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Interactions between Meloidogyne javanica (Treub) chitwood and rhizobia on growth of Sesbania sesban (L.) Merr.
Applied Soil Ecology 29 (2005) 252–258 www.elsevier.com/locate/apsoil
Interactions between Meloidogyne javanica (Treub) chitwood and rhizobia on growth of Sesbania sesban (L.) Merr. Johan Desaeger a,*, David Odee b, Joseph Machua b, Milton Esitubi b b
a World Agroforestry Centre (ICRAF), P.O. Box 30677, Nairobi, Kenya Biotechnology Laboratory, Kenya Forestry Research Institute (KEFRI), P.O. Box 20412, 00200 Nairobi, Kenya
Received 26 May 2004; accepted 13 December 2004
Abstract A screenhouse experiment was conducted to evaluate the effects of inoculation with Meloidogyne javanica (uninoculated nematode control, low level [1000 eggs pot 1] and high level [10,000 eggs pot 1]) and rhizobial strains (uninoculated rhizobial control, strains KFR 647 and MBS1312) on nematode infection, nodulation and growth of Sesbania sesban (L.) Merr. (sesbania) provenances (Kisii and Kakamega). Heavy nematode infection of sesbania was noted for all three rhizobial inoculation treatments 2 months after sowing, and nodulation and plant growth were affected negatively at the high nematode inoculum level. Shoot dry weights showed negative and positive relationships with nematode infection (gall index) and number of effective rhizobium nodules, respectively. At 3 months, negative effects of the high nematode inoculum level on nodulation and plant growth were observed for rhizobial strain KFR 647 and the uninoculated control, but not for strain MBS 1312. Low nematode inoculum levels did not negatively affect nodulation or plant growth. The two sesbania provenances had similar growth at the high nematode inoculum level, but the Kisii provenance showed better growth than the Kakamega provenance at the low nematode inoculum level. # 2005 Elsevier B.V. All rights reserved. Keywords: Improved fallow; Nodulation; Root-knot nematode; Sesbania provenance
1. Introduction Sesbania sesban (L.) Merr. (sesbania), a fast growing, N2-fixing and deep-rooting tree with good * Corresponding author. Present address: University of Georgia, Department of Plant Pathology, Coastal Plain Experiment Station, Tifton, GA 31794, USA. Tel.: +1 229 386 3160; fax: +1 229 386 7285. E-mail address: [email protected] (J. Desaeger).
quality foliar biomass, is very well suited for replenishing soil fertility and fodder production for small ruminant animals in sub-Saharan Africa. Sesbania has traditionally been used for fodder and firewood in Kenya and Tanzania (Onim et al., 1990). Considerable research has been conducted in the last 15 years on sesbania for improving soil fertility and fodder production in Zambia, Tanzania, Malawi and Kenya (Onim et al., 1990; Kwesiga and Ngugi, 1996; Niang et al., 1996).
0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2004.12.005
J. Desaeger et al. / Applied Soil Ecology 29 (2005) 252–258
A potential constraint for sustainability of sesbania fallows is the fact that the tree is a good host of rootknot nematodes (Meloidogyne incognita and Meloidogyne javanica) (Karachi, 1995), supporting nematode population increase, particularly in light-textured soils (Desaeger and Rao, 2000a,b). Negative effects of root-knot nematode have been observed with several Rhizobium-legume symbioses (Hussey and McGuire, 1987; Taha, 1993). Previous field experiments in western Kenya have shown similar negative relations with sesbania, and nematodes were found to invade sesbania root nodules (Desaeger and Rao, 2000a,b). The potential for soil N replenishment through biological N2-fixation by sesbania could therefore be greatly reduced by rootknot nematodes. A large pool of effective rhizobial strains is currently available for sesbania but their interactions with root-knot nematodes are unknown. Inhibition of nodulation on Acacia seyal by M. javanica was noted for only some rhizobial strains (Duponnois and Cadet, 1994). Duponnois et al. (1999) also noted that some bacterial strains decreased mortality of Meloidogyne-infected Acacia holosericea seedlings. Previous pot experiments with sesbania and rootknot nematodes in Kenya have shown considerable differences between sesbania provenances in their nodulation and tolerance to the nematodes (Desaeger and Rao, 2001). The main objectives of this study were (1) to evaluate the effect of inoculum levels of root-knot nematode on rhizobial nodulation and growth of sesbania, and (2) to assess the interactions of different rhizobia and sesbania provenances with nematode infections.
2. Materials and methods 2.1. Seed source and pre-treatment Seeds of sesbania provenances were obtained from the Regional Agroforestry Centre (KEFRI), Maseno, Kenya. Uniformly sized seeds were pre-treated by immersing in hot boiling water for 2 min to soften the seed coat. They were surface sterilized in 3% NaOCl for 3 min, washed in three changes of sterile distilled water, and then soaked overnight.
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2.2. Preparation of potting substrate and sowing of pre-germinated seeds Moist soil (sand = 83%, silt 11%, clay = 6%) that had been collected locally was heated in an oil drum for 4 h to about 90 8C and afterwards air-dried for 4 weeks before commencing the study to avoid any possible side effects of heating (Anderson and Ingram, 1989). Plastic pots (2.5 l) were filled with sterilised soil and allowed to settle for 1 month before starting the trial. Triple superphosphate was added (3 g pot 1, equivalent to 60 kg P ha 1) and mixed into the soil before sowing. Five pre-germinated sesbania seeds were sown in each pot. 2.3. Rhizobial inoculation Two effective sesbania rhizobial strains were used: (i) KFR 647 originating from Yala swamp, western Kenya, and (ii) MBS 1312 originating from Marigat, Rift Valley, Kenya. Rhizobial cultures were grown to late log phase in yeast mannitol broth (YMB; Vincent, 1970). Seedlings were inoculated 3 days after sowing by watering each with 1 ml suspension of the culture broth containing 1 109 rhizobia. 2.4. Nematode source and inoculation A single egg mass of M. javanica was collected from sesbania on a farm in western Kenya and the nematode was multiplied on sesbania plants in the greenhouse. Nematode eggs were extracted from the rhizosphere of infected sesbania roots and immediately used for inoculation. The pots were inoculated with nematodes through five small holes made around the seeds 3 days after rhizobial inoculation. The sodium hypochlorite method was used to extract nematode eggs (Hussey and Barker, 1973). 2.5. Growth conditions, experimental design and assessments Pots were placed on four raised wooden benches in a screenhouse at the Regional Agroforestry Research Centre in Maseno, western Kenya. The approximate mean temperature was 35/16 8C (day/night) with a 12h photoperiod (natural daylight). Pots were watered every other day to field capacity.
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The experimental design was a 3 3 2 factorial, with three M. javanica inoculum levels (uninoculated nematode control, low [1000 eggs pot 1] and high [10,000 eggs pot 1]), three rhizobial inoculation treatments (uninoculated control and inoculation with strains KFR 647 and MBS1312), and two sesbania provenances (Kakamega and Kisii). Treatments were replicated four times in a randomized complete block design. Seedlings were thinned to two per pot 2 weeks after sowing, and those removed were stained for nematode infection using acid fuchsin (0.03%) in lactoglycerol (Bridge et al., 1982). The remaining seedlings were removed at 2 and 3 months, respectively, after sowing. At each sampling, seedlings were carefully uprooted and soil washed off with gentle running water. Roots were rated for root galling on a 0–10 scale (Zeck, 1971) [0 = no galls, 1 = very few small galls, 2 = numerous small galls, 3 = numerous small galls of which some are grown together, 4 = numerous small and some big galls, 5 = 25% of roots severely galled, 6 = 50% of roots severely galled, 7 = 75% of roots
severely galled, 8 = no healthy roots but plant is still green, 9 = roots rotting and plant dying, 10 = dead plant]. In addition, 1 g of roots was stained for nematode infection using the above lactoglycerol method, and another 1 g was stained for egg masses using phloxine B (Holbrook et al., 1983). At 3 months nematodes were also extracted from soil using a modified Baermann method (Southey, 1986). Nodulation and growth of seedlings were assessed at 2 and 3 months after sowing. Nodules were detached from roots and counted. Shoots, roots and nodules were oven-dried at 60 8C for 72 h, and then weighed. In addition, nodule effectiveness for biological N2-fixation was scored on the basis of internal pigmentation (Beck et al., 1993). Nodulation rate plant 1 was calculated as described by Duponnois et al. (1995). 2.6. Data analysis Analysis of variance (ANOVA), standard error of differences of means (S.E.D.) and linear regression
Table 1 Meloidogyne javanica root infection and growth of sesbania 2 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode infection Gall index (0–10)
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a b
0b 1.5 4.1 0.37 <0.01
Juveniles + females (no. of plant 1)
Egg masses (no. of plant 1)
0b 30 113
0b 12 38
10 <0.01
5 0.01
94 104 49 11 0.01
3.2 2.5 2.7
74 58 84
0.28 0.08
17 0.31
2.5 3.1
60 84
17 32
66 99
0.36 0.09
13 0.07
5 <0.01
8 <0.01
Standard error of difference of treatment means. Not included in ANOVA.
28 23 22
Shoot dry weight (mg plant 1)
5 0.43
69 81 98 8 0.01
J. Desaeger et al. / Applied Soil Ecology 29 (2005) 252–258
analysis were performed using a GENSTAT1 statistical package (Version 5).
3. Results 3.1. Nematode infection Nematode gall indices on sesbania roots averaged 0, 1.2 and 4 for the control, low and high nematode inocula, respectively, (P < 0.05, data not shown) 2 weeks after sowing. Similarly, root infections were 0, 5 and 23 juvenile nematodes plant 1. At 2 months after sowing, nematode infection was greater at the high inoculum level, and shoot dry weight was significantly reduced at the high inoculum level compared with uninoculated plants or the low nematode inoculum level (Table 1). Sesbania provenance Kisii had greater nematode infection and shoot dry weight than the Kakamega provenance at 2 months after sowing. There were also significant interactions: nematode inoculum level provenance for nematode egg mass infection (P < 0.01), and nematode inocu-
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lum rhizobial strain provenance for gall index and shoot dry weight (P = 0.04). At 3 months after sowing, nematode inoculum level and sesbania provenance showed similar (but not identical) trends in their effects on nematode infection and shoot dry weight as those at 2 months (cf. Tables 1 and 2). 3.2. Rhizobial symbiosis All plants, including the uninoculated controls, developed nodules indicating that the heated oil drum treatment did not eradicate the indigenous rhizobia. All stages of root-knot nematode (juveniles, adult females, eggs) were observed inside nodules. In spite of the large number of nematodes found inside some of the nodules (more than 30 juveniles nodule 1), the nodules appeared healthy. Two months after sowing, the number of nodules was significantly lower with the high nematode inoculum level (3.5, 4.2 and 0.5 nodules seedling 1 [S.E.D. = 0.8] for the uninoculated nematode control, low and high nematode inoculum levels, respectively). There was significant
Table 2 Meloidogyne javanica root infection and growth of sesbania 3 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a b
Nematode infection Gall index (0–10)
Juveniles + females (no. of plant 1)
Egg masses (no. of plant 1)
0b 4.2 6.0
0b 36 126
0b 19 71
Shoot dry weight (mg plant 1)
411 499 295
0.3 0.01
34 0.08
12 0.03
4.8 4.8 5.6
66 95 82
48 44 43
412 381 413
0.7 0.50
20 0.40
15 0.95
101 0.94
36 54
341 463
4.9 5.3 0.4 0.39
Standard error of difference of treatment means. Not included in ANOVA.
60 102 15 0.02
7 0.02
56 0.03
67 0.08
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interaction of nematode inoculum level rhizobial strain provenance for nodule fresh weight and nodulation rate (P < 0.05). In addition, there was a negative relationship between the number of effective nodules and nematode infection at 2 months ( y = 5.24 1.03x, r = 0.63**, d.f. = 47, where y is the number of effective nodules and x the nematode gall index). Shoot dry weight showed negative and positive relationships with nematode infection (gall index) and the number of effective nodules, respectively, at 2 months (Fig. 1). At 3 months after sowing, high nematode inoculum level significantly suppressed nodulation of plants inoculated with rhizobial strain KFR 647 or those nodulated by indigenous rhizobia (Fig. 2). Plants inoculated with rhizobial strain MBS 1312 showed no difference in nodulation between nematode inoculated and uninoculated control plants. However, although the high nematode inoculum level significantly reduced the nodulation rate of sesbania plants, it did not reduce the root and nodule biomass (Table 3).
Fig. 2. Number of nodules plant 1 on 3-month-old sesbania seedlings as affected by M. javanica inoculum level and rhizobial strain (values are means for the two sesbania provenances). The nematode inoculum levels were: uninoculated control (black bars), low (1000 eggs pot 1, dotted bars) and high (10,000 eggs pot 1, dashed bars).
The number of effective nodules was positively correlated with shoot dry weight and root fresh weight at 3 months after sowing ( y = 3.77 + 18.99x1, r = 0.76** and y = 4.20 + 8.73x2, r = 0.66*, d.f. = 70, where y is the number of effective nodules, and x1 and x2 represent shoot dry weight and root fresh weight in g plant 1, respectively).
4. Discussion
Fig. 1. The relationships between shoot dry weight of 2-month-old S. sesban and root-knot nematode gall index (A) and number of effective nodules (B) (values are means for the two sesbania provenances) (n = 36) (*** significant at P < 0.001 level).
Root-knot nematodes readily invaded and reproduced inside sesbania nodules. The negative impact of high nematode infection on sesbania growth and nodulation confirms previous results from the area (Desaeger and Rao, 2000a). The highest nematode inoculum in our trial represents a realistic population in field conditions, where population build-up of up to 10,000 nematode juveniles per 100 cm3 soil has been recorded following sesbania fallow (Desaeger and Rao, 2001). The decrease in significance with time, and the negative relationship between growth and nematode infection at 2 but not at 3 months, is also in accordance with published reports that nematode damage to sesbania is most crucial during the earliest stages of growth (Desaeger and Rao, 2001). A possible explanation for reduced nodulation at high rates of nematode infection could be the formation of Meloidogyne root galls and the suppression of lateral root formation and root hairs, which reduces the number of sites available for nodulation and causes nutrient deficiencies (Fazal et al., 1992). Nodulation
J. Desaeger et al. / Applied Soil Ecology 29 (2005) 252–258
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Table 3 Root and nodule biomass of sesbania 3 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a
Root biomass (mg plant 1)
Nodule biomass (mg plant 1)
Fresh weight
Dry weight
Fresh weight
Dry weight
Nodulation rate (mg root 1)
756 1015 708
276 300 223
156 234 129
21 33 19
0.14 0.11 0.04
12 0.51
0.03 0.05
16 32 25
0.09 0.09 0.10
107 0.06 776 739 964 211 0.53 688 965 119 0.03
44 0.28 243 282 275 76 0.86 276 257 61 0.75
80 0.45 124 211 183 37 0.08 168 177 32 0.79
8 0.21 24 24 7 0.99
0.03 0.94 0.10 0.09 0.03 0.92
Standard error of difference of treatment means.
could also be affected through nodule invasion by nematodes, as previously observed with sesbania (Desaeger and Rao, 2000b). Although Meloidogyne species can develop in nodules without disrupting internal tissue, such interactions have often resulted in premature senescence of bacteroids and nodules (Taha, 1993). Nodules compensated for few nodules by increasing in size at high nematode infection. There were also some differences in rhizobial inoculation responses caused by nematode inoculum level. In particular, there was better nodulation and growth of plants inoculated with rhizobial strain MBS 1312 than strain KFR 647 or indigenous rhizobia at the high nematode inoculum level. This indicates that there is scope for selection of a nematode tolerant Rhizobium–Sesbania symbiosis. Rhizobial strain MBS 1312 from Marigat, Kenya, is interesting in that it has previously been shown to be more tolerant to antibiotics and salt stress than other rhizobia originating from the region (Odee et al., 1997). As arid soils are known to favour growth of antibioticproducing organisms (Miller and Pepper, 1988) and are more prone to drought and salt stress, it is possible that rhizobia in Marigat may have developed resistance due to evolutionary pressures (Odee et al., 1997). The same
physical or physiological defence mechanism could also be responsible for the greater tolerance of strain MBS 1321 to root-knot nematode. There is some evidence that nematode tolerance should be regarded in the wider sense as a general non-specific defence mechanism (Wallace, 1989). The superior early growth of the Kisii as compared to the Kakamega provenance at low nematode infection confirms previous data (Desaeger and Rao, 1999, 2000b), and suggests that the Kisii provenance is more tolerant at low nematode levels. Its relatively poorer growth at the high nematode inoculum level was related to higher rates of infection. Although nematode infection, nodulation and growth of sesbania showed some differences due to nematode inoculum level, rhizobial strain and sesbania provenance, further studies are required to establish whether these differences can be exploited to improve sesbania growth under field conditions.
Acknowledgements We wish to thank ICRAF, KEFRI and the Flemish Office for Development Cooperation and Technical
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Assistance (VVOB), Belgium for financial assistance and Elphas Okonda and Violet Achieng for technical assistance.
References Anderson, J.M., Ingram, J.S.I., 1989. Tropical Soil Biology and Fertility: A Handbook of Methods. CAB International, Wallingford, UK, 171 pp.. Beck, D.P., Materon, L.A., Afandi, F., 1993. Practical Rhizobium– Legume Technology Manual, Technical Manual No. 19. International Centre for Research in the Dry Areas, Aleppo, Syria, 389 pp. Bridge, J., Page, S., Jordan, S., 1982. An improved method for staining nematodes in roots. Report of Rothamsted Experimental Station for 1981: Part 1, p. 171. Desaeger, J., Rao, M.R., 1999. The root-knot nematode (Meloidogyne spp.) problem in sesbania fallows and scope for managing it in western Kenya. Agrofor. Syst. 47, 273–288. Desaeger, J., Rao, M.R., 2000a. Parasitic nematode populations in natural and improved cover crops and their effects on subsequent crops in Kenya. Field Crop. Res. 65, 41–56. Desaeger, J., Rao, M.R., 2000b. Infection and damage potential of Meloidogyne javanica on Sesbania sesban in different soil types. Nematology 2, 169–178. Desaeger, J., Rao, M.R., 2001. Effect of field establishment methods on Meloidogyne infection and growth of Sesbania sesban in western Kenya. Crop Prot. 20, 31–41. Duponnois, R., Cadet, P., 1994. Interactions of Meloidogyne javanica and Glomus sp. on growth and N2 fixation of Acacia seyal. Afr.–Asian J. Nematol. 4, 228–233. Duponnois, R., Senghor, K., Mateille, T., 1995. Pathogenicity of Meloidogyne javanica (Treub) Chitw. to Acacia holosericea (A. Cunn. Ex G. Don) and A. seyal (Del.). Nematologica 41, 480– 486. Duponnois, R., Neyra, M., Senghor, K., Baˆ , A.M., 1999. Effects of the root-knot nematode Meloidogyne javanica on the symbiotic relationships between different strains of Rhizobia and Acacia holosericea (A. Cunn. Ex G. Don). Eur. J. Soil Biol. 35, 99–105. Fazal, M., Siddique, Z.A., Imran, M., 1992. Effect of pre-, post- and simultaneous inoculations of Rhizobium, Rotylenchulus reniformis and Meloidogyne incognita on lentil. Nematol. Mediterannea 20, 159–161.
Holbrook, C.C., Knauft, D.A., Dickson, D.W., 1983. A technique for screening peanut for resistance to Meloidogyne arenaria. Plant Dis. 67, 957–958. Hussey, R.S., Barker, K.R., 1973. A comparison of methods of collecting inocula for Meloidogyne spp., including a new technique. Plant Dis. Rep. 57, 1025–1028. Hussey, R.S., McGuire, J.M., 1987. Interactions with other organisms. In: Brown, R.H., Kerry, B.R. (Eds.), Principles and Practice of Nematode Control in Crops. Academic Press, Marrickville, NSW, Australia, pp. 294–329. Karachi, M., 1995. Sesbania species as potential hosts to root-knot nematode (Meloidogyne javanica) in Tanzania. Agrofor. Syst. 32, 119–125. Kwesiga, F.R., Ngugi, D., 1996. Agroforestry research in the Miombo ecozone: experiences of the southern Africa. E. Afr. Agric. For. J. 62, 79–101. Miller, M.S., Pepper, I.L., 1988. Survival of a fast-growing strain of lupin rhizobia in Sonoran desert soils. Soil Biol. Biochem. 20, 323–327. Niang, A., Gathumbi, S., Amadalo, B., 1996. The potential of short duration improved fallow for crop productivity enhancement in the highlands of western Kenya. E. Afr. Agric. For. J. 62, 103– 114. Odee, D.W., Sutherland, J.M., Makatiani, E.T., McInroy, S.G., Sprent, J.I., 1997. Phenotypic characteristics and composition of rhizobia associated with woody legumes growing in diverse Kenyan conditions. Plant and Soil 188, 65–75. Onim, J.F.M., Otieno, K., Dzowela, B., 1990. The role of Sesbania as multipurpose trees in small-scale farms in western Kenya. In: Macklin, B., Evans, D.O. (Eds.), Perennial Sesbania Species in Agroforestry Systems. NFTA Special Publication No. 90–01. Nitrogen Fixing Tree Association, Waimanalo, Hawaii, USA, pp. 167–179. Southey, J.F. (Ed.), 1986. Laboratory Methods for Work with Plant and Soil Nematodes. Reference Book No. 402, Ministry of Agriculture, Fisheries and Food, London, UK, 202 pp. Taha, A.H.Y., 1993. Nematode interactions with root-nodule bacteria. In: Khan, M.W. (Ed.), Nematode Interactions. Chapman and Hall, London, UK, pp. 175–202. Vincent, J.M., 1970. A Manual for the Practical Study of RootNodule Bacteria. Blackwell, Oxford. Wallace, H.R., 1989. A perception of tolerance. Nematologica 33, 419–432. Zeck, W.M., 1971. A rating scheme for field evaluation of root-knot infestations. Pflanzenschutz Nachrichten Bayer AG 24, 141– 144.
Interactions between Meloidogyne javanica (Treub) chitwood and rhizobia on growth of Sesbania sesban (L.) Merr. Johan Desaeger a,*, David Odee b, Joseph Machua b, Milton Esitubi b b
a World Agroforestry Centre (ICRAF), P.O. Box 30677, Nairobi, Kenya Biotechnology Laboratory, Kenya Forestry Research Institute (KEFRI), P.O. Box 20412, 00200 Nairobi, Kenya
Received 26 May 2004; accepted 13 December 2004
Abstract A screenhouse experiment was conducted to evaluate the effects of inoculation with Meloidogyne javanica (uninoculated nematode control, low level [1000 eggs pot 1] and high level [10,000 eggs pot 1]) and rhizobial strains (uninoculated rhizobial control, strains KFR 647 and MBS1312) on nematode infection, nodulation and growth of Sesbania sesban (L.) Merr. (sesbania) provenances (Kisii and Kakamega). Heavy nematode infection of sesbania was noted for all three rhizobial inoculation treatments 2 months after sowing, and nodulation and plant growth were affected negatively at the high nematode inoculum level. Shoot dry weights showed negative and positive relationships with nematode infection (gall index) and number of effective rhizobium nodules, respectively. At 3 months, negative effects of the high nematode inoculum level on nodulation and plant growth were observed for rhizobial strain KFR 647 and the uninoculated control, but not for strain MBS 1312. Low nematode inoculum levels did not negatively affect nodulation or plant growth. The two sesbania provenances had similar growth at the high nematode inoculum level, but the Kisii provenance showed better growth than the Kakamega provenance at the low nematode inoculum level. # 2005 Elsevier B.V. All rights reserved. Keywords: Improved fallow; Nodulation; Root-knot nematode; Sesbania provenance
1. Introduction Sesbania sesban (L.) Merr. (sesbania), a fast growing, N2-fixing and deep-rooting tree with good * Corresponding author. Present address: University of Georgia, Department of Plant Pathology, Coastal Plain Experiment Station, Tifton, GA 31794, USA. Tel.: +1 229 386 3160; fax: +1 229 386 7285. E-mail address: [email protected] (J. Desaeger).
quality foliar biomass, is very well suited for replenishing soil fertility and fodder production for small ruminant animals in sub-Saharan Africa. Sesbania has traditionally been used for fodder and firewood in Kenya and Tanzania (Onim et al., 1990). Considerable research has been conducted in the last 15 years on sesbania for improving soil fertility and fodder production in Zambia, Tanzania, Malawi and Kenya (Onim et al., 1990; Kwesiga and Ngugi, 1996; Niang et al., 1996).
0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2004.12.005
J. Desaeger et al. / Applied Soil Ecology 29 (2005) 252–258
A potential constraint for sustainability of sesbania fallows is the fact that the tree is a good host of rootknot nematodes (Meloidogyne incognita and Meloidogyne javanica) (Karachi, 1995), supporting nematode population increase, particularly in light-textured soils (Desaeger and Rao, 2000a,b). Negative effects of root-knot nematode have been observed with several Rhizobium-legume symbioses (Hussey and McGuire, 1987; Taha, 1993). Previous field experiments in western Kenya have shown similar negative relations with sesbania, and nematodes were found to invade sesbania root nodules (Desaeger and Rao, 2000a,b). The potential for soil N replenishment through biological N2-fixation by sesbania could therefore be greatly reduced by rootknot nematodes. A large pool of effective rhizobial strains is currently available for sesbania but their interactions with root-knot nematodes are unknown. Inhibition of nodulation on Acacia seyal by M. javanica was noted for only some rhizobial strains (Duponnois and Cadet, 1994). Duponnois et al. (1999) also noted that some bacterial strains decreased mortality of Meloidogyne-infected Acacia holosericea seedlings. Previous pot experiments with sesbania and rootknot nematodes in Kenya have shown considerable differences between sesbania provenances in their nodulation and tolerance to the nematodes (Desaeger and Rao, 2001). The main objectives of this study were (1) to evaluate the effect of inoculum levels of root-knot nematode on rhizobial nodulation and growth of sesbania, and (2) to assess the interactions of different rhizobia and sesbania provenances with nematode infections.
2. Materials and methods 2.1. Seed source and pre-treatment Seeds of sesbania provenances were obtained from the Regional Agroforestry Centre (KEFRI), Maseno, Kenya. Uniformly sized seeds were pre-treated by immersing in hot boiling water for 2 min to soften the seed coat. They were surface sterilized in 3% NaOCl for 3 min, washed in three changes of sterile distilled water, and then soaked overnight.
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2.2. Preparation of potting substrate and sowing of pre-germinated seeds Moist soil (sand = 83%, silt 11%, clay = 6%) that had been collected locally was heated in an oil drum for 4 h to about 90 8C and afterwards air-dried for 4 weeks before commencing the study to avoid any possible side effects of heating (Anderson and Ingram, 1989). Plastic pots (2.5 l) were filled with sterilised soil and allowed to settle for 1 month before starting the trial. Triple superphosphate was added (3 g pot 1, equivalent to 60 kg P ha 1) and mixed into the soil before sowing. Five pre-germinated sesbania seeds were sown in each pot. 2.3. Rhizobial inoculation Two effective sesbania rhizobial strains were used: (i) KFR 647 originating from Yala swamp, western Kenya, and (ii) MBS 1312 originating from Marigat, Rift Valley, Kenya. Rhizobial cultures were grown to late log phase in yeast mannitol broth (YMB; Vincent, 1970). Seedlings were inoculated 3 days after sowing by watering each with 1 ml suspension of the culture broth containing 1 109 rhizobia. 2.4. Nematode source and inoculation A single egg mass of M. javanica was collected from sesbania on a farm in western Kenya and the nematode was multiplied on sesbania plants in the greenhouse. Nematode eggs were extracted from the rhizosphere of infected sesbania roots and immediately used for inoculation. The pots were inoculated with nematodes through five small holes made around the seeds 3 days after rhizobial inoculation. The sodium hypochlorite method was used to extract nematode eggs (Hussey and Barker, 1973). 2.5. Growth conditions, experimental design and assessments Pots were placed on four raised wooden benches in a screenhouse at the Regional Agroforestry Research Centre in Maseno, western Kenya. The approximate mean temperature was 35/16 8C (day/night) with a 12h photoperiod (natural daylight). Pots were watered every other day to field capacity.
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The experimental design was a 3 3 2 factorial, with three M. javanica inoculum levels (uninoculated nematode control, low [1000 eggs pot 1] and high [10,000 eggs pot 1]), three rhizobial inoculation treatments (uninoculated control and inoculation with strains KFR 647 and MBS1312), and two sesbania provenances (Kakamega and Kisii). Treatments were replicated four times in a randomized complete block design. Seedlings were thinned to two per pot 2 weeks after sowing, and those removed were stained for nematode infection using acid fuchsin (0.03%) in lactoglycerol (Bridge et al., 1982). The remaining seedlings were removed at 2 and 3 months, respectively, after sowing. At each sampling, seedlings were carefully uprooted and soil washed off with gentle running water. Roots were rated for root galling on a 0–10 scale (Zeck, 1971) [0 = no galls, 1 = very few small galls, 2 = numerous small galls, 3 = numerous small galls of which some are grown together, 4 = numerous small and some big galls, 5 = 25% of roots severely galled, 6 = 50% of roots severely galled, 7 = 75% of roots
severely galled, 8 = no healthy roots but plant is still green, 9 = roots rotting and plant dying, 10 = dead plant]. In addition, 1 g of roots was stained for nematode infection using the above lactoglycerol method, and another 1 g was stained for egg masses using phloxine B (Holbrook et al., 1983). At 3 months nematodes were also extracted from soil using a modified Baermann method (Southey, 1986). Nodulation and growth of seedlings were assessed at 2 and 3 months after sowing. Nodules were detached from roots and counted. Shoots, roots and nodules were oven-dried at 60 8C for 72 h, and then weighed. In addition, nodule effectiveness for biological N2-fixation was scored on the basis of internal pigmentation (Beck et al., 1993). Nodulation rate plant 1 was calculated as described by Duponnois et al. (1995). 2.6. Data analysis Analysis of variance (ANOVA), standard error of differences of means (S.E.D.) and linear regression
Table 1 Meloidogyne javanica root infection and growth of sesbania 2 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode infection Gall index (0–10)
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a b
0b 1.5 4.1 0.37 <0.01
Juveniles + females (no. of plant 1)
Egg masses (no. of plant 1)
0b 30 113
0b 12 38
10 <0.01
5 0.01
94 104 49 11 0.01
3.2 2.5 2.7
74 58 84
0.28 0.08
17 0.31
2.5 3.1
60 84
17 32
66 99
0.36 0.09
13 0.07
5 <0.01
8 <0.01
Standard error of difference of treatment means. Not included in ANOVA.
28 23 22
Shoot dry weight (mg plant 1)
5 0.43
69 81 98 8 0.01
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analysis were performed using a GENSTAT1 statistical package (Version 5).
3. Results 3.1. Nematode infection Nematode gall indices on sesbania roots averaged 0, 1.2 and 4 for the control, low and high nematode inocula, respectively, (P < 0.05, data not shown) 2 weeks after sowing. Similarly, root infections were 0, 5 and 23 juvenile nematodes plant 1. At 2 months after sowing, nematode infection was greater at the high inoculum level, and shoot dry weight was significantly reduced at the high inoculum level compared with uninoculated plants or the low nematode inoculum level (Table 1). Sesbania provenance Kisii had greater nematode infection and shoot dry weight than the Kakamega provenance at 2 months after sowing. There were also significant interactions: nematode inoculum level provenance for nematode egg mass infection (P < 0.01), and nematode inocu-
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lum rhizobial strain provenance for gall index and shoot dry weight (P = 0.04). At 3 months after sowing, nematode inoculum level and sesbania provenance showed similar (but not identical) trends in their effects on nematode infection and shoot dry weight as those at 2 months (cf. Tables 1 and 2). 3.2. Rhizobial symbiosis All plants, including the uninoculated controls, developed nodules indicating that the heated oil drum treatment did not eradicate the indigenous rhizobia. All stages of root-knot nematode (juveniles, adult females, eggs) were observed inside nodules. In spite of the large number of nematodes found inside some of the nodules (more than 30 juveniles nodule 1), the nodules appeared healthy. Two months after sowing, the number of nodules was significantly lower with the high nematode inoculum level (3.5, 4.2 and 0.5 nodules seedling 1 [S.E.D. = 0.8] for the uninoculated nematode control, low and high nematode inoculum levels, respectively). There was significant
Table 2 Meloidogyne javanica root infection and growth of sesbania 3 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a b
Nematode infection Gall index (0–10)
Juveniles + females (no. of plant 1)
Egg masses (no. of plant 1)
0b 4.2 6.0
0b 36 126
0b 19 71
Shoot dry weight (mg plant 1)
411 499 295
0.3 0.01
34 0.08
12 0.03
4.8 4.8 5.6
66 95 82
48 44 43
412 381 413
0.7 0.50
20 0.40
15 0.95
101 0.94
36 54
341 463
4.9 5.3 0.4 0.39
Standard error of difference of treatment means. Not included in ANOVA.
60 102 15 0.02
7 0.02
56 0.03
67 0.08
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interaction of nematode inoculum level rhizobial strain provenance for nodule fresh weight and nodulation rate (P < 0.05). In addition, there was a negative relationship between the number of effective nodules and nematode infection at 2 months ( y = 5.24 1.03x, r = 0.63**, d.f. = 47, where y is the number of effective nodules and x the nematode gall index). Shoot dry weight showed negative and positive relationships with nematode infection (gall index) and the number of effective nodules, respectively, at 2 months (Fig. 1). At 3 months after sowing, high nematode inoculum level significantly suppressed nodulation of plants inoculated with rhizobial strain KFR 647 or those nodulated by indigenous rhizobia (Fig. 2). Plants inoculated with rhizobial strain MBS 1312 showed no difference in nodulation between nematode inoculated and uninoculated control plants. However, although the high nematode inoculum level significantly reduced the nodulation rate of sesbania plants, it did not reduce the root and nodule biomass (Table 3).
Fig. 2. Number of nodules plant 1 on 3-month-old sesbania seedlings as affected by M. javanica inoculum level and rhizobial strain (values are means for the two sesbania provenances). The nematode inoculum levels were: uninoculated control (black bars), low (1000 eggs pot 1, dotted bars) and high (10,000 eggs pot 1, dashed bars).
The number of effective nodules was positively correlated with shoot dry weight and root fresh weight at 3 months after sowing ( y = 3.77 + 18.99x1, r = 0.76** and y = 4.20 + 8.73x2, r = 0.66*, d.f. = 70, where y is the number of effective nodules, and x1 and x2 represent shoot dry weight and root fresh weight in g plant 1, respectively).
4. Discussion
Fig. 1. The relationships between shoot dry weight of 2-month-old S. sesban and root-knot nematode gall index (A) and number of effective nodules (B) (values are means for the two sesbania provenances) (n = 36) (*** significant at P < 0.001 level).
Root-knot nematodes readily invaded and reproduced inside sesbania nodules. The negative impact of high nematode infection on sesbania growth and nodulation confirms previous results from the area (Desaeger and Rao, 2000a). The highest nematode inoculum in our trial represents a realistic population in field conditions, where population build-up of up to 10,000 nematode juveniles per 100 cm3 soil has been recorded following sesbania fallow (Desaeger and Rao, 2001). The decrease in significance with time, and the negative relationship between growth and nematode infection at 2 but not at 3 months, is also in accordance with published reports that nematode damage to sesbania is most crucial during the earliest stages of growth (Desaeger and Rao, 2001). A possible explanation for reduced nodulation at high rates of nematode infection could be the formation of Meloidogyne root galls and the suppression of lateral root formation and root hairs, which reduces the number of sites available for nodulation and causes nutrient deficiencies (Fazal et al., 1992). Nodulation
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Table 3 Root and nodule biomass of sesbania 3 months after sowing, as affected by nematode inoculum level, rhizobial strain and provenance Factor
Nematode inoculum level Uninoculated control Low (1000 eggs pot 1) High (10,000 eggs pot 1) S.E.D.a F probability Rhizobial strain Uninoculated control KFR 647 MBS 1312 S.E.D. F probability Sesbania provenance Kakamega Kisii S.E.D. F probability a
Root biomass (mg plant 1)
Nodule biomass (mg plant 1)
Fresh weight
Dry weight
Fresh weight
Dry weight
Nodulation rate (mg root 1)
756 1015 708
276 300 223
156 234 129
21 33 19
0.14 0.11 0.04
12 0.51
0.03 0.05
16 32 25
0.09 0.09 0.10
107 0.06 776 739 964 211 0.53 688 965 119 0.03
44 0.28 243 282 275 76 0.86 276 257 61 0.75
80 0.45 124 211 183 37 0.08 168 177 32 0.79
8 0.21 24 24 7 0.99
0.03 0.94 0.10 0.09 0.03 0.92
Standard error of difference of treatment means.
could also be affected through nodule invasion by nematodes, as previously observed with sesbania (Desaeger and Rao, 2000b). Although Meloidogyne species can develop in nodules without disrupting internal tissue, such interactions have often resulted in premature senescence of bacteroids and nodules (Taha, 1993). Nodules compensated for few nodules by increasing in size at high nematode infection. There were also some differences in rhizobial inoculation responses caused by nematode inoculum level. In particular, there was better nodulation and growth of plants inoculated with rhizobial strain MBS 1312 than strain KFR 647 or indigenous rhizobia at the high nematode inoculum level. This indicates that there is scope for selection of a nematode tolerant Rhizobium–Sesbania symbiosis. Rhizobial strain MBS 1312 from Marigat, Kenya, is interesting in that it has previously been shown to be more tolerant to antibiotics and salt stress than other rhizobia originating from the region (Odee et al., 1997). As arid soils are known to favour growth of antibioticproducing organisms (Miller and Pepper, 1988) and are more prone to drought and salt stress, it is possible that rhizobia in Marigat may have developed resistance due to evolutionary pressures (Odee et al., 1997). The same
physical or physiological defence mechanism could also be responsible for the greater tolerance of strain MBS 1321 to root-knot nematode. There is some evidence that nematode tolerance should be regarded in the wider sense as a general non-specific defence mechanism (Wallace, 1989). The superior early growth of the Kisii as compared to the Kakamega provenance at low nematode infection confirms previous data (Desaeger and Rao, 1999, 2000b), and suggests that the Kisii provenance is more tolerant at low nematode levels. Its relatively poorer growth at the high nematode inoculum level was related to higher rates of infection. Although nematode infection, nodulation and growth of sesbania showed some differences due to nematode inoculum level, rhizobial strain and sesbania provenance, further studies are required to establish whether these differences can be exploited to improve sesbania growth under field conditions.
Acknowledgements We wish to thank ICRAF, KEFRI and the Flemish Office for Development Cooperation and Technical
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Assistance (VVOB), Belgium for financial assistance and Elphas Okonda and Violet Achieng for technical assistance.
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