters in the same column for each crop differed sig- nificantly; P ~ 0.05 (DMRT) aR, number of rhizosphere bacteria; S, number of bacteria in non-rhizosphere soil.
376
TABLE HI
Effects of cropping system on the number of rhizusphere bacteria in field-grown plants at 7 weeks (x l 0 s g-i soil)
Cropping system Number R : S a ratio
Maize sole-cropped 45.7 c* 1.3 Maize intercropped with cowpea inter-row 68.4 b 1.9 Maize intercropped with cowpea intra-row 82.5 a 2.3 Maize intercropped with melon inter-row 60.4 bc 1.7 Maize intercropped with melon intra-row 53.0 bc 1.5
Cowpea sole-cropped 46.1 b 1.3 Cowpea intercropped with maize inter-row 61.3 ab 1.7 Cowpea intercropped with maize intra-row 69.4 a 2.0 Cowpea intercropped with melon inter-row 72.9 a 2.1
Melon sole-cropped 67.7 ab 1.9 Melon intercropped with maize inter-row 74.0 a 2.1 Melon intercropped with maize intra-row 60.9 b 1.7 Melon intercropped with cowpea inter-row 66.2 ab 1.9
*Only means followed by different letters in the column for each crop differed signifi- cantly; P ~< 0.05 (DMRT). aR, number of rhizosphere bacteria; S, number of bacteria in non-rhizosphere soil.
o f rh izosphere bac te r ia fo r b o t h species. I n t e r c r o p p i n g cowpeas wi th maize in b u c k e t s decreased the n u m b e r o f bac te r ia in the c o w p e a rh izosphere . Shan ta ran and Rangaswani (1967) also r e p o r t e d t ha t i n t e rc ropp ing r educed the rh izosphere ac t iv i ty of s u n h e m p (Crotolaria ]uncea) i n t e r c ropped wi th so r ghum in a conc re t e p o t inside a g reenhouse , b u t increased t ha t o f sor- ghum. I t is poss ible t h a t the close i n t i m acy b e t w e e n maize and c o w p e a roo ts , res t r ic ted wi th in 10 1 o f soil, migh t lead to r educed nu t r i en t u p t a k e b y cowpeas , the weake r c o m p e t i t o r , and t h a t could a f fec t the a m o u n t o f me tabo l i t e s sent to rh izosphere bacter ia . Why this occurs in cowpeas and n o t in m e l o n is n o t clear. However , me lon m a t u r e s m u c h earlier t han cowpeas and m a y no t c o m p e t e fo r resources a t the t ime tha t maize makes a great d e m a n d on the soil.
The results o f the field e x p e r i m e n t did n o t d i f fe r m u c h f r o m those of the p o t e x p e r i m e n t . Rh izosphe re activit ies o f i n t e r c r o p p e d maize and cowpeas were s ignif icant ly grea ter in m i x t u r e s t han unde r sole-crop condi t ions . The bacter ia l coun t s were higher fo r in t ra - row than fo r in te r - row inter- c ropp ing pa t te rns . This indicates t ha t when soil v o l u m e and space are n o t l imi ted, closer in t e rac t ion o f ma ize and c o w p e a roo t s enhances rh izosphere act ivi ty .
Fo r m e l o n i n t e r c r o p p e d wi th ma ize or cowpeas , rh izosphere ac t iv i ty did no t d i f fe r s ignif icant ly f r o m sole-crop results. Howeve r , p lan t ing m e l o n b e t w e e n c o w p e a rows increased c o w p e a rh izosphere act ivi ty . I t is poss ible
377
that melon roots released substances that enhanced the proliferation of bacteria associated with cowpea roots while cowpea roots could not do the same to melon roots. Another possibility is that, since melon was at a more advanced developmental stage than cowpeas at sampling time, and rhizosphere activity decreases with plant age (Dommergues et al., 1973); the contribution of the cowpeas might not have benefit ted melon signifi- cantly.
TABLE IV
Linear correlation coefficients between rhizosphere bacterial counts and nutrient uptake in plants grown in the pot experiment
Crops No. Correlation coefficients of plants N P K Shoot dry weight
*Significant at P~ 0.05. **Significant at P~ 0.01.
The correlation coefficients shown in Table IV reveal that the number of bacteria in the maize rhizosphere was not correlated with nutrient uptake or shoot dry weight. This tends to support the opinion of Mishunstin and Shilnikova (1969) that non-symbiotic N2-fixation in the grass rhizosphere might not be significant enough to be of economic importance despite the findings of DaSilva and Dobereiner (1977). They observed that the maize endorhizosphere contained AzospiriUum species which could fix substantial amounts of nitrogen. If such bacteria were present under the conditions of this experiment, the nitrogen fixed by them could not have formed a significant proportion of the total amount taken up by maize. In melon, rhizosphere activity was significantly correlated with nitrogen uptake, but not with shoot dry weight or P and K uptake. This is suprising, since it suggests that melon rhizosphere might harbour non-symbiotic nitrogen fixers. Further work is needed to characterise the species of bacteria asso- ciated with melon roots. This is necessary because the correlation involved both sole-cropped and intercropped melon. The N2-fixers could be those associated with interplanted cowpeas. However, it is important to note that melon thrives in almost all ecological zones where crops are grown in Nigeria, irrespective of nitrogen fertilization.
Rhizosphere activity of cowpeas was significantly correlated with N and K uptake as well as with shoot dry weight, but not with P uptake. This is not suprising for a legume that nodulates very easily in most tropical soils. All that the result indicated was that Rhizobia bacteria dominated the cow- pea rhizosphere and that they fixed significant amounts of nitrogen. If the
378
vegetative development of cowpeas was affected by nitrogen nutrition and there was sufficient exchangeable K in the soil, it would be natural for K uptake to be affected. Phosphorus uptake might not be affected in the same way since the soil was low in P, and P fixation is a common phenomenon in tropical soils.
Generally, the R : S ratios indicated that the rhizosphere softs contained more than double the number of bacteria in non-rhizosphere soils. With potted plants watered regularly, the ratio went as high as 3 for softs with cowpeas. Solomon (1981) who used many potted tropical grasses, including maize, obtained ratios between 1.3 and 17.3. It seems that in a controlled environment, with regular watering, weeding and pest control, this ratio could be high. The pot experiment also revealed that bacterial counts taken 6 weeks after planting were about 20% higher than those taken 2 weeks later. This could be due to changes in photosynthates sent to the roots as plants aged.
CONCLUSION
This study has shown that: (1) In the field, rhizosphere bacterial counts for maize and cowpeas were
greater when these species were intercropped with each other or with melon than in sole-crop systems. Intercropping did not increase the rhizosphere bacterial count for melon.
(2) Plants intercropped in alternate stands within the row had greater bacterial counts than those intercropped between the row.
(3) With potted plants, bacterial counts were higher than they were under field conditions, but the effects of intercropping did not quite reflect what happened in the field with normal stand geometry and plant popula- tions. For practical applications, it may not be prudent to depend on green- house findings only.
(4) Rhizosphere activity of cowpeas was highly correlated with N and K uptake and with shoot dry weight, but not with P uptake.
(5) For melon, only N uptake correlated significantly with rhizosphere activity.
The significant correlation between N uptake by melon and rhizosphere bacterial number deserves further attention, especially as melon thrives in most farm lands in West Africa. Does the rhizosphere of melon roots contain micro-organisms that fix nitrogen? Does melon have any other means of obtaining nitrogen which may encourage proliferation of bacteria in the rhizosphere? These and other questions could be answered in a separate experiment, using probably more samples, involving counting and charac- terization of bacteria and fungal species as well as testing the bacterial isolates for nitrogenase activity.
379
ACKNOWLEDGEMENTS
I am grateful to the University of Ibadan for funding this research through Senate Research Grants. My sincere thanks go to Prof. C.T.I. Odu for allowing the facilities in his soil microbiology laboratory to be used for this study. Finally, I express my indebtedness to Messrs. I. Amugo, John Ekpe and Ijeoma for their help in all laboratory procedures involved.
REFERENCES
Akobundu, O.T., 1981. Weed control in intercropping mixtures. In: S.J. Pandey (Editor), Agronomy Training Manual for Agroservice Agronomist. IITA--National Accelerated Food Production Project, Lagos, Nigeria, pp. 217--227.
Bowen, G.D. and Rovira, A.O., 1973. Are modelling approaches useful in rhizosphere biology? In: J. Rosswell (Editor), Modem Methods in the Study of Microbial Ecology. Swedish National Science Research Council, Stockholm. pp. 443--450.
DaSilva, M.F.S. and Dobereiner, J., 1977. Occurrence of Azospirillum spp. in soil and roots. In: First International Symposium on Limitations and Potentials of Biological Nitrogen Fixation in the Tropics, 14--18 September 1976, Brasflia, Brazil, pp. 64--71.
Dobereiner, J., 1978. Nitrogen fixation in grass bacteria associations in the tropics. In: Isotopes in Biological Dinitrogen Fixation. Proc. Advisory Group Meeting, 21--25 No- vember 1977, Vienna, Organized by FAO and International Atomic Energy Agency, pp. 51--69.
Dommergues, Y., Balandreau, J., Rinauda, G. and Weinhard, P., 1973. Non-symbiotic nitrogen fixation in the rhizosphere of rice, maize and different tropical grasses. Soil Biol. Biochem., 5: 83--89.
Keswani, L.C., Kibani, T.M. and Chowdhurry, M.S., 1977. Effect of intercropping on rhizohium population in maize and soyabean. Agric. Environ., 3: 363--368.
Mishunstin, E.F. and Shilnikova, V.K., 1969. The biological fixation of atmospheric nitrogen by free-living bacteria. In: Soil Biology. UNESCO, Paris, pp. 65--124.
Odu, C.T.I. and Adeoye, K.B., 1970. Heterotrophic nitrification in soils: a preliminary investigation. Soil Biol. Biochem., 2:41--45.
Pathriquin, D.G. and Dobereiner, J., 1977. Bacteria in the Endorhizosphere of Maize in Brazil. In: First International Symposium on Limitations and Potentials of Biologi- cal Nitrogen Fixation in Tropics, 14--18 September 1976, Brasllia, Brazil, pp. 24--36.
Sanni, S.O., 1978. Preliminary screening of maize for Spirillum--maize symbiotic nitrogen fixation. Niger. J. Microbiol., 1 : 59--65.
Shantaran, M.V. and Rangaswani, G., 1967. A comparison of the rhizosphere microflora of mixed crop. Mysore J. Agric. Sci., 1: 7--12.
Solomon, M.G., 1981. Bacterial associations and nitrogen fixation in the rhizosphere of some tropical grasses. Ph.D. Thesis submitted to Agronomy Department, University of Ibadan, Nigeria, 34 pp.
Wahua, T.A.T. and Miller, D.A., 1978. Effects of intercropping on soyabean N2-fixation and plant composition of associated sorghum and soyabeans. Agron. J., 70: 292--295.
